Practical Microbiology for MBBS [5 ed.] 8178558858, 9788178558851

Table of contents :
Contents
METHODOLOGY
PRACTICAL EXERCISES
SPOTS
APPLIED MICROBIOLOGY
APPENDIX

Citation preview

C P BAVEJA

PRACTICAL MICRO BIOLU for

FREE with

COMPLETE

MICROBIOLOGY

FOR MBBS

As per the revised competency

ARYA

based NMC (MCI) Curriculum

PUBLICATIONS

Contents

Please Note!

The chapters in this book are interrelated, therefore, following points must be remembered

while

studying the book. 1.

2.

Unit I 'Methodology' should be studied first as to perform the 'Practical Exercises' of Unit II.

the relevant

portions of this unit will be helpful

Preliminary study of chapter 4 will help the reader in better understanding of Chapter 5.

Unit I:

1-39

Methodology 1.

Introduction

2. Commonly Used Methods 3. Culture Media

General Approach

12

Culture - A Specific Approach

16

4. Identification of Bacterial Culture 5.

Unit II:

Identification of Bacterial

A

41-149

Practical Exercises

43

6. Staining Exercise 7. Stool Examination Exercise Disease and Infectious Endocarditis 8. Rheumatic Heart 9. Diarrhoea and Dysentery

12. Upper Respiratory Tract Infections Lower Respiratory Tract Infections

14. Collection of Specimens II

16. Infection Control Practices 17.

Malaria and Filariasis

18. Confidentiality in Laboratory Results of Infectious Diseases 19. Appropriate Laboratory Tests in the Diagnosis 20. Spots

63

72

11. Meningitis

15. Collection of Specimens

60

67

10. Enteric Fever

13.

55

79

82 85

90

92

102 109

112 117

209 21

Xopul

i n fe c t i o n 品昌晶员

N

G e n r a l A p ro a c h

⼀

C u l t re M d i a I n t ro d u c t i o r

C o m n l y U s e d M t h o Identif ca o B r Cult

云

Identif ca o B r l Cu A Sp ch

9

H a n d yg i e n

H

三

Antige bodysuea DiagnostcBeriolgy CutreMthodAspliedMcrobilgy

N

Unit

U

⼈6olpuaW

Unit

807 L0Z 堂家器美园 9

Bac teria

5

3号号号

Charcteis ofEg GramVegtivCocN GramPositveCcG adDpegAnsodGram GramNegtivBcl

Lac tose Jlua n

品

APpMoresAuaymd pendixVuIParstfcg euopaHINDdsETUO 4uos8eNMTapd HealthcrAsoidInfSterlsaiondDfectAnimrobalSusceptyTg O E L re I y O D I N

X x i p u a d y A p e n d i x I X A p e n d i x VT A p e n d i xV I A x p u a d v x p u a d y A p e n d i x m A p e n d i x A p e n d i x I 4 p e n d i c e s

Introduction

Microbiology includes the following

Practical in exercises for 1.

undergraduate students.

Staining Methods Smears may be provided for staining to identify the bacteria.

working in the Microbiology laboratory

1. Always wear a white coat or gown while working of in the laboratory. It will prevent contamination your clothes.

2. Identification of bacterial culture Bacterial growth is provided for identification on

in liquid medium. Stool/Faeces examination Faeces specimen is given for isolation and identification of two pathogenic findings i.e. ova a culture plate as well

3.

Practical instructions to students for

as

and/or cysts.

4. Spots Different spots

in the

form of specimens, slides,

should be regarded potentially dangerous and capable of producing

2. All material provided

disease. 3. All cuts and burns, how so ever minor, should be

4.

5.

or

chief laboratory

Working bench/table should be kept free of books, papers and eatables.

Do not moisten

the labels, pencils etc. with your

tongue.

glassware, media etc. are kept for identification with relevant questions to answer in one or two

6. Do not touch eyes, nose and mouth with your fingers

of sentences. This may be in the form

7.

objective

and hands, while working in

separately in subsequent chapters. Each exercise is dealt two parts:

perform

the exercise and

1.

The way student has

2.

interpret. be asked by the The possible questions which may examiner and how

to

to answer these.

8.

after use. and are not to

10.

for examination of different simple, hanging drop preparation

and faeces should

it

examined under high power (X40)

objective of microscope while other Gram's stained preparations staining) are (ZN staining, Albert's staining or

observed under oil immersion

objective (X100). Take

at the care that condenser of the microscope should be objective.} highest level while using oil immersion

Certain

precautions must

handling the live laboratory. These

be

followed

while

microorganisms in Microbiology

precautions would help to minimise transmission of infective agents to laboratory

personnel and thus reduce the laboratory health hazards.

acquired

Do

be kept upright in

the racks

be laid on the bench.

not place cotton wool plugs on the bench. Plugs

should be held in that

hand without touching the part

goes inside the test tube.

11. While transferring culture material from wire loop

off on to your slide, take care that it should not fall

be used

be

laboratory.

other contaminated material, immediately report it thoroughly to your batch tutor for disinfecting before discarding. Always flame the bacteriological loop before and

9. Culture tubes should

examination,

(Important Note: During microscopic selecting power students sometimes have a problem in of objective lens to exercises. To make

the

In the event of accidental spillage of bacterial growth or

been described How to deal with above exercises has

the

reported to your tutor

technician.

structured practical examination (OSPE).

in

to you

the

wire loop.

12. If the wire

loop contains infective material, heat it

slowly to avoid spurting.

13. Test tube mouth should be flamed immediately before and after use in case of handling liquid culture.

14.

Put used slides, coverslips and other contaminated material in

disinfectant jar.

15. Do not sit on the

table tops.

16. Do not eat in the laboratory. 17. Students with long hair should tie them

at the

back properly to avoid risk of contamination and catching fire.

e

a

물

록

대

공

z

1

분" 3

를

l

몸음

을

l

경

(

율류

홈을

L

협

1

는

떨

x 육공

이

Unit I Methodology Precautions

use oil immersion (1) Never

power objective (X10).

examine the hanging drop preparation

if it has dried up. Dry preparation

may show

it is

always better

to

repeat the hanging drop preparation for proper

interpretation.

separately, taking care that no

Examine the preparation under the

below).

microscope(Sce

() Faecesin container should be mixed properly

with wooden stick before adding either to saline iodine drops. Sometimes or cysts may or settle downin thecontainer, mixing ensures the uniform distribution of these within the faeces.

Culture Media

ti) If preparation has dried up,prepareanotherslide for examination purposes.

STOOL/FAECES EXAMINATION Stool examination is done to find out ova and cysts of different parasites. Method (Fig 2.3)

Wet preparations of stool specimen are prepared

as

follows:

preparation as well as iodine preparation of stool are made on the glass slide. 2. A drop of normal saline (0.9 per cent) is put one end of a clean glass slide while on the other end of

1. Saline

the

5.

Precautions

false results i.e. motile bacteria may be seen

as non-motile. Therefore,

(saline and iodine) air bubble forms

objective (X100) for

hanging drop examination. (i) Always look for the motility of bacteria under the high power objective (X40) and not under the low

slide, a drop of iodine solution is put.

3. A minute portion of faeces is added in both the drops with the help of a small wooden stick

PREVIEW

Examination

COMPETENCY

I. Compositon of media

1. Examine both

the preparations (saline and iodine) under low power objective (X10)starting from one a particular given direction(Refer to in end another Fig. 2.4).

II.

MI 2.3, MI 3.2, MI 3.4 and MI 5.3 as mentioned in chapter 5,

Identification of Media

Knowledge of this chapter will be applied in competencies of chapters

2. Any suspicious object, if found, should be studied

underhigh power objective (X40)foridentification confirmation o: the ova, trophozoites or cysts. 3. Always show your findings to the examinerunder high power objective

student should be able to learm:

SPECIFIC LEARNING OBIECTIVES

1. To identify various culture media.

(X40).

To apply this knowledge to identify bacteria in chapters 4 and

2.

make a uniform suspension.

Two types of culture media

most commonly

used

(v) Neutral red (indicator)

practical microbiology include solid and liquid

Fig. 2.4 Stool examination:It should be done in the direction of arrows starting from one end

preparation

preparation

Fig. 2.3 Stool preparation

() low show your findings to the examinerunder Neverpower (X10) or oilimmersion lens (X100). not report normal non-pathogenic findings, e.g. cyst of Entamoeba coli.

Undestandandstrictlyfollowalltheworkingstupsofvariousmethodsbecausethesewils usedin different practical

exercises later in subsequent chapters.

2. In Albert's staining,smear is always blot dried and not washed with water. Decolourisation stepis veryimportant1 4.

precautions preparation Erythr allnecesary The Follow proper

during precautionswhile making 2

and during its

1.

I.

COMPOSITON

A.

Solid Media

2.

Nutrient broth

3.

3.

100 mL Peptone

water

Meat extract- 1%

Brain heart infusion (BHI) broth (1) Brain and beef heart

Peptone water

(ii) Peptones

Meat extract-

(ini) Glucose

(iv) Sodium chloride (v) Buffers

Nutrient agar

blood (5-10%)

IL.

MacConkey's agar

(1) Peptone (ii) Lactose (iii) Sodium taurocholate (iv) Agar

Nutrient broth

(in) Glucose (0.5%)

2. Blood agar (i)

Glucose broth (1)

OF MEDIA

(ii) Agar (2-3%)

(11) Sheep

1%

0.5%

(iii) Water to make

Nutrient agar (1)

Peptone

(ii) NaCl

Peptone water Glucose broth

Brain heart infusion (BHI) broth

1.

examination (refer page

(i)

Liquid media

of thehangingarp

anditsexamination (refer page7 and 8). wet preparation of the stool sample

1. Peptone water (pH 7.4)

Chocolate agar

4.

Liquid Media

B.

MacConkey's agar

3.

Essential Points to Remember

6.

It is heated blood agar.

Nutrient agar 2. Blood agar

(ii) Do

100 mL

4. Chocolate agar

Solid media 1.

Precautions Normal saline

0.075 gm

(vi) Water to make

media.

2.0 gm 1.0

0.5 gm 0.5 gm

IDENTIFICATION OF MEDIA

A. Solid Media (Table 3.1) Nutrient agar semi-transparent and pale, yellowish in

colour (Fig

3.1).It is not difficult to identify this medium.

Blood agaris bright red in colour and itis opaque (Fig. 3.2). Two layers are present

in

blood agar medium, one

10

Unit

Table 3.1:

Methodology

Differentiating Features of Three Solid Media

Character

Nutrient

Blood agar agar

agar

1. Colour

2.

Opacity

3. Two

Pale

Bright red

yellowish

(like blood)

Semi-

Opaque

Pink red

Transparent

transparent Absent

Present

Absent

layers

Fig. 3.3 MacConkey's agar

in the

medium

Fig. 3.1 Nutrient

Fig. 3.4 Chocolate agar

agar

Liquid Media (Fig. 3.5) Glucose broth is yellowish in colour while peptone B.

water is colourless or less yellowish than glucose broth. media are dispensed in cotton plugged

Both the liquid test tubes.

Composition and uses of some liquid and solid media

are shown in Table 3.2 and Table 3.3.

Fig. 3.2 Blood agar

above the other. The lower layer is

of nutrient agar and upper layer is of sheep blood. In contrast, MacConkey's agar is transparent and it is pink red in colour (Fig. 3.3). If you keep your finger outside below the culture plate and try to see your finger through the medium, it is clearly visible; whereas the finger cannot be seen clearly through the blood agar. Absence of two layers in MacConkey's agar is another differentiating feature. All the three media are dispensed in petridish.

Chocolate agar is heated blood agar. The colour of medium changes to brown and named chocolate agar (Fig. 3.4).

Fig. 3.5 Peptone water

and glucose

broth

Culture Media Table 3.2

Composition and Uses of Liquid Media Medium

1.

Composition

Peptone water

Uses

Peptone 1% NaCI - 0.5%

Routine culture, Sugar

(pH 7.4)

Peptone water Meat extract-

Glucose broth

Routine culture

1'

10A Isigned

Nutrient broth

Blood culture, Culture of certain organisms e.g. Streptococci

+

Glucose - 0.5% 4.

5.

Brain-Heart Infusion (BHI) broth

fermentation

tests

Water - 100 mL

2. Nutrient broth

3.

11

Brain and beef heart, peptones,

Blood culture, culture of certain

glucose, sodium chloride and buffers

organisms, antibiotic sensitivity testing

Peptone water (pH 9.0)

Culture of Vibrio

Enrichment media (i) Alkaline peptone water (ii) Selenite F broth

Peptone water, Sodium selenite

Culture

of

faeces for salmonellae and

shigellae

Nutrient broth, Sodium thiosulphate, Culture of faeces especially for Calcium carbonate, Iodine solution

(iv) Cooked meat broth (CMB)/ Robertson's cooked meat broth

salmonellae

Nutrient broth, Predigested cooked Culture of anaerobic bacteria meat of ox heart

(RCM)

Table 3.3 Composition and Uses of Solid Media Composition

Medium 1.

Uses

Simple medium (i) Nutrient agar

Nutrient broth,

Routine culture

Agar (2-3%)

Enriched media

(i) Blood agar (ii) Chocolate agar

(iii) Loeffler's serum slope

Nutrient agar, Sheep blood (5-10%)

Routine culture

Heated blood agar (55°C x 2 hr)

Culture of Neisseria, H. influenzae

Nutrient broth,

Glucose, Horse serum Culture of C. diphtheriae

Indicator medium

3.

(i)

MacConkey's medium

Culture of Gram negative bacilli

Peptone Lactose

Sodium taurocholate Agar Neutral red

Selective media

4.

(i)

Deoxycholate citrate agar

(ii) Bile salt

agar (BSA)

Nutrient agar,

Sodium deoxycholate,

Culture of Salmonellae and Shigellae

Sodium citrate, Lactose, Neutral red

Nutrient agar, Sodium taurocholate

Culture of Vibrio cholerae

(0.5%) pH 8.2

Essential Points to Remember 1.

Identify the solid culture medium

2. Remember the medium.

correctly especially the blood agar from the MacConkey's agar (refer pages 9 and

composition of different media (refer page

11) and also

10).

the name of indicator, if any, present in the

음

1

유곡

7

B

N

(# 물s

e

e 입

9 폭

때

일

e

을

을

e화

법

종인

종e

졸역

(

1

로

우

i

i

)

홈;

ss

좋

요 엄

h

E

Unit I Methodology Gram's staining (Fig. 5.12)

small

(1

Identification

Gram's staining (Fig 5.14) Gram negative bacilli

um

Cocci are Gram positive, slightly elongated arranged in pairs (diplococci). They are flame shaped or lanceolate

in size

diameter),

of

Bacterial Culture

Specific Approach

measuring 1-3um x 05um

appearance.

Pin-point

Pin-head

Swarming

Uniform

turbidity A

Fig. 5.12 Gram positive

pairs (diplococci) as seen

100 magnification (diagramatic)

under

Fig. 5.14 Gram negative bacilli of

under X100 (B)

5.

Presumptive diagnosis Streptococcus pneumoniae

Proteus species: (A)

diagrammatic representation

seen

Hanging drop (Fig. 5.15) Actively motile

Gram positive

Draw well labelled diagrams of your observations

5.

Gram's staining

COCCI

Gram positive

in clusters

chains

Non-motile* and Non-motile'

(Take help of Figs. 5.11 and 5.12). Use colour pencils

Hanging drop preparation

Gram positive

Gram

cocci pairs

and Non-

negative bacilli and

motile"

Motile*

lan-motife

to show Gram reaction.

6. Confirmatory tests (1) Bile solubility test: (ii)

(iii)

positive Inulin fermentation: positive

species

Optochin sensitivity: positive differentiate S. pneumoniae

*On

The above tests

will also from viridans streptococci

as follows:

b.

Presumptive diagnosis

Catalase t

Proteus spp 7.

Draw well labelled diagrams of your observations (Take help of Figs. 5.14 and 5.15). Use colour pencils

8.

Confirmatory tests

Medium

Positive

to show Gram reaction.

Blood agar

Colony morphology (Fig. 5.13) 'Swarming' is exhibited on blood agar and characteristic fishy or seminal odour may occur.

Staphylococcus spp.

(i) PPA test - Positive (ii) Urease test - Positive (iii) Glucose - A/G

Growth in peptone water

(iv)

Uniform turbidity with slight powdery deposit and

Streptococcus

Positive

in

both Pr.

vulgaris and

Pr.

Positive in Pr. vulgaris (vii) Ornithine decarboxylase - Positive in Pr. mirabilis but negative in vulgaris (vili) Methyl red (MR) - Positive (ix) Voges-Proskauer (VP) Negative 9. Final diagnosis (vi) Indole

Depending on biochemical reactions, it may

identified as Pr. vulgaris or Pr. mirabilis.

blood

on

blood agar have been summarised in Fig. 5.16.

Confirmatory tests for bacteria grown are shown in

on

Fig. 5.17(a) and Fig. 5.17 (b).

Positive

Negative

Sensitive

CAMP reaction

Other staphylo

coccal species Micrococcus spp.

Group 'B'

Group 'D

streptococci

streptococa

be

Salient features for identification of bacterial growth

showing swarming

pneumoniao (pneumococcus)

Coagulase test

Lactose - Negative

ammonical odour is present.

agal

Identification Scheme of Bacterial Growth on Blood Agar and in Liquid Medium

(i) For Gram positive cocci

solid

medium, growth in liquid medium, Gram's staining and hanging drop preparation, findings should be recorded

Fig. 5.13 Growth of Proteus spp.

hanging drop preparation Fig. 5.16

species

After observing the colony morphology on

3.

Proteus

species

Fig. 5.15 Hanging drop preparation: Motile bacilli of Proteus

B. Proteus spp.

2.

Pneumococcus Streptococcus species (other than (Streptococcus

blood agar

Confirmation by Lancefield technique

Fig. 5.17 (a) Confirmatory tests for

Gram positive coca

21

Unit

Methodology

(ii) For Proteus SPP.

Urease test

If positive

Proteus spp. H,S production

Positive

Indole test

Positive

Negative

Pr. vulgaris

Pr. mirabilis

Fig. 5.17 (b) Confirmatory Tests for Proteus

III. MACCONKEY'S AGAR

A. Lactose fermenter

In general practice, for undergraduate students, important LF bacteria given are Esch. coli and Klebsiella spp. Both the bacteria are discussed simultaneously so that student can differentiate

Working Steps

1. On MacConkey's agar, colonies may be either pink (lactose fermenter, LF) or pale (non-lactose fermenter, NLF). 2. LF colonies may be Esch. coli or Klebsiella spp., while NLF colonies generally given are Salmonella spp. or Shigella spp. 3. Examine the colony characters on solid medium and growth in liquid medium. 4. Gram staining and hanging drop preparation are

Chapter 2, pages and 7). The following features may be observed on Gram's staining and hanging drop preparation. to be

5.

b.

two

the two easily.

Important features of Esch. coli and Klebsiella are shown in Table 5.2 and Figs. 5.18 to 5.23. 3. Presumptive diagnosis: On the basis of above 2.

features, presumptive diagnosis of Esch. coli or Klebsiella spp. can

be made.

(Important note: Klebsiella spp. shows mucoid colonies, short and plump bacilli on Gram staining and they are non-motile.}

made (Refer

(i) Gram staining: Gram negative bacilli (ii) Hanging drop: Motile or non-motile By correlating the colony characters, Gram staining

and

(LF) colonies

1.

4. Draw well labelled diagrams of your observations

hanging drop preparation, the bacteria may be

identified.

for Esch. coli

(Take the help of Figs. 5.19 and 5.20) or Klebsiella spp. (Take the help of Figs. 5.22 and 5.23). Use colour pencils to show Gram

reaction.

Table 5.2: Salient features of Esch. coli and Klebsiella spp. compared Character

1. Colony morphology on MacConkey's agar

2.

Growth in peptone water

3. Gram

staining

4. Hanging drop preparation

Esch. coli

Colonies are pink due to lactose fermentation Colonies are pink or red in colour due to (LF or lactose fermenter colonies), circular, lactose fermentation (LF or lactose fermenter moist, smooth with entire margin, non- colonies), large and mucoid. mucoid.

Uniform turbidity.

Uniform turbidity.

Gram negative bacillus measuring 1-3 um 0.4-0.7 um, long and thin.

Gram negative bacillus measuring

Motile bacilli.

Non-motile bacilli.

1-2 um

0.5-0.8 um short, plump.

Identification of Bacterial Culture - A Specific Approach

23

Fig. 5.18 Lactose fermenter colonies (non-mucoid) of Esch. coli

on MacConkey's agar

Fig. 5.23 Hanging drop preparation: Non-motile bacilli of Confirmatory tests of Esch. coli and Klebsiella spp.:

5.

The fermentation of

carbo-hydrates and other

biochemical reactions

are

very important in

differentiating and confirmation of these bacteria (Table 5.3).

two

Table 5.3: Biochemical Reactions of Esch. coli and B

Fig. 5.19 Gram negative bacilli of Esch coli: (A) as seen under X100 magnification (B) diagrammatic representation

Esch.

Test

coli.

1. Catalase 2. Oxidase 3. Glucose

4. Lactose 5. Mannitol b. Sucrose

7. Indole 8.

Urease

9. Citrate

Fig. 5.20 Hanging drop preparation: Motile bacilli of Esch. coli

10. Methyl red (MR) 11. Voges-Proskauer (VP)

+12

1+ 0I

+ : Positive; - : Negative; AG : Acid and gas

IMViC (Indole, MR, VP and Citrate) are important to remember.

IM

Esch. coli

ViC

++

Klebsiella spp. mentioned biochemical reactions, Esch. coli or Klebsiella spp. can be final diagnosis made but tests such as serotyping should be done for Based on the above of

Fig.

5.21 Lactose fermenter colonies (mucoid) of Klebsiella

spp. on

MacConkey's agar

B. B

A

confirmation.

Non-lactose fermenter (NLF) colonies 1. For

undergraduate students, two important

NLF bacteria provided are Salmonella spp. and

Shigella spp. Both the bacteria are discussed simultaneously sO that student may not find any difficulty in differentiating the two. 2.

Important features of Salmonella spp. and Shigella spp.

3.

negative bacilli of Klebsiella spp (A) as seen X100 magnification (B) Diagrammatic representation

Fig. 5.22 Gram

under

are shown in Table 5.4 and Figs. 5.24 to 5.29.

Presumptive diagnosis: On the basis of above features, presumptive diagnosis of Salmonella spp. or Shigella spp. can be made.

Unit

24

Methodology

Table 5.4: Differentiating Features of Salmonella

MacConkey's agar 2. Growth in 3. Gram

peptone water

staining

4. Hanging drop preparation

Shigella SPP.

Shigella spp.

Salmonella spp.

Character

1. Colony morphology on

and

2-3 mm in diameter, Colonies are 2 mm in diameter, circular, translucent, smooth, smooth, convex, colourless (NLF) except in case of Sh. sonnei which forms pink colonies due to late lactose fermentation. convex, colourless (NLF).

Colonies are

circular, translucent, low

Uniform turbidity

Uniform turbidity

Gram negative, short, bacilli about 1-3 um x

Gram negative bacilli measuring 1-3 um x 0.5 pm

0.5 um

Non-motile bacilli

Motile bacilli

{Note: Important feature which differentiates the two is motility.} 4.

Draw well labelled

diagrams of your observations

of Salmonella spp. (Take the help of Figs. 5.25 and 5.26) or Shigella spp. (Take the help of Figs. 5.28 and 5.29). Use colour pencils to show Gram reaction.

Fig. 5.27 Non-lactose fermenter colonies of

MacConkey's agar

Shigella spp. on

Fig. 5.24 Non-lactose fermenter colonies of Salmonella spp

on MacConkey's agar

A

Fig. 5.28 Gram negative bacilli of

Shigella spp.:

(A)

as seen

under X100 magnification (B) diagrammatic representation

A

B

Fig. 5.25 Gram

negative bacilli of Salmonella spp.: (A) as seen under X100 magnification (B) diagrammatic representation

Fig.

5.29 Hanging drop preparation: Non-motile bacilli of Shigella spp. 5.

Confirmatory tests of Salmonella spp. and Shigella spp.: The fermentation of

Fig. 5.26

Hanging drop preparation: Motile bacilli of Salmonella

carbo-hydrates and

other biochemical reactions are very important to differentiate these two bacteria at species level

(Tables 5.5 and 5.6).

Identification of Shigella

are

spp.

anaerogenic with few exceptions.

They ferment carbohydrates by production of acid only. They are catalase positive (with some exceptions) and

oxidase negative. They are urease, citrate, H,S and KCN negative.

Bacterial Culture A

have been summarised

in

made

A. Vibrio cholerae

summarised in Fig. 5.30.

IV. GROWTH ON ANY OTHER MEDIUM If bacterial growth has been provided on the medium other than nutrient agar, blood agar and MacConkey's agar, the basic method of processing will remain the same as discussed in Chapter 4. In case of bacteria other than those mentioned in this chapter, identification will depend on the basis of colony morphology, Gram staining, hanging drop preparation and further by confirmatory tests. After isolating the bacteria, specific questions related to that bacterium may be asked, however, general questions related to culture media, Gram staining, hanging drop will remain the same.

Table 5.5: Biochemical

Catalase

2.

Oxidase

follows:

You may be provided with the bacterial growth on one

of the selective media (solid media) such as Thiosulphate citrate bile sucrose (TCBS) agar or Bile salt agar (BSA) and as well as

in the liquid medium such as alkaline

peptone water (APW). After observing the colony morphology on solid medium, the growth in liquid medium, Gram's staining and

hanging drop preparation, the findings should be

recorded as follows: 1. Medium TCBS or BSA 2

Colony morphology On TCBS, the colonies are large, circular, convex and yellow coloured.

Reactions of Salmonella species

Test 1.

Fig. 5.31.

Some examples of growth on any other medium are as

Salient features for identification of bacterial growth on MacConkey's agar and liquid medium have been

S. Paratyphi A

S. Typhi

S.

Paratyphi B

S. Paratyphi C

S. Typhimurium +

+

AG

AG

AG

3. Glucose

AG

4. Lactose

AG

AG

5. Mannitol 6. Sucrose 7.

Indole

8. Citrate

9. Methyl red

(MR)

10. Voges-Proskauer (VP)

11. H,S production Positive; -

Negative; A : Acid; AG : Acid and gas

Table 5.6: Biochemical Reactions of Shigella species Test 1.

Sh. dysentriae

Sh. flexneri

A

A

Glucose

A

3. Mannitol 4. Sucrose

5. Ornithine decarboxylase Positive; -

Sh.

boydii

Sh. sonnei

A

A*

2. Lactose

+

25

Principal features for identification of bacterial growth on any media (other than nutrient agar, blood agar and MacConkey's agar) along with liquid medium

On the above mentioned biochemical reactions, the diagnosis of Salmonella species or Shigella species can be

but serotyping should be used for confirmation.

Specific Approach

Negative; A: Acid, *

Late fermenter

때

Be일

1

9

R

l바z

3

입 BBs

폐

3

인 중

33

입

대

없

음

3 트

g

을

(

클

좋g

배

베

!a

i

I

1

3 3 Be

홈

좋

'

입

8

6g

출e

.

용

를

!

경

스 #e

홍 트

영

를

z

을

를

8

불를

등

2

1 g

종n

l E

#

를

류의

6

료 8

의 s a

ttla

용 분종

h l

영

방

를

요

을e

e

캐

sed33

(

입

를

인

e 중을

5

9r

4

중

E르

pEF메

/HII

베a

요

배

융

l

인

을

5

평

대

일 e

때

입

(

(동합

임

e

을

i

를

요

음

인

음

폭

g 없

e

은

3

g

읍 플

입 준입

읍

9

입k

율

트

I

3을

i

을

콜

I

f

m

요

입

e

번

요

콜

3 폭로

1

읍

을

1을

올

5

의

g

좋

*

e국

베

을

요

i

클

을

i를

e

을

월

올

e

배

업

o

1

음

동

출

g

a

음

유 9

Practical Exercises 6. Staining Exercise

43

7. Stool Examination Exercis e. .P........................l.

55

8.

Rheumatic Heart Disease and Infectious Endocarditis

9. Diarrhoea and Dysentery ...................

63

67

10. Enteric Fever ...........................r.

72

11. Meningitis ..............................

79

12. Upper Respiratory Tract Infections 13.

60

82

Lower Respiratory Tract Infections

85

14. Collection of Specimens

90

15. Collection of Specimens IT

92

16. Infection Control Practices p.......... .... .t.........g.u.... 102

17. Malaria and Filariasis

18. Confidentiality in Laboratory Results

19. Appropriate Laboratory Tests

in the

Diagnosis of Infectious Diseases ........we.o.m

109

112

117

20. Spots

Staining Exercise PREVIEW I.

COMPETENCY

Gram Staining

The student should be able to

II. Ziehl-Neelsen (ZN) Staining or Acid-Fast

MI 1.2

Staining

Perform and identify the different

causative agents

of infectious diseases by Gram stain, ZN stain and

III. Albert's Staining

stool routine

microscopy.

this chapter, staining methods have been described. However, stool/faeces examination has been described in chapter 7. In

SPECIFIC LEARNING OBJECTIVES The student should be able to learn: 1. To

identify various bacteria on Gram staining.

2. To identify Mycobacteria on Ziehl-Neelsen (ZN) staining. 3.

Fixed

To identify Corynebacteria on Albert's staining.

unfixed smear may be provided for Gram staining, Ziehl-Neelsen staining or Albert's staining. This exercise is the or

The commonly given smears include Gram positive

cocci and Gram negative bacilli. In case of Gram positive cocci, observe the arrangement of cocci which

to be performed independently by

student.

I. GRAM STAINING

Morphology of some important bacteria

staining is shown in Table 6.1.

A fixed bacterial smear is provided for Gram In case of

staining. unfixed smear, fix the smear by gently heating

Table 6.1 Characteristics of

the slide from underneath and then stain it.

S.

Procedure 1. 2.

3.

1.

Staphylococcus aureus

smear

under oil immersion

Report your observations.

2.

Different Bacteria

Gram positive cocci Non-motile

Arranged in grape like clusters Streptococcus

Gram positive cocci Non-motile 0.5 um in diameter

Arranged in chains 3.

the smear, these may be cocci, bacilli or coccobacilli.

Streptococcus pneumoniae

On combination of Gram reaction and morphology; the following possible observations may be present.

Gram positive cocci Non-motile I

um in

diameter

Arranged in pairs

Gram positive cocci. Gram

Gram

lum in diameter

objective (X100) of microscope.

In case of Gram staining smear, Gram negative or Gram Positive bacteria are observed. Depending upon bacteria in

•

on

Morphology

No.

Apply Gram staining method as described in

chapter 2, page 5. Examine the stained

Bacteria

Observations

•

may be

in clusters, in chains or in diplococci.

4.

negative bacilli

Neisseria

meningitidis

Gram negative coccobacilli Gram positive bacilli

Gram negative cocci

Non-motile

0.6 um-0.8 um

in

diameter

Arranged in pairs (diplococci)

Gram negative cocci

with adjacent sides flattened

43

Contd.

44

Unit II Practical Exercises

Table 6.1 (Contd.)

Morphology

Bacteria

S

No.

5.

Corynebacterium • Gram positive bacilli diphtherine

The bacilli look green

and

metachromatic granules appear bluish black when Albert stain is used.

Arranged in Chinese letter pattern 3-6 pm 0.6-0.8 j1m 6.

Escherichia coli

A

Gram negative bacilli 1-3 pm x 0.4-0.7 pm

7.

Shigella spp.

Gram negative bacilli

•

Salmonella

ACID-FAST STAINING

Gram negative bacilli

Vibrio cholerae

10.

Pseudomonas

11.

Haemophilus influenzae

from underneath and then stain it.

Gram negative

•

Curved or comma shaped bacilli

Procedure

um x 0.2-0.4 um

1. Apply the method of Ziehl-Neelsen (ZN) staining as described in Chapter 2, b.

• Actively motile (darting motility) Gram •

negative bacilli

2.

Motile

1.5-3 um x 0.5 um •

•

3.

Gram negative bacilli 1.5 pm x 0.3 pm

4.

Non-motile

Non-sporing Draw a well

labelled diagram of your observations

using colour pencils (take help

of

objective (X100) of microscope. Grading of

smear (Table 6.2) is done

Report your observations.

No. of AFB seen in

oil

mainly on the

20

10-99 AFB/100 1-9 AFB/100 No AFB/100

B

in

Report

of fields

examined 1-10 AFB/field

magnification (B) diagrammaticclusters (A) seen under X100 representation

(ZN) Smear according

Number

immersion field*

>10 AFB/field

Fig. 6.1 Gram positive cocci

immersion

to RNTCP (now NTEP). Gradethe givenaccording smear.

RNTCP (now NTEP)

Figs. 6.1,

this exercise is

page

Examine the stained smear under oil

Table 6.2 Grading of Ziehl-Neelsen to

6.2 and a good of 63i field focus stained Always your smear because the emphasis of

staining technique.

staining. In

unfixed smear, fix the smear by gently heating the slide case of

•

1.5

STAINING OR

A fixed smear is provided for Ziehl-Neelsen

Motile

9.

6.3 Gram

II. ZIEHL-NEELSEN (ZN)

Non-Motile

1-3 um x 0.5 pm 8.

B

negative bacilli (A) as seen under X100 (B) diagrammatic representation Fig.

Motile

as

*

objective

50

fields

fields

fields

10X eye piece

Positive 3+

Positive 2+

100

Positive

100

1+

Doubtful

positive 100

should be used

Negative, repeat

along withoil immersion

Observations In

shte of Mycobacteriumtuberculosis,baciiae

bright red (acid-fast) seen blue (Fig. 6.4). These while the tissue cells are stained bright red bacilli are called acid-

fust bacilli (AFB).

Draw a well labelled diagram of your observations using colour

Fig. 6.2 Gram

positive cocciin chains (A) magnification (B) diagrammatic

as

seen under

representation

X100

pencils 6.4). Always focus (take help of Fig. good stained field of your smear

because the emphasisor

staining technique.

this exercise

is

mainly on the

Staining Exercise

45

Observations In case

of Corynebacterium diphtheriae, green

bluish black metachromatic granules

bacilli with Pus

are observed. Bacilli

cell

are

arranged

cuneiform arrangement (Fig. 6.5).

field of help of Fig. 6.5). Always focus good stained this exercise is your smear because the emphasis of

B

mainly on the

Fig. 6.4 Mycobacterium tuberculosis on Ziehl-Neelsen

staining technique.

staining: Acid-fast bacilli (AFB) (A) as seen under X100 magnification (B) diagrammatic representation

presentation

LSEN (ZN) STAINING

well labelled

diagram of your observations using colour pencils (take

Acid-fast bacilli

tive bacilli (A) as seen under X100

in Chinese letter

Draw a

ALBERT'S STAINING

OR

STAINING

ovided for Ziehl-Neelsen staining. In ear, fix the smear by gently heating

lerneath and then stain it.

A fixed bacterial smear is provided for Albert's staining. In case of unfixed smear, fix the smear by gently heating

the slide from underneath and then stain it. Procedure

Apply the method of Albert's staining as described

in chapter 2, page 6.

of Ziehl-Neelsen (ZN) staining Chapter 2, page b.

thod

tained smear under oil immersion

of microscope.

ear (Table

6.2) is done

Examine the stained smear under oil immersion

Fig. 6.5 Green

3.

objective (X100) of microscope. Report your observations.

X100

according to

Essential Points to Remember

TEP) Grade the given smear.

servations.

Ziehl-Neelsen (ZN) Smear according

Number

Corynebacterium diphtheriae on Albert's staining: bacilli with metachromatic granules (A) as seen under magnification (B) diagrammatic representation

2.

1.

Follow the working steps of staining of the smear correctly (refer pages

2.

Always examine the stained preparation under oil

3.

Report a good stained field

4.

Draw a well

and 6)

immersion objective (X100).

of the smear to the examiner.

labelled coloured diagram of special staining.

Report

of fields

examined 20

Positive 3+

50

Positive 2+

100

Positive 1+

Questions Related to Gram Staining Q.1. How will you clean new glass slides? Ans. New glass slides can be made grease free by rubbing the surface with cotton cloth and then by heating over a flame. 0.2 What are the

100

Doubtful

Ans.

positive 100

of fixation of

a

smear are as

follows: (i) The smear gets fixed to the slide.

Negative, repeat

be used along

advantages of fixation of a smear?

The advantages

with oil immersion

(ii) Bacteria gets killed thus safer

and become non-infectious,

to work with.

Q.3 What should be

tuberculosis, bacilli are seen

right

red bacilli are

as

stained

called acid-

well labelled diagram of your our pencils (take help of Fig.

od stained

field of your smear

this exercise is mainly on the

immersion

Why oil is used for examination under oil objective? to Ans. Immersion oils have a refractive index similar to be light glass, thus, use of these oils permit more Q.4

incorporated in the image resulting in improving the resolution power.

2.5 What do

"resolution you understand by the term

power of the

microscope"?

Resolution power is the ability of the lens system to distinguish two objects as separate rather than one.

Q.6 Describe the method of Gram's staining. Ans. Refer

0.7

Chapter 2, page 5.

Can you use stain other than crystal voilet in Gram's

staining? Ans. Methyl violet or gentian violet may be used. Q.8 Can you use decolourising agent other than acetone in Gram's staining? Ans.

the position of condenser while

immersion lens? examining the smear under oil Ans. Condenser should be at the highest level.

thile the tissue cells are

Ans.

Alcohol may be used.

0.9 Can we use counterstain other than safranin in

Gram's staining? fuchsin may be used.

Ans. Carbol

Q.10 Who invented Gram's staining? Ans. Christian Gram. Q.11 Why are bacteria classified

as

Gram-positive or

Gram-negative on Gram's staining? Ans.

Gram positive: They resist decolourisation and retain the colour of primary stain (crystal violet) i.e. violet colour.

8

川

*

雅M

【美美

9

⾨% .『

38ee5

【 &

国下

2年 : :s

加

開

&

:『 &

『ii1

開関語

&:

川#

浅s⽉ e

『e

좋을 플

z

을

으

.

2

S

율

물

(

옮

없 B

5

를

경역

#

윤 음

2

바

독 등

좋문

중

8

을

움

8

(

움

H e

을

육

음 s B

#

마

l

.

입

율

요

본

( 2

hil

유

1

배

우

Be

좋 좋

6

문

중

lt

좋 e

순3

대베베

대 대법

2

1

바

#

f

배

i

을

B

e

1

IE "

I| 1

E

매 !

1 I

i

(

문

을

1

대

플

문 유 유을

립

3

(

을열유

에

약

B3 z 일 g

7

e 3

"

을

3

줄

육

좋

#

:

움

(

을

없

#

통

g

a

울

율

물

를

좋을

(5

야

베

움B문 l

를

을

입e

물

육8을

분

공

에

a

적

메

바

올

패를

대

대

베

비z P

"

z

유

Lo d s u e I

1 T L OA

号

G

u o m a l fo

N

8

Fa e c e s

Specimens

S

Ro u t i n e D a g o s i L A B O R T Y D I G N S

E P 三

9

e g m e n ts

Entamoeb Hhist ly ca

Bac teria

t n p m d s ay r s e r d

Suigel a sp

Table9.2CustivAgnofDyer austivegnofdrhy w S

三

6 C u l t re

9

o cholera

5

can

三 美

i n f a e c s H owe v W D i S t a i n g

9

ondit ons

r e c t a l sw b

百

ueap

5

larv e of sar e d

每 ≥

万

uonDa p

3

puop

万

disease Saline e

t re a d i l y v a b l e

Mdo s bnu d i re c t

3

pInbr

9

C o l u r fo l w i n g

u l t re i p a u

9

onaj rp out

una d wmpuo s di

dasulodspnCystou prabeli MPLD

1 Ro t av i r u s

SalIser

Sa nILA

Solid Media X v l o s e y i n e s u c r o e TC B S a g r l a c C o n k e y a g r

sTulaodsv ormaoirus

EIEC

Diarthoea

PP

srisnp

SGrampositvebclwith taplyocusGrmitveco

blo d ag r n a e m o l ys i O n b l o d

Motile

media

er f inge s Clostrid m

a u re u s

Salmonel h

n

c u l t u re

Slide ag lutina o

fermntaio NLF coluresdctloniesarcul

c u l t re

Nn eun 13pm 0.5um Gramnegtiv baclCiapsulatedNonmotile46umx1umsubterminalspoe Arangedi1BrpIumlkeindametNornmotile n e g a t i ve b a c i l

Shigel a sPp

P

c u l t u re

g

cholera f om

motile dar ng u n T 0 Z

C u l t u re

Table9.3ChrctisFuofmBPgnDy

3 7 C fo r 1

H

p ayo u a m D 1 o n E y u o P I

三

e

Isouie d

1g en ts

E

D

SaDL 6 nedia

PHIOS C

g

S e l n i t F b ro t h

b ro t h

g

DoLE

incubal

done

E n r i c h m e r L i q u d M e i a 1 8 24 h o u r s

Teruaporg

eipou ATLSIH

hIdentifcaoThbrgw ThesubcltrdoimaDCAXL oursandtebclurLiqudcltremanb reitSaDLofAqu e JoyunpaAmePs Thiosulfatecrbsl DeaxycholtirgaDc P L a p e g Aq s u w r

Indoletspwivehacdngas Isolatinfbacteri Slideagutino selctivmedifaaecsbyultreon suosela fbacterilgowth

fe r m n t a i o L c s e c o l n i e s a r c u l

bacil i

Gram negativ M o r p h o l g y

u nie L Motile

1 .5 u r b a c i l i

Vbrio ch lera B a c t e r i a

E te ro l i n

NS

S

Non t v p h i d a l E T SR JUIPHO ES k n re p d u n 4 Calciv ns Norulk o i rs 3 Ad e n o w r u s t y p 4 0 a d 1

BCurvedocmashped SAarethnicubd37Cfo1824s elnitFbrohDCAXLadPWTBS AlkaineptowrPWfMbch TCBSagrAnothelTsargunpAsSXoTeuva xycholateXLDgrsiv OnMacCokey'sagr lactosefrmntaio NLF OnMacCokey'sgar Cngrow dinary meatbrohCtMcBbaengdrowicked convexpaquwithdbemrculaSsmoYthI HS colni ermntcoliesLF mucoidpnketlas OnMacCokey'sgarnow bldagr isuedforlatTiCnBSorbilesatgSeBlSActivmdasuch mediaconts f CHangidopf ly olnymrphoegidntfbysuo G r a m s t i n g o f c l y u s e d f o r a n t i b c v y g B H O I 9 A u V e n s i t v y g O U I S p e c i f C a u s t v d i f u s o n m e t h r c g a p coluresdtnaiSgcolvuexBhmartiwndyeIsolfbactri BiochemalrctionsIolatinfbceray onCMBadbloIsgtirnfbaceyultr CouglasetCpoilvaestpocsuilvrenbodagIsrltinfbacery Carbohydtefmnaio Biochemalrctions Isolatinfbcerom tbrohCMBundeaaronbicerobicaterz DiarhoendystrdiarhoeysntwTcbl9.3furomeaiphgns ortaA OPVITESKglutinaospecfrty ianseAuomtdhc三品XLienfatorThpslumDvSdIPgbceina Diarhoendystr

1oculated

号 9

amediau sSMonyaunerobic taForidentfcaio BacterilCue

万

e L a p e g pas oD ud

a

⾔

⼾ P N

Clastrun m 1 Vbriochlera Table9.1CusativeS I E T I O L G Y B a c i l k e s r u VtrinpheCamiupsylobcterjimEshcaoliEHC Table9.1ndTable9 PlesiomnahgelAoirdmnashydirYoeplsan teroScalpiyncrusm Clastriode fcilcrfmgensWGbarSSinTeyphimlsurGSamonelEtrids epaea PP

b a c t e r i l u s o f p m D i r e c t M o s p y b y a d i n g v r o a t s e v3lodugestivfynatrerysolincherCaonsitecyFrmd f MacrospiExmnato w o n u d a l n o r t T h e f o l w i n g m t d s a r u i H a n g D r o p P e t i o d n e p r a t i o ifaecspeimnreuopqasdxcepl dngyeastclCSuspectbdifngalPrGmoiv SuspectdhlraPnofm doneithflowng cholerawinbfmdytIs1 golVir alsouedfrOHD1Ftphzi POLT0I94HY JfucsSgetivonlamrydh B10jolpaveuThfcsimnxd

ontainer o l e c te d

specimnshould asterilconaer sapeJspecimn VomitLesufl RectalswbIfec re L D l u S E H L D E SO C

uo pads 53 B transpor ted

9

5

im ediat ly

5

cmediuThMonytraspcb品liueultnreoifrtadyomhnwspq otherspcimnuldbte rontaieMksuh worte

u n i p o y d s e r u S t a ' s n p o r mediu Ca y B l tr nspo mediu

choleraATeDruP

⻝

pe tone Ja eM

su pectedOUTEHIY

号 W

8

a p ro c h t i d e n f y

8

g 品

p o D a ro s 品 n o te n d r

I001S

克

6

Ans ELISA

9

S ⾔

万

6 u re y L P I Y Ms a p d mo s p Cy tois p ra b e l i

PS 8

專

alge

you

m

aid eyD

品

5

9

S

ofk

g

SQIV

g

s p e c i f c Chapter

Bac terial IM4 D

9

9

t

Y

C u l t u re

a

used

e

Sunrasu

E

5

t r a n s fe d h o u g l e

品

ofentricvTabl10.

aeJoTup

av i l a b l e

雪

E

S

viruses

品

Tdentifcaon Astroviue Sarlik 2 Identifcaon

LDirectmunofls atexglutaion ofEntamcbhisolytca raditngfomawidtevhrusb Wlhaped Cr yptos rid um p a r s i te s 9

o c ys t M o d i f e d

3

a

F

Susnep t y h o i d W R B r a p A i n d C 号 ter m c f v

g TOW I g

g

5

dcentofcase eclinshraftewekand60prcet TnI 34078

∞

uo ul s r a t y p h i u I o s Pa r t y p h i

g

thes bo tle pInous

r e m a i n OA R I S O

常

Aygrsod X0aM vercent

号 lood 号 電

g

呂

9

001 0 M AqyAursod

⾖

D uogeina

Fa e c s u l t r e B l o d c u l t r e Fa e c s u l t r e B l o d c u l t r e B l o d c u l t r e o n e u r x a u a p a d s

Widal TPIM

3rd We k 2nd We k 3 M IST OSEaSIP

TasepticurohlbaottelesSbfguccnooegbrtkhhiBelufRbSrdtocanshtransfeiiogbldcbutloreydvnpudrsubidencofpyrxia 5ofcaseinrtwkvphd SpecimnsatDfrPhoEFv Caps

suo Puo

10 mL p o Iq

ZUUT

9

3

以

总

5po号rgus5aaSlnPeSorgmoJpqpue nso后

P 6

u a r p d s o n ye l E q A R L

t e o w re p

号 S fadeup chapter 9

E

AnsRefrTabl9.1inWhatrepasitc RefrTable9.1

g 家2

5

⾔

3 三 F

D ys e n t r a d B O L I e K Q u e s t i o n P e r a d

6

TaeM

E

号 e l v a n t e s t p r ov i d e yo u

⾃

9

tec ion of

Dif er nt

g

uone u xa

9

p u eA a f y

CIF LEARN G OBJ T V S

SI3OuSep

再

t o h e d u r a i n f l s H P y u a l e 再 A q p I T E y 2 9 D a 4 P I N OY S H U R M L L D N E I d O D

S

S

c u l t u re

w i t h b l o d 2 D ys e n t r y

6

JaA J Enteric

号告

a

p r ov i d e 1 yo u

⾼ 6

Identify

⽩

⼀

®SUVTILAROJsSuvRefrTabl9.2inchaptelSENPrseoRpne8usnyo宝A®rdName Whatrebcilusofd yourbsevaaitgiuoondsKqser oudAyqapI LruopigtsmMSuoRredpBactrilgoPwouhp1JarseMtolpcimnISos8edaur受滋三mcsAYHR SEuCogeNsqmSpfepdAqaluusewAreatoqrsuaporgOLs4PMueSyEdwatoIrnmHWMl8TEAPR bac terial

1 BuM

g

g row t h i

号

9

品

E n t e r i c f v e r p r o u d A 4 m q s a p n u l 1IsolatinBcl Demonstraifbdes1Isolatinfbc udKre uBos 1SalmoneTyphi万SLNHVTAIn SursneDartyphoid INTRODUCIN bensuoapo Thestudnolba109u PractilExercs abortsouBerd CaustiveAgns Introduction VExamplesofPrcti MalATd Othernospcif1LI te l1BORATYDIGNS e

used instead

Aq a u o p

8

fa e c s u r i n e T h i s m a y I

中D

T h e y b l o n g f a m i E t r c D e m o n s t r a i f c u l g p e c i m n s 零 b l o d r v a t e p n s f i l A I T L P M u m p o r t a n s 三 美 b y d i g E a c t e r i o l g d n s f v o f ve r t b a s w h i c n m l d g a re n p u o s S L i m l y t

醫

S

c u l t re 1 2 u M 路2

Q u e s t i o n R l a e d S T L I A E 1 O g p a s n e 5 S u V n p o d s y h 1 a r v m s a n e p q r d u y o N c i t r a e g D C A L TO P x d N a m e s l c t i v d u J D 1 A o S 4 9 D u r tSpecifc amnigHaing Nametsdonfrig c POyNZiehlsn tagupodsArqanebvmdiagnosi fdut Ie8DHTOupAxaPXylosine otherslcivmdauf 四SaDLreHsnqoAPO1T umportansiecgdyN

nyd e u D o s p u Re l a t d S u o n a a n d s u y e D o i p a L B D 8

c u l t re m diu

may

Procesing OuosfayuoDper曾DIPLAa品PSFor example 電

ELISA test

S I D M u E p l e s oy n r 1 d a W q F o r s p e c i f d n t a E h l S g 8 C h a p t e r 4 I d n i f c o B l u ⼀ A E W S r C i s p o e n r a m F l e c t r o n m i s p y d a l o r h g

HO4eDyAuop 亨 upeoudy uOEDyAuOPL

HM

sHoupadsToprusnoPmAerapdkuuepyyoseM IMuPriodeput MOFpuSorenacygrowthDeailsLpPAOICDUJuS refsopadunecis bacterilgowth Forpcesingadtfo UnLaDeqrod124ETlate maybeprovidwth providewithPRACTILEXSONG ImunochratgpCirypteossidInaec G s specinfCaercypsto ridhnm GinrdspecWtmuPSa ofcertainvus gdarthoe UnitIPraclExeis HeoOLPTJArnsytedwih SIDaXETVLOd BrauoDApchSIULtedlswign bacterifer relvantdigoscbhmweatrilgonphsecrSalmoin somerlvantbichmslodutremclinasBDhtory ntamoebhrislyc f Fordetcion parvumC9yacclidosfpstrangolpecimf ovandcystegs withsorpkes

乐

andcultreAopsuPHaLpolgAueBy ibdomen

B a o y u r e d S T d W v x a A X a m i n t o g i ve n a t h e n d

号

1

포

e

# 베

and

Meningococcal meningitis

age. H. influenzae

11 Meningitis

III.

COMPETENCY

viruses are the

meningititis Table 11.2:

A.

SPECIFIC LEARNING OBJECTIVES

Measles

INTRODUCTION

Acute

Meningitis is an inflammation

It is caused by bacterial agents except Mycobacterium tuberculosis. It is serious and fulminating type of meningitis. It is characterised by elevated polymorphonuclear cells in CSF except for Listeria

of the membranes

(leptomeninges) surrounding the brain and spinal cord. It may be caused by viruses, bacteria, fungi or protozoa. Meningitis of bacterial origin may be caused by pyogenic organisms (acute pyogenic meningitis)

monocytogenes in which lymphocytes are predominant or by M. tuberculosis (tuberculous meningitis). Aseptic in CSF. The causative agents involved in different ages term used for meningitis caused by

are

by bacteria (other than

shown in Table 11.1.

Meningitis

Age Group

Meningitis where the infectious agent could not be identified was named aseptic meningitis (aseptic meaning no infection). Now it is possible to identify most of the infectious agents by molecular techniques. Therefore meningitis are named as bacterial meningitis, viral meningitis and chronic meningitis. Chronic meningitis includes most of the causative agents of aseptic meningitis. However the term aseptic meningitis is still used in clinical practice.

1.

Neonates and infants

Causative agent

Group B Streptococci (Streptococcus agalactiae) Escherichia coli Klebsiella spp. Listeria monocytogens H. influenzae

Streptococcus pneumoniae 2. In children and Streptococcus pneumoniae adults

(pneumococcus)

Neisseria meningitidis

Meningitis is classified as:

(meningococcus)

bacterial (pyogenic) meningitis

Listeria monocytogenes

Chronic meningitis 72

B. Collection CSF: CSF is obtained by lumbar puncture

count, biochemical analysis and bacteriological

(HSV-2), more frequent

cause

When CSF is allowed to stand in container, fibrin web (cobweb) often develops in case of tubercular meningitis. 2. Other Specimens: Blood is collected in a sterile container by venepuncture under all aseptic conditions.

in CSF although elevated neutrophils in CSF

observed in some of

may be

these cases. Chronic meningitis

•

The CSF should be examined immediately after

collection. If delay is expected then place CSF in the incubator at 37°C (never refrigerate the CSF as H. influenzae may die). For virus isolation, CSF may be kept in the freezer.

be caused by bacterial, fungal or parasitic infectious (Table 11.3). Fungal meningitis occur in

After

Bacteria The most common cause

Borrelia burgdorferi Treponema pallidum

test

D. Biochemical Analysis and Cell Count Biochemical analysis and cell count give an indication

the type of meningitis (Table 11.4).

Acute pyogenic meningitis: The CSF usually contains more than 1000 leucocytes per cu.mm and as many as 90-95% of which are neutrophils. The total protein content is elevated and the glucose level is markedly diminished or even absent.

Leptospira interrogans Fungi

Cryptococcus neoformans -The most common fungal cause. Candida albicans Histoplasma capsulatum

refrigerator until

performed

also occur by non-infectious causes.

11.3: Causative Agents of Chronic Meningitis

serum collection from blood specimen,

it should be kept in a

immunocompromised persons. Chronic meningitis can

2. Tuberculous meningitis: The CSF in tuberculous meningitis shows moderate rise in cell count (50-

500/cu.mm) and predominant cells are lymphocytes.

Blastomyces dermatitidis Coccidioides immitis

There is moderate rise of total protein and sugar is slightly reduced.

Parasites

Toxoplasma gondii

sterile

C. Transport and Storage

Chronic Meningitis

Acanthamoeba

: detection of

Urine is collected in a screw capped container under all aseptic conditions.

Haemophilus influenzae

2. Viral meningitis

Blood (for blood culture, :

virus

M. tuberculosis

important

examination.

virus-2

Chronic meningitis is defined as inflammation of meninges which persists for more than 4 weeks. It characterised by predominantly elevated lymphocytes

Table

The most

antigen or antibody in serum) Urine (for antigen detection)

can

Table 11.1: Causative Agents of Acute Bacterial (Pyogenic)

those responsible for acute pyogenic meningitis), fungi and protozoa. Tuberculous meningitis is also included as a part of aseptic meningitis.

3.

of

Other specimens

West-Nile

Bacterial (Pyogenic) Meningitis

episodes

under strict aseptic conditions. collected in three sterile containers, one each for cell

Epstein-Barr virus (EBV) Paramyxoviruses Mumps virus

To identify the microorganisms on the basis of direct microscopy of stained CSF smear, colony morphology and biochemical

To interpret the abnormal results of CSF analysis.

acute

ideal specimen

2.

usually mild.

Varicella zoster virus (VZV)

tests

Specimen 1. Cerebrospinal fluid (CSF);

most common cause. Viral

Causative Agents of Acute Viral Meningitis

Herpes viruses - Herpes simplex than HSV-1

occurrence of multiple

III. LABORATORY DIAGNOSIS

- Polio virus . Coxsackie viruses Echoviruses

The student should be able to learn:

3.

When the

Enteroviruses

V. Examples of Practical Exercise

1. Acute

has

73

Recurrent Meningitis

menuingititis, each of which is 2 hours in transport then specimen should be refrigerated. Preservatives can be used if specimen is not

refrigerated. Boric acid (1.8%) is the commonly used preservative.

most

1II. PRACTICAL EXERCISE You may be asked to demonstrate the appropriate method for collection of any one of the specimen

Frank pus/exudate collection (Fig. 14.5) from abscess. Swabbing of pus/exudate.

Essential Points to Remember

2.

Ans.

What are the steps for collection of specimen? Depending upon specimen, you should know the steps for collection of that specimen according to the method of collection of that particular specimen e.g blood, urine

(i) Pus

Oozing pus should be directly sterile test tube/container.

Collection of Specimen Q.1

Specimen

Method of collection of specimen.

Questions Related to

in a sterile container

Wound swabbing is the most widely used method.

Collection of

Depending upon the specimen, demonstrate the collection of that specimen according to the method already mentioned in this chapter.

The materials required for collection of the specimen.

Specimen

Pus or exudate may be collected from wound. Frank pus or exudate is better than pus swab. Pus/exudate, can be collected by following methods:

Pre-sterilised disposable swab

port and collect

without interrupting the urine flow, mid-portion mentioned above.

other for culture.

Fig, 14.4

a small

debris prior to collection of specimen

rinse the mouth with water.

Swab is first rubbed firmly over both the tonsils Rejection criteria and then rolled over the posterior pharynx.

case

swab should

chances of recovering organism.

Early morning specimen should be collected.

(i) Throat swab

the

moistened with sterile saline to increase

Woundshould be cleaned for removal of excess

Instruct patient to take

Tracheobronchitis

wound or in

specimen,

amount

remove.

Specimens

Lower respiratory tract infections

Clamp the catheter below the

the urine above it.

the sampling port with an alcohol impregnated swab to reduce the risk of

not touch the surrounding skin area

• In case of a dry

2. Collection of Lower Respiratory Tract

Laryngitis

89

Urine Collection from Catheter A sampling port can be found on the tubing of the catheter drainage bag. Urine should only be collected from this point. Clean

pharynx.

Tonsilitis

•

Pus swab (Fig. 14.4) should be collected by rotating the tip of swab over cm area of open for 5 seconds.

(i) Nasopharyngeal swab Tilt the patient's head backwards. Insert a flexible fine nasopharyngeal swab into one of the nares under proper light. Guide the swab backwards and upwards alone the nasal septum until it reaches the posterior

B. Respiratory Tract Specimens

Collection of Specimens

(ii) Swab

collected

in

etc.

0.2 What are indications for collection of specimen? Ans. The indications for collection specimen vary with different specimens. You must know the indications for

collection of each specimen e.g. blood, urine,

Specimens

not in bare

15 Collection of

Specimens I1

potential

should be transported in carrier hands.

hazard

to

A. 1.

MI 8.11

III. Information on the label of specimen container

Demonstrate respect for

the laboratory for performance of tests il

IV. Transport and Storage

patient samples sent

the detection of microbial agents causing

I. GENERAL

INSTRUCTIONS

Diagnostic test to be performed Clinical diagnosis/history

These general instructions for collection of any specimen must be taken care 1.

2.

IlI.

Specimen should be of adequate quantity. Optimal time of collection of specimen. Specimen should be collected before

administration of antimicrobial agents. 4. Specimen must be collected from actual

the Label of specimen should include:

site of

infection with minimum contamination from normal flora of human body. Appropriate container should be choosen for

•

collection of a particular specimen. 6

Proper labelling of specimen container must be done.

7.

Specimen handling should

not be risky to health

care workers. II.

INFORMATION ON THE LABORATORY FORM

It should include:

Name, age, sex of patient Registration number OPD/IPD status Specimen type e.g. sputum, urine etc. Diagnostic test to be performed Time of collection

IV. TRANSPORT AND STORAGE

The objectives of proper transport include: To maintain the specimen as near to its original state as possible. • To maintain the integrity of specimen even in adverse

(OPD)/Indoor patient

It is recommended that the specimen should reach the laboratory as soon as possible. delay is inevitable then this delay should not be more than hours. In

(IPD) status e.g; medicine OPD, medicine case of expected delay, specimen shouhal be stores department ward number per

Specimen type e.g. sputum, urine etc.

prescribed guidelines and transport media may be transportation of specimens to the laboratory.

used for 90

Use a sterile container

with minimum delay, if this is not possible, the specimen is to be refrigerated at 4°C or stored by adding boric acid (1.8%). H. Faeces

Use a clean sterile, screw capped, wide mouthed,

in transport then refrigerate

Nasopharyngeal swab

• Use sterile nasopharyngeal swab. Specimens for viruses should be transported in viral transport media (VTM) and immediately placed on ice or cold packs.

Specimens can be kept at 4°C for no longer than 72 hours. If delay is expected in testing then store the specimen at -70°C or below.

D. Sputum Use a wide mouth sterile container. In case of

Urine specimen

leak proof container.

delay, refrigerate the specimen.

E. Cerebrospinal fluid (CSF) Use a sterile container.

Specimen container should be leak proof.

environmental conditions.

of patient Name, age, Registration number

Out patient department

INFORMATION ON THE LABEL OF SPECIMEN CONTAINER

C.

In case of delay the specimen should be kept at

G.

urine is good culture medium, specimens after collection should reach the laboratory

Throat swab

the specimen.

transport the specimen. 3. About e information that should be written in the requisition/laboratory form. The storage methods used in case of delay in transportation.

pus swab

laboratory as early as possible.

Use sterile swab.

To demonstrate how to

or

2°-4°C in refrigerator

systems)

If delay is suspected

Haemophilus

Wound specimens must be transported to

Use sterile blood culture bottles (conventional

be stored at 2°4°C in a refrigerator for 10 days. Store it at -20°C/-70°C in a deep freezer if storage is required for a long duration

The student should be able to learn: To choose the correct container for the collection of specimen.

temperature kill

Blood culture specimens should be transporte

Serum can

B.

91

not be

Use sterile container/swab.

Use sterile plain vial or test tube.

infectious diseases.

SPECIFIC LEARNING OBJECTIVES

2.

F. Pus

2. For serological tests

V. Practical Exercise

delay the specimen should

influenzae.

For culture

blood culture specimens

The student should be able

Il. Information on the laboratory form

it is

Blood specimen

of

refrigerated but kept refrigeration tends to

specimen handlers In certain

at room temperature and do not refrigerate. delay in transport is inevitable then incubate

COMPETENCY

PREVIEW

• In

This precaution will prevent the

delicate organisms such as Neisseria gonorrhoeac important to use transport media.

or for automated

General Instructions

Collection of Specimens II box and

In case of faeces specimen for bacterial culture, if a delay of more than 2 hours anticipated, the specimen should be collected in a suitable

transport medium. Cary Blair transport medium, Stuart's transport medium, Amies transport medium may be

used.

For detection of viral antigen; faeces can be

refrigerated at 2°-8°C.

V. PRACTICAL EXERCISE L. You may be asked to choose a correct container for collection of specific specimen. 2. You may be asked the methods of transport for a specific specimen. Choose the correct container for collection of specimen. Describe transport methods for different specimens as already mentioned in this chapter.

Essential Points to Remember 1. Which container/swab is to be used for which specimen. 2. In case of delay, the methods of

transport and storage for each specimen.

Infection Control Practices

3.

After a procedure or body fluid exposure risk

5.

After touching patient's surroundings.

touching a patient 4. After

16

of

hand hygiene techniques

Types Routine hand washing

Hygienic hand care Surgical hand washing or hand scrub

Infection Control Practices 1.

hands with

It involves washing PREVIEW

The student should

IV.

able to

MI 8.7 Demonstrate infection control practices anduse of

Personal Protective Equipment (PPE) IlI

Personal Protective Equipments (PPE).

Spill Management Biomedical Waste Management

hand care.

most transient microorganisms from the hands.

following conditions. (a) Before and after contact with patients, (b) Before wearing and after removing gloves,

Besides hands, wrists and forearms are also

devices

patient.

SPECIFIC LEARNING OBJECTIVES

2.

patient care or having in

A. Personal Protective Equipment (PPE)

and/or body fluids which may contain pathogens PPE also protects the patient by preventing the transmission

Hygienic Hand Care Wash hands with soap and water by rubbing

1. To perform independently the steps of hand hygiene. 2. The five moments of hand hygiene. To perform independently the donning and doffing of Personal Protective Equipment (PPE)

PERSONAL PROTECTIVE EQUIPMENT

and around Personal protective equipment (PPE) are used to protect Health Care Workers (HCWs) from exposure to blood

(i) Hand wash method

The student should be able to learn:

included for washing. For drying, sterile towels are used. I1.

(c) After wound dressing, (d) After contact with body fluids of patients,

for (e) After handling contact with inanimate objects

V. Practical Exercise

hands in the six movements by covering all surfaces of hands as shown in Fig 16.2.

of organisms from HCWs hands to patient during sterile and invasive procedures.

1. Components of PPE

Rinse hands with water.

About blood and body fluid spill management under supervision. 5 The

soap and water.

3. Surgical Hand Washing or Hand Scrub requires more time for washing than hygiene

Routine hand washing should be performed in the

COMPETENCY

Hand Hygiene

Duration of hand rub method is 40-60 seconds.

Routine Hand Washing

removes

Gloves (Non-sterile): It is used when a risk

Dry hands with single-use towel Duration of hand wash method is 20-30

segregation of biomedical waste (BMW).

93

(i1) Hand rub method When alcohol-based hand rub solution used instead of soap, dry hands are required. Rest above with the of the procedure is same difference that no towel is required for drying as alcohol evaporates in the environment.

infection

to

HCW only e.g. touching blood,

body fluids.

seconds.

I. HAND

HYGIENE

Hands have been known to be

the

involved in many

infectious disease outbreaks especially in nurseries, other hospital settings. This happens because hands of medical neonatal units, intensive care units and

personnel may be transiently infected with pathogenic organisms from infected patients and thus these can

or environment, be transmitted to other patients.

Therefore, hand hygiene IS very important in keeping

health care associated infections at minimum. The

World Health Organisation (WHO) has published the standard guidelines when hand hygiene should be performed. There are five moments for hand hygiene which are as follows: Five Moments 1. 2.

of Hand Hygiene

(Fig. 16.1)

Before touching a patient Before any aseptic procedure

Soapy water droplets

Step 1 : Wet hands and apply soap. Rub hands palm to palm

Step 2: Rub each palm over the back of the other palm

with interlaced fingers.

Step 3: Rub palm to paim with

fingers interlaced.

clean/aseptic procedure Before touching patient

After touching patient

Step

After bodyfluid exposure risk

After touching patient's surroundings

ig. 16.1 Five moments for hand hygiene 92

Rub backs

Step 5: Rotational rubbing

your

of your thumb in

fingers to opposing palms with fingers

opposite 1 vice-versa.

interlocked.

Fig. 16.2

and

Hygienic hand care- standard method

Step 6: Rub in circles backward: and

forwards with clasped fingers of right hand in left palm and vice-versa; on and then rinse dry y

r hands.

Unit

94 .

Practical Exercises

Donning

Doffing

Perform hand hygiene

Gloves

of

when a risk Gloves (Sterile): It is used

infection is to both HCW and patient e g. during invasive procedure or surgery. Plastic apron: It surgery.

Coverall: Coverall are whole

By

Goggles or face shield

Mask or respirator

designed to cover

Gown

Mask

the

body and provides 360° protection.

using appropriate protective clothing

biomedical waste bins. Yellow bin: GoT or coverall,

droplets precautions while handling patients. It is triple layered mask, fluid resistant, provide protection from droplets of infection. It is also

Red

Donning and

hands get contaminated.

doffing of different components of Ppr

ith airborne infections e.g. tuberculosis, have been shown in Figs It is

respiratory protective device with high filtration efficiency to airborne particles. These masks are cup-shaped structured design which does not collapse against the mouth. If correctly worn, the tighter air seal than surgical masks, they protect wearer from inhaling airborne particles. Cap, goggles, face shield: It is used when spillage

The

or during aerosol generated procedure. Hence goggles/face shields are an integral part of

standard and contact precautions.

goggles should provide good seal with the skin of the face, covering the The flexible frame of

eyes and other areas.

Wearing the gown

COVID-Care

in

following components of PPE are used.

Goggles and faceshield. Both

goggles and faceshield should be used.

2. Mask Mask

N-95 mask

Triple layer medical mask If N-95 mask is not available, triple layer medical

of blood is likely to be expected e.g. cardiac surgery. Contamination of mucous membranes

eye, nose and mouth may occur during droplets generated by cough, sneeze of an infected person

16.3 and 16.4.

Personal Protective Equipment (PPE)

B.

filtration capacity of these masks exceeds that of

surgical mask. Since these masks have a much

mask can be used. 3.

Selection of appropriate PPE It is based on the level of risk associated during I specific patient care procedure or intervention 0

part of standard precautions).

Route of

transmission of suspected pathogen

e.g. contact, droplet, airborne (as a part

transmission based precautions).

Checking the fitting of mask

Nitrile gloves •

Latex groves

Two pair of gloves, one over the other, may be used 4. Coveralls/Gowns Goggles

Coveralls

as to environment.

(as

Wearing the mask

Without the mask

Gloves

are designed to cover the whole body and Surgical shoes: It is used in operation theaters provides 360 degree protection. By using protective and intensive care units (ICUs) to prevent clothing, it creates barrier to eliminate or reduce contact and droplet exposure. transmission of organisms to HCW and as well

2.

Hand wash

respirator, cap,

Plastic apron, goggles or face shield, gloves.

the steps also

named as medical mask.

Ebola virus, SARS-CoV-2 etc.

Hand rub

Note: During doffing, perform hand hygiene in between

N95 mask: It is used when handling or examining HIN1 virus,

mask or

of

3. Donning and Doffing of PPE

Donning (wearing) and Doffing (removing) of PPE must be done in a particular sequence mentioned below, to minimise the risk of infection

shield

Face shield

Goggles

Gowns can also be used instead of coveralls. Both gowns and coveralls can also be worn. 5. Shoe covers

Shoe covers should be made of impermeable fabric similar to fabric of coveralls. These are used over shoes

to facilitate personal protection and decontamination. Gloves

6. Head cover

Coveralls usually cover the head. When using gowns, head cover should be used to cover the head and neck. Hair and hair extensions should fit inside the head

95

Hand hygiene

Gloves Perform hand hygiene During doffing, discard PPE componentsinto appropriate

is

droplet exposure. Surgical mask: It is used during surgery and for

patients

Goggles or face shiela

expected during

and possible to eliminate or reduce contact

•

Gown

used during surgery.

Gown: It is used when soiling

Infection Control Practices

Inspect that no pinholes or tears are

present in the gloves

Insert fingers

Put the har

the glove by alligna

with the fingers and thumb of the glove.

into the cut nd pull the cutf over t

cover. Cap may be used as head over. Fig 16.3 Donning of PPE

Check the proper

Repeat

steps for the

other hand.

The cuft should il around the whist.

Unit II Practical Exercises

SELECTION OF

Infection Control Practices

PPE

97

community. Biomedical waste is a broader term which

The selection of appropriate PrE is based on level of risk includes Waste meneraies in the diagnosis, treatment

Glove.

Pull forward

glove

Pull off glove

turning it

Pull off glove further.

inside- out

Ball up glove

n gloved hand

Dispose of glove

Slide fingerull under o f f (containing cuff a glove c anothe

hand covering

removed glove

another

glove inside)

appropriately

how risk, moderate risk and high risk) associated during specific Fprocedure intervention. The components PPEmay beselected accordinglyasdescribedearher under headingcomponents of personal protective equipment The complete PPE mentioned above is used at high risk

microbiology laboratory for RT-PCR testing,

area

COVID-19 patient care ward.

III. SPILL MANAGEMENT Goggles face shield

Spill Management of Blood and Body Fluids The following basic steps are to be followed for management of blood and body fluids. Spillage should be attended immediately. • •

area.

Entry to the spill area should be restricted to persons

involved in management. • Bring he spill kit containing all material to who are

Face shield

Goggles

Mark the spill

of spillage. Wear appropriate PPE (gloves and gown).

•

paper towels and leave it for 15 minutes. 15 minutes, remove the paper towels; put fresh discarded as infectious waste.

Wash the area with soap and

water.

Remove PPE.

the gown inside-ou disposing it appropriately

in 2018 and 2019. According to new rules, categories of biomedical waste have been described according to colour code of bag (Table 16.1). Table 16.1 Categories and Colour Coding of Bags for Segregation of Biomedical Waste

Category

All PPE material and paper towel etc. are to be discarded as infectious waste in appropriate waste container.

Type of container

Yellow coloured non-chlorinated plastic bag or container Red

coloured non-chlorinated plastic bag

or container

White

Puncture

proof, leak proof, temper proof

(Translucent) container Blue coloured puncture proof and leak

Blue

proof container

paper towels to clean the area. Then all these towels

are to be

Wipe the area with 0.5% sodium hypochlorite(freshly

Taking off the gown inside out

(BMW)

The new Biomedical waste (BMW) rules 2016 have been notified on 28th March 2016 to replace the earlier rules (1998). The amendments were made in BMW rules 2016

Red

prepared) Untieing the straps

Categories of biomedical waste

the site

Cover the spillwith paper towels to absorb the spillage. Pour 0.5% sodium hypochlorite (freshly prepared) After

B.

Yellow

top of

Gown

and immunisation of humans or animals or in research activities or in the production of biologicals

Categories 1. Yellow: For infectious non-plastic waste Red: For infectious plastic waste 3. White (Translucent): For sharps including metals 4. Blue: For broken or discarded glass items and metallic body implants.

C. Steps of Waste Management

Perform hand hygiene. Record the incident.

The best concept of waste management is that of '3Rs' namely reduce, recycle and recover. The most useful Note: The above steps are basic. However hypochlorite among these is to prevent or reduce the production

solution percentage and its period of contact may Mask

Taking off the mask

through one string.

The mask was taken off and

CDC etc). CDC recommends 0.5% hypochlorite for large

of waste to minimum. The next option is to reuse as

such or after recycling. The compost can be produced

from waste, for use in agriculture. This is named none of these 3Rs is available then spill >10 mL) and 0.05% hypochlorite for small spill recovery. (1200°C. cytotoxic drugs

Incineration or plasma pyrolysis or encapsulation.

(f) Chemical liquid waste"

Pretreated before mixing with water and then disposal.

other waste

Non-chlorinated chemical disinfection followed

Discarded linen, mattresses, beddings, contaminated with blood or body fluid, routine mask, cap, gown and shoe cover.

by incineratic n or plasma pyrolysis.

(h) Microbiology, biotechnology other clinical Pre-treat to sterilize with non-chlorinated laboratory waste, blood bags and live chemicals on-site per WHO guidelines vaccines. on safe management of wastes from health care activities and WHO Blue Book 2014 and

thereafter s Red coloured

non-chlorinated plastic bag or container

incineration.

Contaminated Plastic Waste (Recyclable): Autoclaving or microwaving/hydroclaving Waste generated from disposable items such as followed by shredding or mutilation. Treated tubing, intravenous tubes, catheters, urine bags, waste to be sent to registered or authorised

syringes (without needles and fixed needle

recycler

syringes); vaccutainers with their needles cut, Plastic waste should plastic apron, goggles and gloves. sites. White (Translucent) puncture proof, leak proof, temper proof container

Waste

not be sent to landfill

sharps including metals: Needles, Autoclaving or dry heat sterilisation followed

syringes

with fixed needles, needles from by shredding or mutilation or encapsulation in

needle tip cutter or burner, scalpels, blades or metal container or cement concrete and then any other contaminated sharp object. final disposal to iron foundries (having consent to operate from

the

state pollution

control boards or pollution control committees) or sanitary landfill or designated concrete waste

sharp pit. Blue coloured Puncture proof and leak

proof container

Glassware:

Broken or discarded and

contaminated glass including medicine vials and ampoules except those contaminated with cytotoxic wastes microscopic slides.

Metallic body implants. Separate collection system leading to effluent treatment system is used.

autoclave.

(in) Microwaving

Radiations produced by the microwave are involved to

break apart molecular chemical bonds and thus disinfect

infectious waste. The microwaves heat

to temperatures of 979

Microwaving Hydroclaving

to

the waste

100°C. Cycle time

IS

40-45

and minutes. It has advantage of disinfecting the there are no hazardous emissions. However, it cannot be used to treat body parts and tissues.

pyrolysis

Chemical treatment Effluent treatment plant

of

Autoclaving is used for microbiological waste, blood and blood products, body fluids and used sharps. It is not recommended for pathological waste. Autoclaved material is typically land-filled, therefore, it has a large

(iv) Hydroclaving The hydroclave is an expansion of the autoclave technology. Unlike in the autoclave, steam is introduced into the hollow walls of the hydroclave. The steam does very high temperatures Incineration burns waste and thus reduces its volume. The double chambered not come in direct contact with the waste. Volume (ETP)

(i) Double Chambered Incineration

(e) Chemical waste

g)

for treatment of infected

Double chambered incineration Autoclaving

(v) Plasma

container

used for validation test

strain on land fill capacity

material.

Type of waste

Type of bag

treatment and Disposal

Waste Treatment

(i)

Table 16.2 Segregation, Collection, Treatment and Disposal of ammendments in 2018 and 2019

Vacuum autoclave time period should not be less than

45 minutes. Biological (Bacillus stearothermophilus

transport or in the storage area.

filled to 3/4th of their

2. Bags must be labelled with

by

99

BMW should le transported within 24 hours sporesy or chernica indialars lsr pbrapptbe hopiu The by dedicated trolley to central storage area of

transport

laboratory wastes, used sharps, pathological wastes (samples, tissues etc.), bedding and other wastes like

liquid isteshould be residence time of not less than 60 minutes, while Pre-treatment oflaboratory done either with 1% hypochlorite solution

4

Disinfection (by soaking washed glass waste after cleaning with detergent and sodium hypochlorite (1%) treatment) through autoclaving or microwaving or hydroclaving and then for recycling.

reduction of waste is much more than autoclave. Cycle incinerator contains two chambers. Waste is burnt one chamber (primary chamber) at 800°C. Combustion time is one hour. of gases emitted from the first chamber, occurs in the Hydroclave contains a series of large rotating rods, second or secondary chamber. This chamber has which spin slowly, putting the material into steam high temperature of 1000°C. The negative pressure is chamber as it is loaded. The rods continue to spin and maintained inside the incinerator by the system, thereby rupture the waste bags and ensure complete exposure

forcing the end-gases out of the chimney.

The incinerator has an advantage of dealing with all

pathological and cytotoxic wastes. Body parts, animal waste, microbiological waste and soiled dressings can be treated with this technique. The disadvantage of incinerator is that it generates highly toxic gases (e.g dioxins and furans, if PVC plastics are

present). It

adversely affects the health of the community. Another disadvantage is that recycling and reprocessing of materials cannot be done. Burning of plastic waste or

sharps is also not

recommended.

(ii) Autoclaving

Autoclave relies on the circulation of steam through the infectious waste to decontaminate it

There are two kinds of autoclaves: the Gravity autoclave and the Prevacuum type. In the Prevacuum type, steam is created outside the chamber loaded with waste. Air in the chamber is then gradually removed and steam is injected in This type of autoclave eliminates 'cold spots' and air pockets' (where the steam is unable to

to the heat. After the sterilisation, the material is passed shredder. The waste can then be safely recycled

into or

land filled. All items including pathological waste

can be treated.

(v) Plasma Pyrolysis

Plasma pyrolysis is an innovative technology disintegrates organic compound into

which

gases

and

non-leachable solid residues in an oxygen starved

environment. Plasma torches are used in this technique, to generate high temperature. Plasma is the state of matter obtained by breaking down atoms into ions and electrons by ionisation. Plasma can easily reach temperature of 10,000°C or above and is used for dissociation of molecular bonds. Plasma pyrolysis can be used to dispose of all types of waste including

plastic waste in a safe and reliable manner. The toxic

compounds such as dioxins and furans in the emissions below the prescribed limits.

Plasma pyrolysis

has

been accepted as an alternative to incineration. This is also included in the category of non-burn technology.

penetrate) by creating this vacuum. This ensures quicker (vi) Chemical Treatment hypochlorite heating. A temperature of 121-C and pressure of 15 Chemical treatment ensures disinfection. 1% waste such used. It is done for liquid discarded blood and body fluids.

pounds per square inch is used For gravity autoclave, solution the waste material should be subjected to autoclave

Unit II Practical Exenises

of COVID-19 facility Report opening time to Stay Pollution Control Boards (SPCBs) and respectiv, the area. CBWTF located in

isid) Effluent Treatment Plant (ETP)

The liquid waste(effluent) isfirstsubjected to chemical

treatment and then it is drained 2.

Depute dedicated sanitation workers separately

into FTP.

for biomedical waste and general solid waste sothe and transferred waste can be collected

Disposal

for Land filling, deep burial and sewage are used after treatment can be disposal. Infectious waste disposed of by land-filling or deep burial. Besides treatment, incineration is also a method of disposal.

Segregation, collection, treatment and disposal of

biomedical waste are shown Table 16.2. Biomedical waste rules 2016 and its ammendments (2018) documents may be referred for details of various aspects of biomedical waste

F. Biomedical Waste Management Guidelines in COVID-19

In addition to existing BMW Management Rules 2016 the

following specific guidelines are to be followed. These

are according to guidelines for handling, treatment and disposal of waste generated during treatment/

diagnosis/quarantine of COVID-19 patients-Rev. Keep separate colour coded bins/bags/container for proper segregation of

waste as per BMWM, 2016 as

temporary

Collect and store biomedical waste separately prior to handing over the same to CBWTF. Use a dedicated collection bin labelled as COVID-19 to store COVID19 waste and keep separately in temporary storage room prior to handing over Common Biomedical

storing infectious waste must

V. PRACTICAL EXERCISE 1. You may be asked to perform the steps of hand hygiene practically.

Ans. Perform

steps of hand hygiene as shown in Fig

16.2 of this chapter. 2. You

may be asked to explain the five moments

hand hygiene.

Ans. Explain the five moments of hand hygiene as shown in Fig. 16.1 of

3. You

chapter.

may be asked to perform donning and doffing

of PPE with correct sequence of various components

of PPE. Ans. You

should be able to perform this exercise

correct sequence as

into

4.

CBWTF collection van.

•

Ans.

be trained

with

shown in this chapter.

may be asked to select components of PPE as per situation.

You

In addition to mandatory labelling, bags/containers Ans. For example, collection of blood requires only biomedical waste should be labelled as sterile gloves. Handling or examining patients with airborne infections (e.g. tuberculosis, H1N1 Virus) General waste not having contamination should be requires N95 mask. used for

"COVID-19 Waste".

disposed

as

solid waste as per SWM Rules, 2016.

Maintain separate record of COVID-19 generated. Use dedicated trolleys and collection bins.

•

waste

label

"COVID-19 WASTE" should be pasted on these items

5.

spill

6.

also.

The inner and outer surfaces of bins/containers/ trolleys used for storage of COVID-19 waste should be

disinfected with 1% sodium hypochlorite solution daily.

You may be asked to perform under supervision the

spill management of blood and body fluids. Ans. You should be able to perform the 'management of as

described in this

culture plates, syringes etc as biomedical waste and

Ans.

askedtosegregatein differentcolour coded containers. You may segregate the various items as described in

Table

16.2 in this chapter.

Essential Points to Remember 1 2.

Steps of hand hygiene.

Five moments of hand hygiene.

Donning and doffing of PPE. Steps of spill management.

5. Segregation of biomedical waste.

chapter.

You may be provided with items such as gloves,

Infection Control to

Refer to

Infection Control Practices

0.1 Ans.

timely th

programme.

Waste Treatment Facility (CBWTF). Biomedical waste

collected can also be lifted and directly deposited

storage area.

G. Waste Management Programme All laboratories should develop waste managemen. programme according to the specific needs of the individual laboratory. The policies and procedum should be incorporated in the laboratory's operating manuals. Emphasis should be on waste minimisaties (by reducing waste, reuse and recycling), Proper segregation, and health and safety of the workers

amended.

As a precaution double layered bags (using 2 bags) should be used for collection of COVID-19 waste.

waste

Questions Related

0.3

What are different steps of hand hygiene?

Refer to Fig

What are

16.2 in this chapter.

tive moments of

hand hygiene?

Refer to Fig 16. in this chapter.

Describe donning and doffing of PPE

Practices

101

Donning and Doffing of PPE in this

chapter.

Q4 Describe the steps of "spill management'. Refer to 'Spill management' in this chapter. Q5 What are methods of treatment and disposal of

Ans.

biomedical waste? Ans. Refer Table 16.2 in this chapter.

(e.s.

17 Malaria and Filariasis

before staining.

Thin and slide

Two drops of

thick blood smears

blood are taken

on the sameslide

made smaller area.

of blood

PREVIEW I.

Il.

MI 2.6 Identify the causative agents of malaria and

Identification of Parasites

filariasis.

Detection Microfilariae IV. Exercise

The student should l 1.

stained with

body and flagella stain red.

able to learn:

A. Malaria

Erythrocyticstages and Gametocytes of different species plasmodium are shown in Fig, 17.1 and 17.2 ' avale. Among these P. vivax and P. falciparum are more

important.

stain, Leishman's stain, Field's stain and laswant Singh, Bhattacharjee (SB) stain. These stains P vivax of methylene blue and eosin. They combination a are also contain oxidation products of methylene blue named azures. The azures provides further contrast in peripheral blood stained by these stains. Cytoplasm of

protozoal parasites stains blue whereas nuclei, parabasal

SPECIFIC LEARNING OBJECTIVES

I1. IDENTIFICATION OF PARASITES

Malaria is caused by mainly four types of malarial parasite; Plasmodium vivax, P. falciparum, P. malariae and are

Romanowsky's stains. Examples of these stains include

e student should be able to

Methods

smear on

smears

Staining thick blood smears Thin or

To demonstrate the staining methods of peripheral blood smear.

3. To identify the different stages of malarial parasite in the given peripheral blood 4. To identify i microfilaria in a given smear,

I.

solution which fixes

with buffered distilled

A thick blood smear may be prepared by taking 4 small drops of blood and joining the corners of the drops

with a needle. It may also be prepared by taking a thick

drop of blood on a slide and

spreading with a needle or

with the corner of another side to form

thick smear in cm square area. The smear is allowed to air-dry. The thickness the smear should be such as to allow about

Three types of

1.

peripheral blood smear

a newsprint to be read through the preparation. Thick blood smear is many cells thick as compared to thin

peripheral blood smears are used:

Thin blood smear

blood smear which is

Thick blood smear

one cell thick.

Thin and thick blood smears or

the same slide

to

Thin blood smear

The pulp finger or lobe of an ear IS wiped with spirit and allowed to dry. iS pricked with

a sterile cutting needle under all aseptic conditions. A drop of blood, about the size of pin head, taken on

be observed

in

microscopically even when lying deep the smear. With water based stain (e.g., Giemsa),

dehaemoglobinization occurs when the stain poured on the

102

smear while for alcohol-based

Ring Form

P. ovale

Schizont

Ring Form

Fig. 17.1 Erythrocytic stages of

water. It is dried in air

and examined under oil (11)

Schizont

mersion.

Male Gametocyte

different species

Plasmodium

Female Gametocyte

Giemsa stain: Prior fixation of blood smear

required as the stain used is in aqueous solution. The smear is fixed by covering it with absolute methyl alcohol for to 3 minutes. Plasmodium vivax

Composition Giemsa stain powder - 0.75 gm Glycerol Methanol

25 mL 75 mL

Procedure: Fixed blood smear IS immersed 1:10 dilution of Giemsa stain in buffered distilled water for 30 minutes. It is washed in buffered distilled water. The stained smear is then examined under oil immersion lens. in

Thick blood smear is dehaemoglobinized prior staining. It is dehaemoglobinized by placing the smear in distilled water in a glass cylinder for to 10 It is used primarily for the definitive species identification minutes. The disruption of the erythrocytes and the of plasmodia and other parasites present inside the loss of haemoglobin permits the blood parasites to erythrocytes. of 3.

1.

Leishman stain for 2 minutes and diluted with twice its volume of buffered distilled water The diluted stain is allowed to remain over the smear for 15-20 minutes. The slide is washed

2. Thick blood smear

microfilariae.

B. Stained

Normal Size of

Schizont

Multiple

as it stains.

Procedure: The blood smear is covered with

should ideally be one cell thick.

A. Wet preparation drop of anticoagulated blood is placed on a clean glass slide and a coverslip is put over it. The preparation iS examined microscopically for parasites such as

Schizont

Ring Forms

Leishman dry powder - 0.15 gm Absolute methyl alcohol - 100 mL

formed with 'tails'. It is allowed to dry. The thin smear

METHODS

Ring Form

not necessary as the stain contains an alcoholic Pmalarino

a grease-free clean glass slide at one end. The drop of blood is then touched with the edge of another slide It is held at an angle 30 degrees and pushed gently to other end, till the blood is exhausted. The smear

rhodesiense, Trypanosoma cruzi, Wuchereria bancrofti and Brugia malayi. Following methods may be used for the examination of parasites in the blood.

Schizonts Enlarged

Ring Form

Composition

The blood is the most common specimen for recovery of various stages of parasites, i.e., Plasmodium spp., Babesia spp., Trypanosoma brucei gambiense, Trypanosoma brucei

Trophozoites

P falciparum

Leishman and Giemsa stains are used to stain both thin and thick blood smears. (i) Leishman stain: Prior fixation of blood smear

To observe and identify the causative agents in the peripheral blood smear examination.

103

stain and • kept for 30 minutes to 2 hours

drop is madeintoa thick smear

on

Filariasis

thin smear is first fixed and after drying, the whole slide is flooded with diluted Giemsa

stain,

the

two different places

the otherinto thin smear. Thin smear preparation and the larger area and the thick COMPETENCY

Malaria and

Leishman), dehaemoglobinization with water

necessary

stains

Plasmodium falciparum

For staining thin and thick blood smears

Plasmodium malariae

on the same slide, the thick smear

dehaemoglobinized and then stained along with the thin smear. A line with a glass-marking pencil is drawn between two smears. The

undiluted Leishman stain is poured over the flooded thin smear and after dilution the stain over the thick smear also.

case of Giemsa

Plasmodium ovale Fig.

17.2 Gametocytes of Plasmodium species

Unit II Practical Exercises For identification of these stages, you should know these of

he morphological features of different stages are plasmodia. Differential features of different stages shown in Table 17.1.

infect humans. Four

Eight species of filarial

species Wuchereria bancrofti, Brugia malayi, causes

Loa Toa and

Onchocerca volvulus are important Table 17.1: Differential Features of

peripheral blood

peripheral blood smear The most important among these are microfilaria W.

bancrofti and B. malayi,

the

causative

agents

lymphatic flariasis. Differentiatingfeatures ofdiffen"

Different Plasmodia of Man

cytoplasm opposite

P. malariae

P. falciparum

Ring forms and

Trophozoites, schizonts and gametocytes

2. Early trophozoite 2.5 jm in diameter, or ring form

of human

P. vivax

Feature

Forms in

important causes. Microfilariae of Wuchereria bancros Loa loa,Mansonella perstans Brugia malayi, B.betimori, detected in M. ozzardi can

B. Filariasis

1.

disease while other four species Mansouell. perstans, M. streptocerca and Brugia timori are

M

gametocytes (crescent

Trophozoites,

schizonts and

ovale

Trophozoites.

schizonts and

shaped)

1.25-1.5 um in diameter, multiple

the

Similar to that to P. vivax

Similar to that of P. vivax

rings in one red blood

nucleus is thicker

Figs 17.3

Table 17.2: Mf. malayi

17.4. Microfilariae may be sheathed or unsheathed

Morphology of microfilariae (Embryos) colourless and transparent with blunt heads and pointed tails. The embryo measures 245-290 um 5-7 um in size

by a hyaline sheath.

The

vacuole present

Compact form, rarely Band shaped, slightly

amoeboid, pigment: collect:

single

amoeboid, vacuole

inconspicuous

Larger (290

Appearance

sheath is much longer (359 um) han the embryo so that the microfilaria (embryo) can move forwards and backwards within it. The somatic cells or nuclei appear as granules i the central axis of the embryo and extends head to the tail-end. The from the granules do

Mf malayi Smaller

Sweeping curves Kinky with secondary curves

Cephalic

the tip of the tail and is a distinguishing feature of Microfilaria bancrofti (Fig. 17.3). Microfilariae (MF) of W. bancrofti should be differentiated from those of B. malayi. Differentiating features of Mf. bancrofti and Mf. malayi are shown in

of Mf bancrofti and

Mf bancrofti

Length

space

Length and breadth equal

Twice

Anterior end

Single stylet

Double stylet present

long

broad

present

extend up

Nuclear

Discrete nuclei

Blurred

column

Tail tip

Two distinet nuclei,

Free

one terminal and the

and Table 17.2. Fig. 17.5

other subterminal

Sheath

cell, form accole

Irregular, amoeboid

Differentiating features

Feature

They

and is covered

105

Malaria and Filariasis

microfilariae found in blood are shown

Faintly stained

Well stained

Compact,

amoeboid, vacuole

Sheathed microfilar

inconspicuous

Unsheathed microfilariae

mass

4.

Schizont

5. Merozoites

6. Gametocyte

in diameter, 4.5 to 5.0 um in diameter, fills two almost completely fills an enlarged erythrocyte thirds of normal to 10 um

12-24 in number

6.5 to 7.0 um in diameter, almost

6.2 um in diameter.

fills a normal size

quarters of slightly enlarged erythrocyt

size erythrocyte

erythrocyte

18 to 24 in number

6-12 in number

Round, size of a red Crescentric (sickleshaped), larger than a blood cell red blood cell Similar to that Spherical, larger than male Sickle-shaped, longer, more slender, P.vivax but smaller gametocyte, cytoplasm stains deep blue, cytoplasm stains nucleus is small and deep blue, nucleus is

Spherical, much larger

than a red blood cell

(i) Female

(Macrogametocyte)

compact.

(ii) Male

(Microgametocyte)

Spherical, smaller than female gametocyte, cytoplasm stains light blue or pale blue, nucleus large

P.vivax but smaller

8. Infected

Enlarged, pale,

erythrocyte

Schuffner's dots present

Age of the

Young

erythrocytes

10

Duration of erythrocytic

48 hours

Nucle

not

up to tip

up

Round, size of a red Two nuclei at the tip of tail

Similar to that of P. vivax but smaller

Nuclei present continuous

up

Mansonella

Mansonella Streptocerea

Onchocerca malayi

Similar to that of P. vivax but smaller

Fig, 17.3 Differentiation of different microfilariae

light blue, nucleus is diffuse

SHEATHED MICROFILARIAE

Dark-brown

Dark-brown

UNSHEATHED MICROFILARIAE

Dark yellowish-brown

Size unaltered, Normal size and Slightly enlarged, oval Maurer's dots present dots, Ziemann's dots shaped and James's on prolonged staining dots present All

ages (young and

Old

Young

72 hours

48 hours

old)

infected

Nuclei extend up

up to tip

blood cell

diffuse

Malarial pigments Yellowish-brown

Nuclei do not extend

6-12 in number

bancrofti

Similar to that of

Tail nuclei

fills about three

compact

Sickle-shaped, broader, shorter, cytoplasm stains

nuclei

hours

schizogony

BANCROFTI

MALAYI

Fig, 17.4 Differences of nuclei at tail

ips of different microfilariae

Unit II Practical Exercises

106

You may be

Body curves small

irregular and kinky

Body curves large,

regular and smooth

as follows:

provided witha case history of patient

vear old man A 30

spikes as

stylets

of shaking chills followed by fever morpholigy of W. bancrofti (Fig. 17.5). 104'F and then profuse sweating

40 year old man presented with swelling of

he had either no fever or

blood specimen was collected and sent to the laboratory for peripheral blood smear examination You have been

right leg with fibrosis was present. Left leg was normal. Blood specimen was collected in night at 11.00 pm for examination. Giemsa stained peripheral blood smear is provided to you for identification of parasite.

In between

provided with peripheral parasite.

and squeezed

A

whole right leg up to the foot. He also had feeling of

heaviness and discomfort. Hardening and thickening

these episodes,

low grade fever, myalagia, nausea and vomiting. On examination, he appeared very fatigued and pale. His

Body nuclei blurred

and wel

high as

always felt exhausted. These severe episodes had been occurring every other day

longer than broad

Body nuclel round

You may be provided with a stained peripheral blood with complaints of smear for identification of microfilaria You should days. observe the stained smear under microscope for typical

After these episodes he

•Cephalic space is

Cephalic space as broad long

107

Filariasis

chills, nausea and vomiting for the last 4 He had episodes fever

Single stylet

presented

Malaria and Filariasis 2.

blood

mear to identify the

of skin was

present. He had no history of cancer

or

radiotherapy or surgery. On examination, bulky swollen

together

separated

Essential Points to Remember

Preparation of peripheral blood smear thick and thin.

Staining methods of peripheral blood smear. Differences between stages of P. vitur and P. falciparum. Sheathed and unsheathed microfilariae

3.

Differences between microfilariae No nuclei at tail-tip

nuclei at tail-tip, terminal d other

Questions Related to Malaria

subterminal Mf. bancrofti

Fig.

17.1

. malayi

smears are then examined under the microscope for the demonstration of microfilariae. When the microfilaria low, the concentration

membrane

techniques such

as

filtration technique and sedimentation

the tail portions.

technique may help to detect microfilariae in blood.

the

Microfilariae can also be demonstrated in microscopic mounts of anticoagulated blood (unstained preparation)

by their undulating motion, displacing the

from side to side

Acridine

with their movement.

erythrocytes

in other specimens such as chylous urine, exudate of

Diethyl carbamazine (DEC) provocation test cases

of

nocturnal periodicity of

microfilariae may in

induced to appear in the blood by giving diethylcarbamazine (hetrazan) single dose (hetrazan provocative test) of 2mg per body weight. Usually peak numbers are reported

to occur after

be

15-20 minutes

intake.

for demonstration of microfilariae. A microhaematocrit tube contains heparin, EDTA and

acridine orange. The blood is incorporated in this tube and then centrifuged. Due to centrifugation, the

1.

of

be visualised through the glass wall

of the tube. The

DNA of the parasites is stained byacridine orange and the morphological characteristics

can

be examined by

fluorescent microscopy. A speciesidentification can also

Ans.

B. timori

Q3 What is the vector for Q4

Q.4 How will you differentiate between P CIVIY and Ans.

Ans.

of

Elephantiasis Lymphangiovarix

(i) Cerebral malaria (ii) Algid malaria

Hydrocele

Q5 What is occult filariasis?

(iv) Black water fever examination, what

hypersensitive reaction to microfilarial antigens. Patient develops massive eosinophilia (30-

Ans. It is due

You should be able to demonstrate the method of staining and also to identify the different stages microscopic

80%], hepatosplenomegaly, generalised lymphadenopathy and pulmonary symptoms.

Rapid diagnostic tests (RDTs)

Q.7 What are rapid diagnostic tests? Rapid diagnostic tests (RDTs) are based

Ans.

Vanious manifestations may include:

Lymphangitis Lymphadenitis

malaria caused by

peripheral blood smear

What are manifestations of classical filariasis caused

by Wuchereria bancrofti?

Refer 'Table 17.1' on page 104 in this chapter.

0.5 What are complication P. falciparum?

lymphatic filariasis?

Ans. Mosquito is the vector

are other diagnostic tests for malaria? Ans. • Quantitative buffy coat (QBC) test

parasite.

examination.

Brugia malavi

(i) Giemsa stain (ii) Leishman's stain

0.6 Besides

may be provided with a stained peripheral blood smear for identification of malarial

of parasite (Fig. 17.1 and 17.2) on

Q2 Name the causes of lymphatic filariasis. Wuchereria bancrofti - The most common

Ans.

(iv) Jaswant Singh Bhattacherjee (JSB) stain

(1) You

(ii)

102

this chapter.

03 Name the Romanowsky's stains

diethylcarbamazine

Malaria

parasites become concentrated in the buffy coat and can

bancrofti, Brugia malayi, loa loa, Onchocerca volvulus, Mansonella ozzardi, M. perstans, M. streptocra and Brugi timori.

Ans

the parasite,

during the day kg

Ans. Wuchereria

prepare peripheral blood smear?

Ans. Refer 'stained peripheral blood smear' on page

lymph varix and hydrocele fluid.

prange-microhaematocrit tube technique IV. EXERCISE

can also be used

0.2 How do you in

Besides blood, microfilariae may also be demonstrated

In

Q1 Name the parasites causing filariasis? P.

ovule.

be done by observing the characteristic nuclear pattems

The microfilariae appear in large numbers in peripheral in

Questions Related to Filariasis

Name the parasites causing malaria in humans? malariae and Ans. Plasmodium vIvax, P. falciparum 0.1

Differentiating characteristics of Mf. bancrofti and Mf. malayi

blood at night. Hence, blood film should be made in night between 10 P.M. and 2 A.M. Thin and thick smears are prepared. The thick smear is dehaemoglobinised and both the smears are stained with Giemsa stain. These

density

bancrofti and B. malavi.

Two

Q6 on the

detection of antigens using immunochromatographic

methods. These tests take about 15 minutes and thus are rapid. Histidine rich protein (HRP-II) and parasite lactate dehydrogenase (PLDH) can be detected by

Which microfilaria can be found in blood of patient?

timori and Loa loa can be found in blood. Q7 Name the sheathed microfilariae. Ans. Microfilariae Wuchereria bancrofti

Unit II Practical Exercises

remain in the

Brugia malay! Loa

0.8 What are the differences

as

between microfilariae

of

Brugia malayi? Wuchereria bancrofti Ans. Refer Table 17.2 in this chapter

0.9 What is nocturnal periodicity? Ans.

The microfilariae appear about

after midnight (10 PM to 2 period

microfilariae is nocturnal. Ans. • Wuchereria bancrofti

which

This is

periodicity.

Brugia malayi and

0.11

peripheral

the rest of the 24 circulation. They disappear for hour

filarial worms Q.10 Name the

B. timori

2 hours before

AM) in

pulmonary circulation.

nocturnal periodicity.

from the peripheral circulation

and

What

is Diethyl carbamazine (DEC) provocat

test?

Ans.

Confidentiality in

18

Laboratory Results

Refer 'Diethyl carbamazine (DEC) provocative in

this chapter.

PREVIEW I.

COMPETENCY

Introduction

The student should be able to

I. The Rights and Responsibilities of Patients A. Patient's Rights Patient's Responsibilities ITI.

The Rights and Responsibilities Staff A.

MI 8.14

Demonstrate confidentiality pertaining to patient identity in laboratory results.

Laboratory

Rights

B. Responsibilities IV.

Ethical Issues

SPECIFIC LEARNING OBJECTIVES The student should be able to learn:

About the confidentiality pertaining to patient identity in laboratory results. 2. About the importance of confidentiality. . INTRODUCTION Confidentiality

2.

Right to

person should be discriminated by laboratory staff the basis of their illness, HIV status or

important ethical principle in Confidentiality is medical practice. It is an important legal and medical gender, age,

obligation which all health professionals are supposed to

obey. Matters regarding patients should not be

disclosed to others. are

is

Confidentiality, trust and privacy

interconnected. If there three principles which privacy and trust then there will not be any

confidentiality. II. THE RIGHTS AND

RESPONSIBILITIES

OF PATIENTS A.

Patient's Rights

have, which protected by our laws, while responsibilities are duties or things that

1.

do.

Right to confidentiality

The laboratory report should be kept in strict confidentiality from everyone except the patient and

their caretakers. Unless it is an exceptional case where sharing the information is "in the interest of protecting other or due to public health considerations."

their

religion, caste, ethnicity, sexual orientation,

geographical or social origin. 3.

Right to informed consent

All

laboratory tests carried

out on

patient require

the informed consent of the patient. When patient presents himself or herself with a request form and willingly comes for laboratory test, the consent can be inferred. However in some invasive procedures, written consent may

be required. The patient should

be informed about the procedure and

its

possible

complications.

Rights are freedom we should

non-discrimination

No on

All HIV

laboratories under National AIDS Control

Organisation (NACO) take informed consent before collection of blood specimen. Pre-test counselling is also done. The result of HIV test should be kept strictly

confidential. The report is given to patient after posttest counselling. In

emergency situation, when consent may

possible then the procedure may be carried

patient's best interest. 109

be

out in

Unit II Practical 4.

Right

Exercises

laboratory tests to choose The patient has the right

Ethical Conduct

3.

to choose the source for obtaining

Do

Do

•

protection of participants involved

in biomedical research

mobile phones during the course of testing

I patient is involved in a biomedical research, their

Maintain strictly the confidentiality

format consent needs to be taken in a written

Right

to be

heard and seek redressal

Every patient has the right to address his grievances and give feedback. They have the right to seek redressal in are aggrieved.

case they

B.

Maintaining of Confidentiality

Patient's Responsibilities and for

informing

the

health centre or

laboratory in advance when unable to keep the Patients are responsible information about them.

3

the autonomy

To respect

for

of

giving

results unattended. Do not

•

To treat laboratory staff with respect.

III. THE RIGHTS AND

OF A.

has

or

•

6.

Laboratory personnel should know the importance of ethics and should follow the ethics during various stages of laboratory testing. 1. Ethics

2.

is a set of principles of right conduct.

Importance of Ethics

Diagnosis, prognosis and treatment of patient are

based on resultsandinterpretations of laboratory tests.

Erroneous laboratory tests may harm the patient.

of

2. Beneficence: To act in the best interest of patients to maximise benefits and minimise harm. It is also called non-maleficence.

Justice: The duty to treat patients equally and to distribute fairly what is due in terms of benefits, risk and cost.

These principles can be applied to all three phases pre-analytical phase, analytical phase and post-

timely manner.

Follow universal interpretation of results, residual specimen storage and (a) Respect for persons: The confidentiality of results should be maintained. Patient or patient

laboratory should be able to Justice: provide access to a variety of laboratory tests The

(c)

Patients have expectation that their specimens will

be used solely for the laboratory tests requested for. Any further testing of residual specimens

should be approved by a local ethical committee and patient consent may (b)

older tests.

include name of the

Respect

for

persons: Patients

limits.

Turn around time (TAT) should be as short possible. However achieving this TAT should not compromise the validity of the results.

have right to

decline to have their specimens analysed even after

processing of collected specimens. Confidentiality

Withholding of laboratory results should not

should be respected.

laboratory should be possible analytical result.

case of non-payment, it may harm the patient especially in emergency condition.

done in

(b) Beneficence: The aim of to provide the best This is

achieved by adopting good laboratory professional standards. Good

(c)

practice and

maintained by rigorous laboratory practice quality assurance programme which comprise

Justice: The reporting of laboratory results should be consistent for all patients. Rapid reporting may be

required for

Confidentiality is an

important principle in

medical practice

some laboratory results. Rapid

reporting must apply regardless of patient's ability to pay.

of quality control testing, proficiency testing and Essential Point to Remember

1.

test performed, normal values age and gender specific, unit of

measurement and when possible to indicate whether the test is with in or above normal

laboratories.

The roles of laboratory personnel include:

qualified laboratory personnel

should interpret the reports. The test result should of the test for

(a)

required.

results can lead to patient harm. To minimise this harm, only

Examination (analytical) phase Confidentiality, quality and competence are vital for all

be

Beneficence: Misinterpretation of laboratory

at a

reasonable cost. When better tests are available, the laboratory should discontinue

Pre-examination (pre-analytical) phase appropriate

approval from appropriate

family is regarded as legitimate recipients of patient's laboratory results.

of specimen is preserved.

analytical phase of laboratory testing.

method.

of specimen.

ethical committee. The specimen should be transported in a manner so that the integrity

the ethical issues in laboratory practice depends on

() Proper identification of the patient.

analysed accurately and

All tests should

the patient and

but because they are careless. Sometimes they with good intentions.

(ii) Collection I appropriate specimen by

process including data entry.

Justice: Discrimination in the analysis of patient racial specimen on the basis gender, age origin is an injustice. All specimens should be

Additional specimens should not be collected for research without informed consent from

not falsify results.

1. Respect for persons: Acknowledgement autonomy.

3.

(POCT).

specimen. Properly trained personnel should be involved in collection of specimen.

in your

three principles:

Do not falsify results.

IV. ETHICAL ISSUES

Ethics

All

for these

personnel should perform point of care testing

(c)

as laboratory personnel during collection of

People often violate ethics not because they mean act

has

5. Maintain strictly the confidentiality of patient information and laboratory test results.

Do

to,

B. Responsibilities key role for maintaining the quality of laboratory test. 2. Always conduct in professional manner. 3. Respect the autonomy of patient. 4. Safeguard the diginity and privacy of patient.

process

Do not take shortcuts for test results If a test

•

appropriate policy

such specimens is compromised. Only qualified, properly trained and regularly e-accredited

precautions to protect the patient as well data access.

procedure requires 30 minutes incubation, then wait for the full time before recording and reporting of laboratory test results.

laboratory staff,

or

Post-examination (post-analytical) phase harm e.g. infection or pain from the collection The post analytical phase includes reporting and

patient information with friends

kits which are approved

labelling

benefit the patient. Specimen collection should not cause

Follow Standard Operative Procedures (SOPs)

Provision of safety and protective measures for

Laboratory staff

step of the

(b) Beneficence:

country.

LABORATORY STAFF

environment.

laborator

co-worker's Private

poor

specimens when integrity or identification

Confidentiality must be maintained at every

relatives.

Use only

there should be

testing.

5. Ethics in Laboratory Testing

RESPONSIBILITIES

good comfortable working

confidential

or

deficiencies. In case of precious and difficult to obtain' specimens (e.g. cerebrospinal fluid) may be considered as a special case. However,

of a patient's autonomy versus the benefits of

been sought.

Do not discuss the

Rights

2. Provision of a

your

integrity, incorrect

such as intravenous drug users, prisoners and before blood donation. In these exceptions, laboratory personnel should consult the

information with staff or patients unless permission •

staff.

5. To preserve his laboratory reports.

disclose

practice, refuse to analyse or report a result when there poor specimen

guidelines. They must compare the risk of loss

Do not leave patient information and

•

true

the laboratory

medical records

For good laboratory

cases of compulsory testing in certain groups

documents.

appointments. 2

Patiens

responsible manner.

4.

Handle

Patients are responsible for keeping appointments

of

information and laboratory test results. Safeguard the dignity and privacy of patients Perform duties in an accurate, precise, timely and

in

regard.

for persons:

Consent should be obtained prior to specimen collection. The consent should be informed L.e. the patient should know what laboratory test being done and why. The patient's right to refuse to be tested should be respected. There are

Alwaysconductin a professional mannersud wearing of clean laboratory coat, turning off i

•

Results

laboratory accreditation. 'A wrong result than no result should be guiding principle

handling o thespecimen until testing

done. (a) Respect

practices

5. Right to

specimen. is

not indulge in financial, undue commen

Confidentiality in Laboratory

Appropriate identification and labelling of the

(iv) Appropriate

integrity.

done.

6.

(in)

confidencein laboratory competency or operatine

any diagnostic

centre or laboratory for getting laboratory investigation

If

not get involved in activities which diminia

Appropriate Laboratory Tests in

Tests

Appropriate Laboratory in the

19

Diagnosis of

Specimen

Cholera

13

Acute

14.

Culture

Antigen detection Chancroid

15.

Introduction

MI 8.15

Choose and interpret the

laboratory tests used in infectious diseases.

Blood

results of the

the diagnosis of

V. Examples of Clinical Cases

Syphilis

Blood

21.

Scrub typhus

Blood

22.

Chlamydial infections

1.

To choose and interpret the results of

appropriate laboratory

test used in the diagnosis of infectious disease in a clinic

Table 19.2:

II. APPROPRIATE LABORATORY TESTS

INTRODUCTION

Infectious diseases can be diagnosed by various Appropriate laboratory tests in bacterial viral, and parasitic diseases are shown in Table laboratory tests. These tests have been described in 19.1 to 19, respectively. different chapters of this book. However the appropriate

laboratory test for

diagnosis of some infectious diseases

described here.

Table

19.1: Appropriate Laboratory Tests

Appropriate Laborato

1.

•

2.

4.

Streptococcal sore throat

Throat swab

Throat swab culture

Sputum

Sputum culture

Meningitis Gonorrhoea

Diphtheria

Urethral discharge 9

•

Throat swab

Suspected food

Pseudomembranous enterocolitis

11.

Enteric fever

12.

Food poisioning

Blood

5.

.

6.

Dengue

Blood

• Immunochromatographic test for detection of NS1

fever

for IgM antibody (antibody appears 5 days

of symptoms). ELISA for specific IgM antibody ELISA for specific IgM antibody

Blood

Rubella 9.

Blood

• ELISA for detection of HAV antigen in faeces

Hepatitis A •

Blood

•

ELISA for specific IgM antibody in serum

Hepatitis B

Blood

11. Hepatitis C

Blood

ELISA for HBsAg in serum RT-PCR for detection of HCV-RNA in blood

12.

Hepatitis D

Blood

ELISA for detection of IgM anti-delta antibody

Culture

13.

Hepatitis E

Gram

14.

HIV

15.

Human papilloma

10.

• Gram staining Culture

serum

Albert staining staining

Culture

.

Blood

virus

Severe Acute Respiratory Infection (SARI)

Blood culture

17.

Severe Acute Respiratory

Syndrome (SARS)

18. Rotavirus diarrhoea Contd

ELISA for detection of IgM

Blood

Tissue

Cell smears

•

antibody in serum

Rapid tests or ELISA for detection of antibodyin seru Histopathological staining of tissue biopsy Papanicolaou (pap) smear prepared from cervical scrapings

16.

Culture

112

ELISA for specific IgM antibody ELISA for specific IgM antibody

Blood

after onset

Faeces culture

Food

Reverse transcriptase polymerase chain reaction (RT-PCR)

•

Measles

Faeces culture Blood

Laboratory Test/Tests

ELISA for specific IgM antibody ELISA for specific IgM antibody

Nasopharyngeal swab

Mumps

• ELISA for demonstration of toxin

9.

Dysentery

Influenza

7.

Blood culture

Botulism

10.

Blood

ELISA

Gram staining/CSF culture

0

6.

Chickenpox

antigen (detects antigen on the first day of before antibodies appear).

Pus culture

Pneumonia

Blood 5.

3.

Blood

Gram staining

Pus

Tissue culture on McCoy cells Immunofluorescence

Specimen

Herpes simplex infection

Laboratory Test/Tests

Specimen

Staphylococcal pyogenic infection

Serology for antibody detection • ELISA

Tests in Viral Diseases

Viral Disease

in Bacterial Diseases

Bacterial Disease

S. No.

ZAN staining

Vaginal specimen Cervical specimen

S. No.

I.

ZN staining

Urethral specimen

SPECIFIC LEARNING OBJECTIVES The student should be able to learn:

Serum agglutination test (SAT) Culture

Culture

Skin slit smear

IV. Clinical Case Presentations

Culture

Sputum

Leprosy 20.

Gram staining •

Blood culture

Respiratory secretions

Tuberculosis

18.

The student should be able to

Appropriate Laboratory Tests Diagnosis of Infectious Diseases

Aspirated material from bubo

Whooping cough

Gram staining

Culture

Scraping of ulcer

Brucellosis

COMPETENCY PREVIEW

113

Laboratory Test/Tests

pyogenic meningitis

Direct microscopy I

16.

III.

Diagnosis of Infectious Diseases

Bacterial Disease

Infectious Diseases

II.

the

Table

Oropharyngeal swab Nasopharyngeal swab

Nasopharyngeal swab

Nasopharyngeal aspirate

RT-PCR for detection of RNA of SARS-CoV-2 RT-PCR for detection of RNA

• ELISA for detection of rotavirus in faeces

Unit II Practical Exercises Table

19.3: Appropriate Laboratory

Tests

AppropriateLaboratory Tests

in Fungal Diseases

Fungal Disease

Septicaemia due to candida species Histoplasmosis

Laboratory Test

Specimen

Blood culture

Blood

Latex agglutination test for antibody

Blood

Latex agglutination test

Culture

Cryptococcal meningitis

Table 19.4: Appropriate Laboratory Tests for Parasitic Specimen

S. No. Parasitic Disease 1.

Amoebiasis

detect. antibody detern

Diseases

Laboratory Test

2.

Giardiasis Trichomoniasis

Vaginal discharge Urethral discharge

Malaria

Blood

Peripheral blood smear examination for various stages of malar

Stool

Modified Ziehl-Neelsen (ZN)

parasites.

Cryptosporidiosis

6.

Intestinal taeniasis • Stool

7.

Trichinellosis

of Cryptosporidium parvum.

staining of stool for acid-fast oocysts

Muscle biopsy

Biopsy specimen examination for detection of spiral larvae of Trichings,

III. DIAGNOSIS OF

2

Gram staining may show Grain positive cocci in cluster

while culture on blood agar may show golden yellow

INFECTIOUS DISEASES

Staphylococcus aureus which has characteristics ofGram

infectious diseases have been described.

positive cocci in clusters, catalase positive and coagulase

IV. CLINICAL CASE PRESENTATION

positive.

Appropriate laboratory tests described in this chapter will be very useful for choosing the laboratory tests

clinical cases. Clinical case reports are provided to students with history, clinical examination, other relevant findings of patients and the student is asked to in

choose the appropriate laboratory tests and to interpret results of these tests

Some examples of clinical case presentations are

described below, for selecting appropriate laboratory

tests their

interpretation.

V. EXAMPLES OF CLINICAL CASES CLINICAL CASE 1

CLINICAL CASE 2 25 year old male presented with dysuria and urethral discharge. He had history of sexual exposure with a

commercial sex worker. Urethral discharge was sent to the

laboratory for examination.

Which tests would be appropriate for diagnosis?

2. How will you interpret the results? Answers 1. Gram

50 year old man presented with complaints of a painful

localised swelling with pus discharge. On physical examination, the local area of the lesion was red, warm

and tender. Pus was aspirated and specimen?

Answers

Gram staining of pus and culture on blood agar should

culture on chocolate or modified

Gram staining of urethral discharge may show pus cells withintracellular Gram-negative diplococci. The culture may show typical translucent colonies which are oxidase positive.

A 6 year oldchild was broughttothe emergency department with high

grade fever, pain in the throat and difficulty i

swallowing. On examination, a white patch was presenton the fauces. He had history of incomplete immunisation. 1.

Which specimen and tests

diagnosis?

antibody to HCV is detected by ELISA. The gold standard is to detect HCV RNA by RT-PCR HBsAg detection by ELISA will diagnose hepatitis ELISA and HCV infection. Antibody to HCV

2.

RNA by RT-PCR are useful in diagnosis of hepatitis C infection.

CLINICAL CASE 4

E

year old male presented to STD clinic with a single,

Pa examination,

exposure with a commercial sex worker.

On

the nodes was hard and indurated. The penile ulcer were enlarged,

and inguinal lymph Blood specimen was collected and sent ion-suppurative. diagnosis.

painless, fir

CLINICAL CASE 7

A 30 year old asymptomatic man visits ICTC for testing of Human immunodeficiency virus (HIV) infection. 1. Which is the appropriate test for HIV diagnosis?

to the laboratory 2.

2. How will you interpret the results? 3 What other requirements for HIV

Which test will e appropriate for diagnosis? How will youinterpret the results?

1. Rapid tests for detection of

Venereal disease research laboratory (VDRL) test or rapid plasma reagin (RPR) test is appropriate for diagnosis. test may be 'reactive' It is a flocculation test 2. VDRL

2.

diagnosis of syphilis RPR test may be positive in syphilis.

for

CLINICAL CASE 5 30 year old male presented to

the hospital with high

fever, severe headache, loss [ appetite and pain around the eyes for last four days On examination, the temperature was 103°F and maculopapular rash was observed on the chest and upper limbs. The blood specimen was collected and sent to microbiology laboratory for examination.

1. Which laboratory test will be appropriate for diagnosis? How will you

2.

interpret the results?

will be appropriate for

HIV

antibodies

are

for diagnosis. appropriate If first rapid test negative, it is reported negative. In third rapid case first test is positive, then second tests (total three rapid tests) ire required for diagnosis asymptomatic HIV diagnosis in This is strategy Ill

cases. ELISA can also be performed as alternative to rapid test.

and confidentiality. Pre-test counselling, are required for HIV testing in the laboratory. Laboratory report should be given only after post-test

counselling. CLINICAL CASE 8 A 3 year old boy was brought to the emergency department of the hospital with fever, vomiting and loose motions for one day. He had profuse, watery stools. His classmates

similar complaints recently. No adult member of the family was affected. On examination, the temperature was 100°F and he had tachycardia. His

at day care centre alsc had

Answers 1.

testing?

Answers

Answers

A

115

confirm the diagnosis. For hepatitis infection, HBsAg is detected in serum by ELISA. For hepatitis 1 infection,

ELISA

test for dengue NS1 intigen is an appropriate

test

the basis of clinical history and examination.

Since patient is having fever for last 4 days, antigen detection in blood is appropriate. After 5 days, IgN

antibody can also be detected in serum by ELISA.

eyes appeared

sunken. His abdomen had active bowel

sounds. Stool specimen was sent for direct microscopy and bacterial culture. Direct microscopy was negative for red blood cells and pus cells. Bacterial culture of stool had

growth. The presumptive diagnosis of viral gastroenteritis was made.

CLINICAL CASE( A 40 year old woman

presented with

past 12 days. She had received multiple blood transfusions over the past five months. She had been passing high coloured urine. She had elevated levels of serum bilirubin

and liver enzymes.

What laboratory tests will you perform to confirm the

diagnosis? 2.

Which appropriate

1.

How will you interpret the results of laboratory test?

test will

you perform to diagnose

viral gastroenteritis?

loss of appetite,

nausea, vomiting, abdominal pain and persistent fatigue for

CLINICAL CASE 3

to perform on this

How will you interpret the results?

be performed.

staining and

sent tc the laboratory.

Which tests would be appropriate

Albert's staining of smear from green coloured bacelliwith granules, Culture on tellurite blood agar may show black colouredcolonies of Corynebucteriumdiphtheriae 25

Diagnosis of Infectious Diseases

Antigen and antibody can be detected in serum to

throat swab may show bluish black

2.

Thayer-Martin medium should be performed. 2.

the

Answers

and culture

beta-haemolytic colonies. Identification of bacterial colonies may be done by Gram staining, catalase testand coagulase test. These tests may confirm the bacterium &

Diagnosis of infectious diseases may require performance of more than one type of tests. However, in this chapter the most commonly used appropriate tests for various

interpret theresults?

anhinedmndoeotbr .iomd me tireny wandhchasedebeexamination.

Stool examination for the eggs of Taenia species. spiralis.

the

1.

Stool examination for cysts and trophozoites of Entamoeba histoly

Stool examination for cysts and trophozoites of Giantialamblia for Wet mount' preparation trophozoites of Trichomonas Daginali

ill you

How

Answers

2.

3.

5.

2.

2

How will you interpret the results of this test?

Answers

ELISA test may be done to detect rotavirus antigen 1 stool specimen.

2. Detection of rotavirus antigen in stool is a diagnostic test. Rotavirus is the common cause of viral gastroententis in children.

116

Unit II Practical Exercises 2 How

CLINICAL CASE 9 29 year old

male with known AIDS

presented to the

last 3 days. On examination, 100°F. Neck temperature other vital signs

he

Which appropriate testswill you perform fordiagnosis?

agglutination

for

on Sabouraud dextroseTyptocony, antigen and culture agar neoformans.

rigidity was observed but

ail aseptic conditions. CSF was sent to the laboratory for diagnosis of fungal causes.

results?

should be examined by Gram staining, Indias test

preparation

the

were normal. Clinically, meningitis was due to fungal cause was suspected. A lumbar puncture performed to collect the cerebrospinal fluid (CSF) under

1.

CSF

and fever for

emergency department with headache confused and

will you interpret the

Answers

(SDx Cryptococous Gram staining may show Gram positive, round

20

for

2.

CE India ink

reparationmay reveal budd,

calmiy bepositive Culture

of Cryptococcus neoformans.on stDA may

clear

Spots

revealpond

be in the form of culture media without The spots may growth and with growth, biochemical reactions,

Glassware, equipments, swabs, specimens of parasites,

animal pathogenicity tests, ticroscopic slides showing some organism, wet preparation of stool for ova or

cysts of parasites and some miscellaneous items such as

anaerobic jar, nichrome wire or

loop, microtitre plate, VDRL slide, agar-agar shreds etc. The spots which are generally given in the examination are as follows. These

Urease test (Fig. 20.28)

Citrate utilisation test (Fig 6. Phenylpyruvic acid (PPA) test (Fig. 20.31)

Triple sugar iron (TSI) agar (Fig. 20.32)

7.

IV. GLASSWARE 1. Universal container (Fig, 20.33) 2. Bijou bottle (Fig. 20.34) 3. Medical flat bottle or McCartney bottle (Fig.

MEDIA

1. Nutrient agar (Fig. 20.1)

2. Blood agar (Fig. 20.2) 3. MacConkey's agar (Fig. 20.3) 4. Chocolate agar (Fig. 20.4)

20.35)

5. Potassium tellurite blood agar 6.

Glass syringe (Fig. 20.36)

Tuberculin syringe (Fig. 20.37) Petridish (Fig. 20.38) Graduated pipette (Fig. 20.39)

Loeffler's serum slope (Fig. 20.5)

Lowenstein-Jensen (LJ) medium (Fig. 20.6)

Cooked meat broth (CMB) (Fig. 20.7) 9. Selenite F broth 10. Blood culture set containing glucose broth

7.

8.

taurocholate broth (Fig. 20.8)

9. Desiccator V.

DISPOSABLE ITEMS . Pre-sterilised disposable container (Fig. 20.41)

II. CULTURE MEDIA WITH GROWTH of

Tissue culture bottle (Fig. 20.43) 4. Pre-sterilised disposable swab (Fig. 20.44)

3.

VI.

EQUIPMENTS/INSTRUMENTS 1. Incubator (Fig. 20.45)

Streptococcus,

2. Hot-air oven (Fig. 20.46) Autoclave (Fig. 20.47)

Staphylococcus or Proteus spp. (Figs. 20.14-20.16) 2. Nutrient agar with Pseudomonas spp. (Fig. 20.17) 3. MacConkey's agar with growth of Esch. coli, Klebsiella spp. or NLF colonies. (Figs. 20.18-

4. Inspissator (Fig. 20.48) 5. Centrifuge Water-bath

6.

20.20)

Potassium tellurite blood-agar with growth of C. diphtheriae. (Fig. 20.21) 5. Lowenstein-Jensen (LJ) medium with growth of M. tuberculosis (Fig. 20.22) or atypical

Pre-sterilised disposable syringe (Fig. 20.42)

2.

Peptone water (Fig. 20.12) 15. Glucose broth (Fig. 20.13)

14.

agar with growth

Pasteur pipette (Fig. 20.40)

8.

and

11. Automated blood culture bottle (Fig. 20.9) Castaneda medium (Fig. 20.10) 13. Sabouraud's dextrose agar (SDA) (Fig. 20.11)

1. Blood

growth

4. Indole test (Fig. 20.29)

examination (OSPE).

I. CULTURE

with

III. BIOCHEMICAL REACTIONS Glucose with Durham's tube (Fig. 20.26) 2. Carbohydrate fermentation tests (Fig. 20.27)

spots may be asked in the form of 'objective structured

practical

Sabouraud's dextrose agar (SDA) of Aspergillus niger (Fig. 20.25)

8.

7. VDRL rotator

4.

8

Lovibond comparator

9. Micropipettes (Fig. 20.49) VII. SWABS

mycobacteria.

6. Sabouraud's dextrose agar (SDA) with growth of Candida spp. (Fig 20.23) 7. Sabouraud's dextrose agar (SDA) with growth

1.

Sterile swab (Fig. 20.50)

2.

Pre-sterilised disposable 20.44)

3. NIH swab (Fig.

of Aspergillus fumigatus (Fig. 20.24).

4.

117

Postnasal swab

20.51)

swab

(Refer Fig.

Unit II Practical Exercises VIIL. SPECIMENS OF 1.

Housefly (Fig. 20.84) Sandfly (Fig. 20.85)

ADULT PARASITES

Hydatid cyst (Fig.

6.

20.89) 7. Soft tick (Fig 8. Mite

20.53)

SPECIMENS OF ANIMAL

PATHOGENICITY TESTS

Specimen of Guinea-pig showing 2. Animal pathogenecity

of C. diphtheriae toxin

XIV. MISCELLANEOUS ITEMS

Candida albicans (Fig. 20.58) Corynebacterium diphtheriae (Fig 0.59) Mycobacterium tuberculosis (Fig. 20.60)

McIntosh-Filde's ar

10. or

falciparum (Figs. 20.62-20.65) 10. L.D. bodies (Fig. 20.66)

Microfilaria bancrofti (Fig. 20.67) 12. Rhinosporidiosis (Fig. 20.68) 11.

Cryptococcus neoformans (Fig. 20.69)

14. Negribodies

XI. IMMUNOLOGICAL TESTS Radial immunodiffusion (Fig. 20.70) 2. Counter immunoelectrophoresis (Fig. 20.71) 3. HIV-comb test (Fig. 20.72) 4. HIV-Tri-dot test (Fig 20.73) XII. WET PREPARATION OF STOOL SPECIMEN

Egg of Roundworm (Fig. 20.74) 2. Egg of Hookworm (Fig. 20.75) 3. Egg of E. vermicularis (Fig. 20.76) 5. 6.

7.

XIII.

Egg of H. nana (Fig. 20.77) Egg of Trichuris trichiura (Fig.

20.78) Cyst of E. histolytica (Fig. 20.79) Cyst of G lamblia (Fig. 20.80) MEDICAL ENTOMOLOGY

1. Mosquito

1.1 Anopheles (Fig. 20.81)

1.2 Aedes (Fig.

20.82)

1.3 Culex (Fig. 20.83)

P.

Salient features: 1. Uses:

(Fig. 20.99)

(1) For growing

Gas-Pak (Fig 20.100) Candle jar (Fig. 20.101)

organisms

Seitz filter (Fig. 20.102) 12. Syringe filter along with Syringe (Fig 20:103 N-95 Mask (Fig. 20.104)

Agar-Agar shreds

15. Specimen of pocks on chorio-allantoic membran

agar

Stokes method of antibiotic sensitivity

18.

testing (Fig. 20.107) Epsilometer or E-test (Fig. 20.108)

(Fig. 20.106)

The

1. Uses:

For growing intestinal organisms To differentiate lactose fermenter (LF) colonies from non-lactose fermenter (NLF) colonies 2. Classified as differential or indicator medium. 3. Contains neutral red indicator. (1)

(11)

above list contains the most commonly asked

spots in the examination. Colour plates and diagrams d some spots are also included to make the identification their characteristic

features.

Questions generallyasked are:'give one use of the spot, 'identify the bacterial growth on culture media', name

Fig. 20.3 MacConkey's agar

the 'indicator used in the culture medium', 'sterilization procedure used for glassware and swab', 'identif

will surely solve the problem. All the spots listed above

examination.

For

growing organisms such

as

Neisseria,

Pneumococcus and Haemophilus

but study of corresponding chapters

(except microscopi

Composition: Refer Chapter 3 (page 9). Sterilisation: By autoclaving,

1. Uses:

and so on. It is not possible to include all the questions

also asked in

5.

Salient features:

whether biochemical reaction is positive or negative related to spots

4.

Salient features:

mycology

casier. Try to identify these spots by

3. Composition:

testin

Kirby-Bauers method of antibiotic sensitivin

19. Slide culture test used

Classified as enriched medium. Refer Chapter 3 (page 9). Sterilisation: By autoclaving.

2.

Fig. 20.2 Blood agar

(CAM) (Fig. 20.105)

16.

most of the pathogenic

(in) To differentiate haemo-lytic colonies from nonhaemolytic colonies (nii) In the preparation of potassium tellurite blood

13. 14.

For preparation of blood agar (in) To determine antibiotic sensitivity 2 Refer Chapter 3 (page 9). Composition: 3 Sterilisation: By autoclaving.

20.96)

6. CAMP reaction (Fig. 20.97) raigie's tube (Fig 20.98)

Germ tube of

vivax

VIDRL slide (Fig.

5.

3. Intracellular diplococci (Fig. 20.56) 4. Candida albicans (Fig. 20.57)

1.

Fig. 20.1 Nutrient agar

3. Haemagglutination plate 4. Latex agglutination tile (Fig. 20.95)

1. Gram-positive cocci (Fig. 20.54) 2. Gram-negative bacilli (Fig. 20.55)

13

(1) For growing common pathogenic organisms

Nichrome wire loop (Fig. 20.93) Microtitre plate (Fig 20.94)

X. MICROSCOPIC SLIDES

Different stages of Plasmodium

1. Uses.

Cyclops (Fig. 20.92)

testing.

8. Mycobacterium leprae (Fig. 20.61)

Salient features:

8.1 Trombiculid mite (Fig. 20.90) 3.2 Itch mite (Fig. 20.91)

caseous

necrosis due to M.tuberculosis

9.

119

L. CULTURE MEDIA

5. Rat flea (Fig 20.87) Hard tick (Fig. 20.88)

2 Hookworm

3. Tapeworm

IX.

Spots

Louse (Fig 20.86)

Roundworm (Fig. 20.52)

3.

Classified as enriched medium. Itis prepared by heating the blood agar at 55°C for

5.

Sterilisation: By autoclaving.

2

slides and wet preparations) are

2 hours. Chocolate in colour

"Grand-viva' which is part of practial

Fig. 20.4 Chocolate agar

Unit II Practical Exercises

Spots

Salient features:

121

Uses:

i) For growing C. diphtheriae and other corynebacteria

For demonstration of meta-chromatic

(ii)

Salient features: 1. Itis a readymade culture medium usedin autornated blood culture system such as BACTEC system

granules.

Classified as enriched medium. is a solid medium without agar. 4. Serum

used in its

agent.

5. Sterilisation: All the constituents

of this medium

sterilised separately and then mixed. Solidificatie

Fig. 20.5 Loeffler's serum slope

2.

preparation as solidifying

of the medium is done by

etc.

Fig.

Salient features: 1. t contains both liquid medium

2. Used for diagnosis of brucellosis. 3. Any growth in liquid medium can be subcultured over solid medium by tilting the bottle in such

For growing M.tuberculosis and atypical

mycobacteria

(ii) For antibiotic susceptibility testing of A solid medium without agar. its preparation

3. Egg is used in

4. as

solidifying

agent.

Fig, 20.10

Castaneda medium

4. Malachite green is selective agent. of

well as the solid

medium.

1. Uses:

5. Green colour

specimens such as blood, ascitic fluid

20.9 Automated blood culture bottle

inspissation.

Salient features: (i)

It is used for

a manner that liquid medium comes in the contact surface of solid medium. The above method of subculture reduces chances of infection of hazardous bacteria to laboratory personnel.

the medium is due to malachite

green.

Fig.

Lowenstein-Jensen (LJ) medium

6.

M.tuberculosis takes 3 to

6 weeks to grow

medium.

on this

Sterilisation: By inspissation. Salient features: 1. Uses: For growing fungi.

Salient features: 1.

2. This medium has acidic pH.

Uses:

3. Fungi can take weeks to grow but yeasts grow in 24-48 hours on this medium.

(i) For growing anaerobic organisms (ii) For preservation of stock cultures of aerobic organisms 2. It contains cooked meat.

Sterilisation: By autoclaving. It i: also named as Robertson cooked meat (RCM

broth

Fig. 20.7 Cooked meat broth (CMB)

Fig. 20.11 Sabouraud's dextrose agar (SDA)

Salient features: 1. Uses:

(1) For growing most pathogenic organisms (i) For making hanging drop preparation (in) For testing indole production (iv) For preparation of sugar media (v) Growing bacteria for anti-biotic susceptibility

Salient features: 1.

Uses: For blood culture in origin (PUO).

2.

is specially useful in enteric fever septicaemia.

3.

pyrexia of unknown and

testing

Teach bottle 50 mL of liquid medium is inoculated

2. Composition: Refer Chapter 3 (page 9) 3. Sterilisation: By autoclaving.

with 5 mL of blood under aseptic conditions. Glucose broth

Taurocholate broth

Fig. 20.8 Blood culture set

Fig. 20.12 Peptone

*

gHV

9

unIpauBADoIaPsTuEoODYeIqse nmeq1SeJpe5 u n p a s n A u m o r s m o r p q P u e H 8 n o 2

Sre8polqnaauumDosyo yAuds mIpaur

6

s

5

JeSporqDunIpy serodBUWApD

C co

neD

gsoru DapranrngeayPpMuolarq8suroeesgaURIoLMaBHd1DE9 JayO SOIMBLgnWapDhrl aSYayMsrno aSrIpOenusDoaroByqenAl刊J0WjuMoO8eIDAns

s a l xd r e 4 p e l d S

6

E

u npau

So r nal d wm sog fiu Z 'O UBSRHUPISBMoT

di co ⼨

SeaxBoAAHnIIsOddWaqEDySwIrepsSsHnhguu8oAyypn4sTrneroJdjuo8aAHsneD

sa mneaj nue TeS

5

asonx p em q SMID A UP W EZ'O

Vas

⽐ agal

8

saruoT

psnauyprmodamorpuars

E

co

e

su om Srad e A n p g 9

®

电 EEE

S

KroegluropodreuJ0 c

sonelAUOITES4un r18odSsMy nusruLnpouSDA saestaseutnor8adsSv dse3AISELI叉 品MycotikerasSinusitsSinceAsprglufmiat acertinsphogcPTO1 f As p e rg i l u m a t s WMOLIVasF'Z ⽐

125

S1odS

F e a t u re s Salient 1

S l m d y t i c f g o n s r u p a e q S M 8 y A s g u B I 1 8 U OW T D b c o n t a m i r e p d s l A u y j 0 q t o a c e r i n s p h g o l e 4 Since

m e d i u m S A9 e 1 0Z DHO3AN i O t o myc o s i

This Vas

Diseases e

F i g 2 0. 5 S D A w t h r o f s p e l n

I L B OCHEM A R TI NS luarres sa mmgay

uon p d se8

PuO I e S f a t u re s

O

usru esuc

⼲

mDeg

spp r

Ylou l s

MTPinkcolurfthesdap ousaqmZrd8y天IOEPUSpeLVan104⼲IVSa1dutxzrqofnebci ueJoabiltyrpsnITdhLfermntaspcibohydse8pu10PDEanodZIndicatoruseA'lfympSLPIM01hdoxiPinkcolurfthesdarubpeSEnaspAyoqJ uosadJrnpe8STMUI4eusdtoinfymGraasgqvny103YpEeLs u o r e a j A q f 0 S O 3 n1 8 w i t h o u f e r m n s a

t u i b e s m a WM S O n I D 9Z 0 8 r 1

313

d s naold s

syanuoeLPAOqTD20.71

出, e

기출

3요

음1

요

:

.

1 . :

ao

Unit II Practical Exercises IV. GLASSWARE

Salient features: 1.

Uses:

(i) Specimens collection. (ii) For preparation of media such

as

LJ

RCM broth.

medium,

2. Sterilisation: By hot-air oven. 3.

Capacity: 30mL

Fig. 20.33 Universal container

Salient features: 1. Uses:

Specimens collection, such as CSF. (i) For preparation of media, such as LSS (i)

medium.

(iii) Fig. 20.34 Bijou bottle

2.

3.

For preparation of urease medium.

Sterilisation: By hot-air oven. Capacity: 6mL.

Salient features: 1.

Uses:

(i) For preparation

(ii)

Fig. 20.35 Medical flat

bottle or Mc Cartney bottle

of

glucose broth and

taurocholate broth used in blood culture (Also refer Fig. 20.8)

For preparation of Castaneda medium used for

diagnosis of brucellosis (Also refer Fig. 20.10) (iii) For collection and storage of sterfie solutions in the (iv)

laboratory

Capacity: 120 mL

2. Sterilisation:

By hot-air oven

Salient features: 1. Uses:

(i)

Fig, 20.36

For

collection of blood via (ii) To collect fluids and pus. (ini) To collect blood from animals(sheep, rabbit), which may be used for

Glass syringe

venepuncture.

agar.

2.

(iv) For

preparation of blood

injecting medicine to patients.

Sterilisation: By

hot-air oven.

Spots

129

Salient features: 1.

One mL

2. Uses:

syringe (glass/plastic).

(i) In tuberculin test.

(ii) Lepromin test. (iii) BCG vaccination. (iv) To inject a given dose of insulin. (v) To inject small amount of animals.

test material into

3. Sterilisation:

(i) Glass syringe by hot-air oven. (ii) Plastic syringe by gamma radiations.

Fig. 20.37 Tuberculin syringe

Salient features: 1. 2.

For preparation of culture media such nutrient agar, blood agar, MacConkey's agar. Use:

Sterilisation: By hot-air

as

oven.

Fig. 20.38 Petridish

Salient features: of fluid used 1. Use: For measuring the quantity

serological tests or 2.

Fig. 20.39 Graduated pipette

other tests.

Sterilisation: By hot-air oven.

in

130

Unit II Practical Exercises

Salient features: 1. Use: To deliver solutions

or

reagents

in test

tubes, during the various procedures. By hot-air oven.

containers etc. 2.

Sterilisation:

Fig. 20.40 Pasteur pipette V. DISPOSABLE ITEMS

Salient features: 1. Uses: For specimen collection such as urine, pus, body fluids etc. 2. For 3.

one

time use

Sterilisation:

(i) By ethylene

only and then disposed of.

oxide.

(ii) By gamma radiations.

Fig. 20.41 Pre-sterilised disposable container

Salient features: 1.

Uses:

(i)

For collection of blood via venepuncture.

(ii) To collect fluids and pus.

(ii) To collect blood from animals (sheep, rabbit), which may be used

for

agar.

(iv) Fig. 20.42 Pre-sterilised

disposable syringe

2.

For

preparation of blood

injecting medicine to patients.

For one time use only and then disposed of.

3. Sterilisation:

(i) By ethylene oxide. (ii) By gamma radiations.

Salient features: 1.

Uses: For

preparation of

growing viruses. 2.

Pre-sterilised one-time use disposable bottle.

3. Sterilisation:

Fig. 20.43 Tissue

culture bottle

various cell lines for

By gamma radiations.

Spots

131

Salient features: 1. Uses:

(i) For collection of specimens from throat, cervix, local lesions.

(ii)

For preparing lawn culture of bacterial growth

as in antibiotic sensitivity testing. 2. It is available commercially. 3. Sterilisation: By gamma radiations.

Fig.

20.44

Pre-sterilised disposable swab

VI. EQUIPMENTS/INSTRUMENTS

Salient features: 1. Use: For incubating culture plates and liquid media 2.

at specified temperature for growth of bacteria. for growing bacteria is 37°C.

Temperature used

Fig. 20.45 Incubator

Salient features: as glass 1. Use: For sterilisation of glasswares such syringes, petridishes, flasks, pipettes and test tubes.

Dry

heat

2.

Sterilisation principle:

3.

Operation requirements: At 160°C for

sterilisation

method.

two hours

160°C. holding time i.e., after attaining Precautions:

(i) It should not

be

overloaded.

The material should be arranged in which allows free circulation of air.

a manner

must be allowed to cool before (iii) The oven doors since the glassware may opening the crack

Fig. 20.46 Hot-air

oven

by sudden cooling.

132

Unit II Practical Exercises

Salient features: 1.

Use: For

sterilisation of culture media, rubber

material, gloves, gowns, dressing etc.

2.

Sterilisation principle: Moist

heat

method.

3. Operation requirements: 121°C at for a period of 15 minutes.

sterilisation

15 pound

pressure

4. Precautions:

(i) The air must be allowed

to

escape from the

chamber as temperature of air steam mixture is lower than that of pure steam.

(ii)

Materials should be arranged in such as to

ensure

chamber.

manner

free circulation of steam inside the

Fig. 20.47 Autoclave

Salient features: 1.

Uses:

inspissation (80°C for 30 minutes daily for three consecutive days).

(i) For (ii)

Loeffler's serum slope (LSS) and Lowenstein-

Jensen (LJ) medium are sterilised by inspissation in this instrument. 2.

Principle

The first day heating destroys the vegetative forms while the spores (which are germinated) are killed by subsequent heating. of bacteria

Fig. 20.48 Inspissator

Salient features: 1. Single channel micropipette volume

2.

can deliver single

of liquid with accuracy of

microliter (ul).

Multichannel

micropipette can

fraction of

deliver multiple

samples of a single volume into multiplewells. 3. Micropipettes of different capacities available. Uses: are

4.

(i) Commonly used for performing ELISA technique.

(ii) Can

A.

Fig.

B.

20.49 Micropipettes A.Single channel B. Multichannel

be used for other techniques where

microliter volumes are to be delivered such as Polymerase chain reaction (PCR).

Spots

133

SWABS

Salient features: 1. Uses:

(i) For collection of specimens from throat, cervix, local lesions.

(ii)

For preparing lawn culture of bacterial growth as in antibiotic sensitivity testing. 2. Sterilisation: By hot-air oven. 3. Precaution: Unplugged swab tubes should never be used.

Fig. 20.50 Sterile swab

Salient features:

1. Use: To collect a sample for demonstration of eggs of Enterobius vermicularis from the anal region.

Fig. 20.51 NIH swab

VIII.

SPECIMENS OF ADULT PARASITES

Salient features: 1. Scientific name: Ascaris lumbricoides. 2. Tail end

of male worm is curved ventrally in the hook, while in female worm it is conical

form of a 3. 4.

Fig. 20.52 Roundworm

and straight. Route of infection: By ingestion. Infective form: Embryonated egg.

134

Unit II Practical Exercises

Salient features: 1.

2.

Causative agent: Echinococcus granulosus.

Thecyst wallconsists of two endocyst.

3. 4.

5. 6.

Fig. 20.53 Hydatid cyst

Hydatid fluid is used for casoni test to patients of hydatid cyst. Man is the intermediate host.

diagnose

Infective agent: Eggs, in dog's faeces. Mode of infection: By ingestion of eggs.

7. The most

X.

layersi.e.ectocyst and

common

organ involved is liver.

MICROSCOPIC SLIDES

Salient features:

1. Violet in colour Cocci may occur either in

2.

pairs.

3.

Examples: Staph. aureus (in clusters), (in chains),

Fig.

Strept. pneumoniae (in

4. Also refer Appendix V.

20.54 Gram-positive cocci

clusters, chains or in

Strept.

pairs).

Salient features: 1.

2. 3.

Red in

colour.

Examples: Esch. coli,

Also refer AppendixKlebsiella spp., Proteus III.

spp. etc.

Fig. 20.55 Gram-negative bacilli

Salientfeatures: 1. 2.

3.

Fig.

20.56 Gram-negative

intracellulardiplococci

Red in colour

Present

and are

foundin pairs. intracellularly but be present some

extra-cellularly. diplococci may also Examples: Neisseria gonorrhoeae, Neisseria meningitidis.

Spots

135

Salient features: 1. Gram positive yeast.

2. Opportunistic fungus. 3.

Causative agent for oral thrush and vaginitis.

Fig. 20.57 Candida albicans

Salient features: 1. Germ tube test 2

is used to identify Candida

This test is also named

as

Reynold-Braude

phenomenon.

3. Also refer Fig. 20.57. Fig. 20.58 Germ tube of Candida albicans

Salient features: 1. Albert's staining is 2.

used to identify C.diphtheriae. Causative agent of diphtheria.

3. Selective medium: Potassium tellurite blood agar. 4. Other medium used:

Fig. 20.59

Loeffler serum slope (LSS).

Corynebacterium diphtheriae

Salient features: 1.

Acid-fast bacilli (AFB).

2. Causative agent of tuberculosis. 3. Commonly used medium for its culture is LJ

medium. 4. It takes 3-6 weeks

Fig. 20.60 Mycobacterium tuberculosis

to grow on

L) medium.

if

(

(

1

분

를

1

号

Slua B s

SuTEu0D

后

a n te o r f e l s n d e l i k struc e

中

8

9

2 0. 8 C y s t o f G i a r d L m b l

品

9 中

⾺

P™

ex t r a i n t e s

2 0. 7 9 C y s t o f E r i l c a

号

t a i n g c ys t

9

e s e u a I n u c l e i 1 T h e Sal ient sa umgej

20.78EgofTrichusta

品

eg s

P

amoebias

Ee

S u re u o

J0BA 号

含 P00y

5

9

2

b

Po e u K I q u E a r o d u p e a

9

F i g 2 0. 7 E g o f H

SEgisnotbleaslaiienndtfaures N F

oute f i n c By g s d o tam f

2

sured

onc spher wit o u

with eg s ho kle sIt contai s

9

3g

choler

Non b i t n s

Sasul

巴

sauedef

U n i t I P r a c l E xe i s

OFI

N

t o s u r fa c e

⽩

n oq u SOxas

E

Samey3suprDTndobJdentifcaoursglAqEPIV alients3.1m8Aoph

1⽶ 1⽶

A U I H P M c ys t wa l

⾃ Ee

9

ADOTNLEVICWX causemlborptinyuDHql ItconaifurleThcystiovanpeSalientfures ArauspqoTuveasproduce Routefinecto EgisblestaSianleiedntfaures R o u t e f i n c y g e s t o n E x t r a i n e s l m o b N T S A H E e 叉 i n g s t o q u a d r i n c l e a t 9suKoynSarApif TheaxostylidgnfrmHLSUIPA eristhmocnlyPaATOUuS

1comnspeci Themonstadfilr

号

N

Mosquito

species

i aodu l

上四

号safIambdeomenyisuoluntpgAa 1the

军

Dengue

3 Eeee

1ssocinted Diseases

aegypti

9中

3

⾃ E

E

9

1a8n

a n d wo h i t e

angle

AorbandechtrofeigH ltheabovgnridfcDsm buolarepiwngsdct

B i te s d u r i n g When a t re s t P u A m o s q u i t o e s a m o s q u i to

N

I d e n t i f c a o e t u r I d e n t i f c a o u r e s 3Gastroe⾃nteriD€ysentyry uDiarhsoeausPaDrtypholidfevDrTyphiosdfevar se Muscadomiet nf1 oSOP ALPTM saume3JSalient WestNil fevrencphalitsBancrofti larDisea ociated Inqyd8gusapovJIdentifcao ursSalientfursCx YelowfvrntiIdavaleyfver1AajEun yDDenguhamoricfve Palpireshotnfmal pofabdmenis t S luaresft Ades Disea octdMlri Whenaoplstricd The b i t n g m a ou d h body sh w a unc k o m e t i s n a d H o t g B E S t a r e p s n t

⽣ 2 0. 8 4 H o u s e f l y

品

号

ve n t r a l y

SR

9

号身

ua opqe

WO e s p o q r d J o 8 台

居

S u r ys i n S u ys r F 单 s i t u a e d o n i t h e r S

aamp e female

posteri bowdeirngsdorsaly

号 g

9 pair

Mo L re u b 6 u y e ⾸品冒 scales

p ar ts he d orax

⿃ 5 Au s n c 營 9 号 ⽣ 品 万 B a u b o d y

⾹

u o p q e x e l o u a e u ay u t 夏

E e

l e n t i f c a o u r e s w i n g s a re c h t d

3 ⾃

号 号

3

syo d s

(

특

B

열

베 e

《e

분로

따'통로

Be

분

F

Be

n

HPLC

号

J E I S P U O JA 6 Z

eLapeq

g

0

B e Lses

D

a

M e t hy l e n e t s

Ouopaq 10 e s n

with

号

Syringe

1en O

中

fluid

8

Syringe f i l te r

um

5

ex a m p l e

品

e q my s a S r e p 8 6 D z

2 0. 1 S e i t z f l

E E sasn

5 3

号

83 ua y I u r

9

以

引

surogpiqaeSn sauneojaures 万softagriuedSJnZ0SalmonetyphieHroD9a4ueoDemonstrai saIme3jlurs 2 0. 1 3 S y r i n g e 号 尋

along

a u As

o re s i ny

居居

rgemoval other end piston of torced

filedwthusfiedcneluosetrs

filter

s1e

E

steril sat on

9

aeDHTIS

9

5

g

3

S a l i e n t sa m 4eaj

F i g 2 0.9 7 C A M P r e a c t i o n w

3

fuo u

y e a n s n a i n e YO B S 0

3

Yaus

E

bac teria

Jer ed BAuend

P

Io rdeJisGroup B

9

g

号

6

S a l i e n t s a u n 4 8 oy

S a l i e n t Sa m 483J W

F i g 2 0.9 6 V D R L s l i d e

5

air tight

T l S E D 2 0. 1

天

中四

1S01 TAC

usec

s a l i e n t s a I m e oy

N

P y u d Ks

esoube P

6

3Ol

CWORLEoISTaushtiveagcnofyphUlosreT2rSTmoHPudprfmVDRL

h p u r a t e y d o l s i p r o d u c t i o n U s e T d t c g ro u p D 3 4 s C a r d i o l p n a t i g e

psepticamndGroupgtBisstecianbl ordnsajuPI0ySauref4OsgownbldareicltpProydasknuMeAFg CMPreactionbdms 品

2 0. 1 C a n d l e j r

A lighted can l d e c r a s e O

G

5

營

a i r t g h J o u ey

Capno hil c

S u L M o1 8 c e r t a i n b

sence of

813 Lo l u p O P H O M P a c t l ys nd h u S a s e 8 1 o u 8 1 s a n y e J

when at r i s d e 0.2E品MPYRSeamTwvblLIistkno lamgedbyhtprocsiun obarqmpisextnguhdbycloa whicrequmoapltsFvyfeidumcrogan Forpvidng510cabxesqu producesanbiautr8AxopIMsqJOPH genrthy xacidnsoumbrteThnreqoaLIPA41dupsySlx meroaqA1suSedhJoypanAmtxs uOHPAH Itcontais uoneAy filter outle Membran filteSrsyringeflt OZTS Syringefilter saIne3J uHS solutinsec Filterdsc ma万 Z1Sflteris Toremvpaticlsfrom Forfiltansmvoue esyringThfludcomtthrougembanmebranHeForsterilsationSy⼀ringf UseFortilan example s u b t a n c e w h i g t asbe to m gnis u u o n e s H T O y S

sample rio

6

吞

Unit I P r ac l So3

sods 5

4동를d

culture Methods Unit

Applied Microbiology 21.

Culture Methods

22.

Diagnostic Bacteriology

153

o..........................

158

168

24. Antimicrobial Susceptibility Testing 25. Sterilisation

and Disinfection

182

186

199

26. Healthcare Associated Infection 204

205

28. Biomedical Waste Management

z 더

떠

.

3

." *

.

7

분

1

은

P

분포

:

i

2

1(

프

2

요

대

를

콜

7

형7

표

홈을

O

1 트트 요

몸

2

동

.

료

,

SSL

spouON iM InD aumban

s a u rg o wn p O P S u o a A x t I e

paxru

as l opn ug b r t s

p u r y q e l sA M

f ro m a u n d

p o u r p a s n P ro c e d u re

bsaoa

s e a JO ro d u c t i o n

1O omu se

w i t h a p s yne c t i g pasn I mer du o

pump

S p owo u

HO

H

⼗

fo

C a t l ys t

hyd ro g e l

I a 8 o I pA H

r e n p s a u u a 8 A xo

the mos r l i a b nd w e y ZDisplacemntofOxygDisrplacemntofxygbrknhitdrogecabdxiemploydOxgnLEDArau q1bythismeodApularn fcg ktiephabdxs1ISDEPnOWMlthraburnigcdlew310sjqousinpa8gx4mtfye uaSAxOJonsqpeldtByEPrUWguSIoavnerobicjFg21.5is ofastuSIUODrepqLdMcnpadhrq T ftubyoes1OMwihPngSHIBLaWq8r 0MsueoDlidBLYanJthpArT41 quyMmsedHIano ctlWYi0gUESJOdAnrepau4osTBLWIMwPD8y1zONctiv paerdsuWIMnboSICPLltaT04eDpuMJqornAls m1L789SaIdOuPepUE iqnBandMhyrogepsuqmH DBA Hy ParnsldpuoqeTSitzI8bAOmDYMHgarn09Ly1uoPesTIEzAS w迁combine01sdrayThtlpuI8gJyeMwuo1SAxTnpsa pDL1gTOoWrensubSwq0JamdAgIOW SIPRLTAEDOUBWMrep4u10o3a9dLSyqsngth2IoueHpaSAxrTqwLCOl9 hi48uno9 JFD uOnETSI0yeasl TeDSolrq1Teuap Suye sMCivaq01pAmoIdngO2DSuEcltresi A n a e r o b i c t g w l y h s f x u n 3 A x o s a r b e y p q P H L d I M O p o L a u n e A q s y I 4 r l s u o p a d T e r P y B D q I J u o A x J n s q r p e a T d I S V J a P o L I A T r e D q u p n i l d 2 a re I p a u r omranou am t I E S a

DIHM

lauqOHe Teulas

by u o n u p

bac teria uo

Sa

o mn

worsuadns Orsar4ogPpasreu Iosard OLSutArasptouro erpou BU

incubated 已 U DoLE T r M o u a s n i 1 p S U L t f c r y aOPIAd1ousapME notpsibleH9Junyo eIPW3unaTJowreq Ipnseua 3rdoe METHODSUnL IgJVN ADNVIST ⼄ D i s p l a c e m n t o f x y g r S p o u r a s d J 8 p M o u r y n p A e o u r d L A q u a x o j r d s q v I S O q r e u V J P T a N 1 P ro d u c t i n f a V m 5 B y r e d c i n g t s a q u r e y p P I o 9

SDagourpsd erno u HOLeSyuRPI 9 un pal i n

LDea

dbopSumsne riumbyHugh pres vation f mineth viableaJOaPInbyr isefolws 1oeMbathat u Pan uo s Lpea01PepIMPaxru te aTuIoSeOu 1u8rsAo1 3dJoudp

Po a rey p h t w i r e O1e nSuO 1 S o HO

Door

seNeus

gumreuopesaud se se uInIperk ait

e l s ow l e u

ioeqnul F'IZ ig

Unit III

Applied Microbiology

strict anaerobiosis with layer the

Pressum gauze Outlet

Electric terminals

Inlet

Lid

Catalyst under the lid

be covered

surface of

CMB medium may

of sterile liquid paraffin.

Interpretation

(Cl. perfringens)turn the colour Saccharolytic anaerobes red while t becomes of

meat pieces into

of proteolytic anaerobes (Cl. tetani). 6.

Anaerobic

black in case

an anaerobic incubation system. It provides oxygen-

environment for inoculating culture medial and free for their incubation. It is fitted with airtight rubber insert hands for

working with specimens. gloves These anaerobic chambers contain a catalyst, desiccant, hydrogen gas, carbon dioxide gas, nitrogen gas and an

Fig, 21.5 McIntosh and Filde's anaerobic jai

III. METHODS OF ISOLATING PURE CULTURES

It is routinely employed in the laboratory. 2 Use of enriched and selective media 3.

indicator. Reduced

methylene blue

IS

generally used as

technique is simple as compared

to

indicator of anaerobiosis in the jar. It remains colourless cylinders in the anaerobic jar. in anaerobic conditions, but turns blue on

exposure to

oxygen.

filling of gases by

been attempted by incubating aerobic organisms along with anaerobic bacteria. Two blood agar plates are an automatic, micro-processor controlled taken- one is inoculated with Pseudomonas aeruginosa system which rapidly removes oxygen from a jar and (aerobic bacteria) and the other with specimen

replaces it with a precise amount of hydrogen gas from i

cylinder. Small residual oxygen left in the jar is removed by palladium catalyst. This is an easier and effective method than McIntosh and Filder's jar method. 4. Absorption of Oxygen Biological Methods

by Chemical or

CHEMICAL METHODS (i) Pyrogallol

Alkaline pyrogallol absorbs oxygen. This method was first introduced by Buchner (1888). A large tube containing

solution of sodium hydroxide and pyrogallic acid placed air-tight jar produces anaerobic conditions. The oxygen is removed by chemical reactions. inside

(ii) Chromium and sulphuric acid A mixture of chromium and sulphuric acid is used for producing anaerobiosis. The two chemicals react in the presence of oxygen and produce chromous sulphate. (iii) Gas-pak It is available commercially as a disposable packet containing pellets of sodium borohydride, cobalt

chloride, citric acid and sodium bicarbonate. It is now widely used for preparing anaerobic jars. These chemicals generate hydrogen and carbon dioxide when

water is added. Hydrogen combines with oxygen in the presence of a catalyst. The catalyst (alumina pallets coated with palladium) used is the same as that in

McIntosh and

Filde's anaerobic jar.

anaerobic bacteria. Two plates are placed one over the other and sealed along the rims and are incubated. This method is slow and

5. By

ineffective.

Reducing Agents

Oxygen in culture media can be reduced by various

agents such as glucose, thioglycollate, cooked meat pieces, cysteine and ascorbic acid. Based on this

principle, the two most widely employed anaerobic liquid culture media are: THIOGLYCOLLATE BROTH It contains nutrient broth and 1%

thioglycollate.

COOKED MEAT BROTH (CMB) It is also known

as

Robertson's cooked meat (RCM)

medium.

contains nutrient broth and minced cooked meat of ox heart. It

pieces of fat-free

Principle 1. Unsaturated fatty acids present in meat utilise

oxygen for autooxidation, this reaction is catalysed by haematin in the meat. 2.

Glutathione and cysteine (both reducing agents) also utilize oxygen. Sulphydryl compounds (present in cysteine) also contribute for reduced oxidation-reduction (OR)

present in meat 3.

Pre-reduced Anaerobically Sterilised (PRAS) Pre-reduced anaerobically sterilised (PRAS) media are

potential.

After the inoculated plates are placed inside an air- Procedure tight jar, the packet of "Gas-pak" with water added, is Before inoculation, the medium i boiled in water bath kept inside and the lid is tightly closed. The "Gas-pak" at 80°C for 30 minutes to For make it

oxygen free

initial sterilisation process to packaging in foil packets. The 'Pre' in pre-reduced refers to that time before culture plate is made. Reduction

done at this step

It is employed for isolating pathogens from specimens with varied flora e.g. faeces.

Pretreatment of specimens Suitable bactericidal substances are used for pretreatment of specimens to isolate a particular bacteria e.g. concentration and decontamination of

7.

prepared entirely under oxygen-free conditions from

BIOLOGICAL METHODS This has

157

oxidised products, which are harmful anaerobic bacteria, from being formed during autoclaving of the culture mediumn

. Surface plating

Chamber

It is an

to

Culture Methods prevent

to

sputum before culture for Mycobacterium

tuberculosis. 4.

By heating liquid medium

Specimens are heated at 80°C to destroy vegetative

forms of bacteria but spore bearing bacteria survive e.g

isolation of tetanus bacilli from dust and similar

sources.

after the incubation period. isolation of the bacteria After

media

22

in

pure culture on

the following studies

Commonly used liquid media

I strain

(i)

bacterial s

Morphology of bacterial colony

(1) Deposit

III.

Conventional Methods

Automated Methods Molecular Methods

Automated methods.

Molecular methods,

such as (1) Conventional methods (2) Automated culture methods (3) Molecular methods (Fig. 22.1).

I. CONVENTIONAL METHODS Conventional methods consist of specimen collection

2.

(iv) Elevation: Colony may be flat, raised, low

Direct Antigen Detection

Latex agglutination test is available for detection o antigen

3

to direct microscopy by Gram staining or other specia

Streptococcus pneumonice, Neisserm

be inoculated on to selective media such as deoxycholate

Mycobacterium tuberculosis and Albert staining of throat swab smear for Corynebacterium diphtheriae.

citrate agar (DCA), xylose lysine deoxycholate (XLD), bile salt sucrose (TCBS) agar

thiosulfate citrate

Blood specimen is directly inoculated into blood culture

nature. (iv)

Low convex

(v) Edge: Entire, crenated, lobate, undulate,

ones.

ciliate

(vi) Opacity: Translucent, transparent or opaque

(viii) Haemolysis: Haemolysin produced by

BACTEC

(v) Biochemical tests

Bacterial species differ in their capacity to break down

(Fig. 22.3)

Bacteria may grow in the form of colonies on culture

culture media are incubated at 37°C for 18-24 hours.

Hanging drop preparation

It helps to distinguish motile bacteria from non-motile

Fig. 22.2 Different elevation of colonies

(vii) Colour: Pigment may be

Automated methods e.g. • BacT/ALERT 3D

by staining

respectively. Both these bacteria are acid-fast i

Convex

bottles without performing direct microscopy. All these

produced by certain

bacteria.

different sugars. Some biochemical tests are

oxidase, urease, gelatinase etc. Some of the widely biochemical test:

bacteria leads to haemolysis around the colony.

1. Catalase Test

Principle

Molecular methods

catalase

e.g. • PCR

Undulate

Modifications of

Procedure

Pick up Staining

morphology

from colony

Ciliate

Hanging drop Biochemical for motility

reactions

Fig.

used

described below.

Certain bacteria have an enzyme catalase which acts on hydrogen peroxide to release oxygen.

Culture

Colony

based

presence of specific enzymes such as catalase, coagulase,

some

(ix) Consistency: Mucoid, friable, firm, butyrous

Specimen Collection

Conventional methods

Flat

Blood agar and MacConkey agar are most commonly

Direct microscopy is generally not done in specimens expected to contain normal microbial flora with some exceptions e.g. acid-fast staining of sputum for

stain is commonly employed for identificationc Mycobacterium tuberculosis and M. leprae which re causative agents of tuberculosis and leprosy

Raised

Culture and Identification

staining methods followed by culture on blood agar and employed for most specimens. Chocolate agar should bd MacConkey agar or other special culture media if required. added for respiratory specimens. Stool specimen should Microscopy

convex, convex or umbonate (Fig 22.2).

(i) Gram's staining: It divides bacteria into Gram for positive and Gram negative. It may be almost all the bacteria. iS employed to identify (11) Albert's staining: Corynebacterium diphtheriae, a causative agent of diphtheria. (iii) Ziehl-Neelsen (ZN) staining: It differentiates acidt bacilli (AFB) from non-acid-fast bacilli This

directly in CSF from acute pyogenic meningits

e.g. antigens of meningitidis.

(described in chapter on collection of specimen) subjected

Direct microscopy

Commonly used stains are as follows:

Size: in millimetres e.g. pin head size characteristic of staphylococci whereas pin point size is a feature of streptococci colonies.

(ii) Shape: Circular, irregular (iii) Surface: Smooth, rough, granular

Identification of bacteria can be done by various methods,

1. Direct

Staining methods are employed to examine smears prepared from the bacterial colony or liquid culture.

colony are studied:

Coventional methods in diagnostic bacteriology. 2.

e.g. Pseudomonas

The appearance of bacterial colony on culture medium is usually characteristic. The following features of the (i)

growth of

(iti) Staining

() Morphology of bacterial colony

The student should be able to learn:

This occurs in

tendency to grow on surface of media due to the surface more content of oxygen present

Vil) Antibiotic sensitivity tests (vili) Typing methods

SPECIFIC LEARNING OBJECTIVES

3.

Biochemical tests Slide agglutination test

(vi)

Il.

and Characterisation of a Bacterial Strain

at bottom:

streptococci, chains of this bacteria being heavier may settle down as deposit. (111) Surface pellicle formation: All aerobes have

Staining

(v)

Uniform turbidity: Most of the Gram negative bacteria grow in this form.

media

drop preparation (iv) Hanging

Identification

water and

peptone

are necessary to characterise a forms:

(n) Growth in liquid

PREVIEW

159

medium from a specimen, it has to be identified. nutrient broth Bacterial growth appears in following

culture .

(i)

Diagnostic Bacteriology

Diagnostic Bacteriology (in) Growth in liquid media

few

Nascent oxygen

colonies of test bacteria with platinum

loop from nutrient agar slope/plate and mix it in a drop

of H,O, (3%) on clean glass slide. A positive catalase

reaction produces gas bubbles immediately. Since blood

22.1 Microbial identification 158

Fig.

22.3 Various types of edges of colory

contains catalase, culture on blood containing media may result false positive reaction. Use of iron wireloop

Unit III Applied Microbiology for picking up bacterial colonies may be another cause of

false positive

Interpretation

Positive test:

Immediate bubbling, easily observed (O,

formed)

Negative test: No bubbling (no O, formed)

Catalase negative: Shigella dysenteriae type 1, Streptococcus,

Clostridium

Catalase test is primarily used to differentiate between genera Staphylococcus from Streptococcus

Diagnostic Bacteriology

decomposed by an by

Peptone

Procedure

Procedure

Urea (20%)

sucrose and mannitol are widely used sugars Sugar media contain 1% sugar. Indicator used is Andraded indicator (a solution of acid fuchsin to which sodit

48-9%6 hours. bcubating it at 37'C for the bacterial

sugar medium and Test organism is inoculated in 37°C for 18-24 hours. Glucose, lactos incubated

Positive and negative bacteria Catalase positive: All members of enterobacteriaceae hydroxide is added). except Shigella dysenterize tupe 1: Staphylococcus, Interpretation Microoncus, Bacillus

tryptophanase produced

specific carbohydrate (sugar) incorporated in a medi producing acid or acid with gas.

certain bacteria

Indole

Positive: Pinkish-red (acidic)

reagent is

into peptone

water (tryptophan rich) and mL of Kovac's

added to

growth

and gently

shaken.

water

Phenol red Interpretation

Positive (Fig. 22.7): Pink colour Negative (Fig. 22.7); Pale yellow colour

10 g 150 mL 50 mL

benzaldehyde

and 22.5)

161

Christensen's urease medium contains

production is detected by inoculating the test Agar

bacterium

Kovac's reagent contain Paradimethyl amino-

Negative: Yellow to colourless (alkaline) Gas production can be seen as bubbles in Durham'® tube (Figs.

enzyme

Amyl or isoamyl alcohol

Conc.

Interpretation

Indole positive (Fig 22.6): A red coloured ring near the

2. Oxidase Test

surface

Principle

Cotton plug

To determine the presence of an enzyme cytochrome oxidase which catalyses the oxidation of reduced cytochrome by molecular oxygen.

of the medium

Indole negative (Fig. 22.6): Yellow coloured ring near the surface of the medium

Procedure

Freshly prepared

paraphenylene

solution of

diamine

1% tetra-methyl

dihydrochloride (oxidase

this test.

(i) A filter paper strip, soaked in the oxidase reagent,

organism. In a positive oxidase reaction the smeared area turns deep purple within 10 seconds. Deep purple colour

Negative Positive

Sugar medium

reagent)is used. There are different methods to perform

Bubble

gas production

Inverted

tube

Fig. 22.7 rease test

Positive and negative bacteria

is smeared with test

Urease positive:

Urease

due to formation of compound indophenol blue. Oxidase positive control (Pseudomonas spp.) should always be included to find out the

(ti)

Negative

Positive

Fig. 22.6 Indole

test

within 10-30 minutes.

This technique is useful pick up Neisseria colonies from mixed growth

Positive: Deep purple within 10 seconds

Positive and negative bacteria

Oxidase positive: Pseudomonas spp., Vibrio spp., Neisseria

22.5 Sugar fermentation tests

Examples of fermentative bacteria Glucose fermenters: All members of the Enterobacteriaceae

Spp., Plesiomonas spp., Glucose and lactose fermenters: Escherichia coli.Klebsica

Oxidase negative. All members of family Enterobacteriacene, Stenotrophomonas maltophila Carbohydrate (Sugar) Fermentation Principle To determine

Fig.

the ability of an organism to ferment

Glucose and mannitol fermenter. Salmonella 4. Indole Production

Principle

To determine the aminoacid

ability of an organism decompos tryptophan into indole Tryptophan

It is the ability of an organism to utilize citrate as the

sole source of carbon for its growth, with resulting

Indole positive: Esch. coli, Proteus spp. other than P.

Procedure

mirabilis, Edwardsiella

Solid (Simmon's) c

Indole negative:

Interpretation

Principle

alkalinity.

Positive and negative bacteria

on culture media. Purple coloured colonies can be subcultured to get pure cultures.

$pp., Aeromonas spp., Alcaligenes

negative: Esch. coli, Providencia spp.,

6. Citrate Utilisation Test

working of

Negative: No colour change

spp.

pestis.

oxidase strip. Another method is to pour oxidase reagent on the surface of colonies, the colonies become purple to

Klebsiella spp., Proteus

enterocolitica, Helicobacter pylori

Fig. 22.4 Inverted Durham's tube showing gas production

Klebsiella spp.,

Proteus mirabilis

Urease Production

liquid (Koser's) media can be used. A bacterial colony is picked up by a straight wire and

inoculated into either

these media. These inoculated

media are incubated at 17 C

for

overnight. Simmon's

citrate nedium contains agar, citrate and bromothymol the medium is To determine the ability of an organisn to produce an blue as an indicator. Original colour of

Principle

enzyme urease which splits urea to ammonia. Ammoni makes the medium alkaline and thus phenol red

indicator changes to pink/red in colour. Procedure

Christensen's urease medium. The test organism is inoculated on the entire slope of

The test is done

in

medium and incubated at 37C. It is examined after

hours

and after overnight incubation.

green.

Interpretation

Simmon's citrate medium

Positive (Fig, 22.5): Growth with an intense blue colour the slant. Blue colour is due to the alkaline pH which blue

results

from utilization of citrate. Bromothymol

indicator) is blue in alkaline conditions

(yellow)butt

Unit III Applied Microbiology Negative (Fig. 22.8): No growth with no change colour (green).

slant/acidic (red) hours.

in

K/A reaction at

18.

Diagnostic Bacteriology K/K (red/red): Neither glucose, lactose, nor

163

fermented

24

K: Alkaline A: Acidic

lactose and/or surcose

Bacteria acid that whole medium large amount produce acidic at hours and acidic pH is maintained (0h) Slide agglutination test turns

at 18-24

hours. Thus medium appears acidic (yellow)

slant/acidic (yellow)

butt or A/A.

on-fermenters not ferment any the sugars present These bacteria doThus there IS no change in medium medium. in the and appears

butt

alkaline (red)

Gas production

Fig.

Phenylpyruvic acid

Some bacteria produce

Principle To determine the ability of an organism to attack speci

Negative

carbohydrates incorporated in a growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide (H,S)

Positive

Fig. 22.8 Citrate utilisation

Koser's citrate medium

production.

Positive: Turbidity due to growth of bacteria

TSI is a composite medium which contains

Positive and negative bacteria

positive: Klebsiella spp., Salmonella spp. S. typhi, Citrobacter spp., Enterobacter spp.

Citrate

except

Citrate negative: Esch. coli, Edwardsiella, Salmonella typhi 7.

Phenylalanine Deaminase Test

three

carbohydrates namely glucose, lactose, sucrose and also ferric salts for testing H,S production. The concentration of lactose and sucrose is 10 times that of glucose in the medium. Phenol red is incorporated as an indicator. The uninoculated medium is red in colour. This medium , widely used. It is in the form of a butt and slant in the test tube.

To determine the ability of an organism to deaminate phenylalanine phenyl pyruvic acid (PPA). This test Procedure is also commonly called as PPA test. Medium is inoculated with bacterial culture by a straight deep in the butt (stab culture) and then

Procedure

wire pierced

A medium containing phenylalanine is inoculated with growth of bacterial culture and incubated at 37°C for overnight. A few drops of 10% ferric chloride solution is added. If PPA is formed, green colour is produced

doing stroke culture on the slant. It is incubated at 37°C

due to reaction of ferric

chloride with PPA.

Interpretation

Positive: Green colour Negative: No colour change

Positive and negative bacteria PPA positive (Fig. 22.9): Proteus spp., Morganella spp.,

Providencia spp

PPA negative: All members of enterobacteriaceae.

8. Triple-Sugar Iron (TSI) Agar

Triple sugar iron (TSI) agar is an important medium used widely for identification of Gram negative bacteria.

in the medium or medium

Certain bacteria produce H,S which is

colourless

ferous sulphide as black precipitate and thus blackening the medium.

fermentation of carbohydrate.

Blackening of the medium (Fig. 22.10): H,S production Various possible combinations of the different TSI

reactions are listed below; remembering that the slant is first, followed by the butt reaction. K/A

(red/yellow): Glucose only fermented.

A/A (yellow/yellow): Glucose fermented, lactose and/ or sucrose fermented.

isolated bacterium

Is

subjected to antibiotic

sensitivity tests in-vitro to select appropriate antibiotic for therapeutic use. Different techniques for antibiotic sensitivity testing are described in Chapter 24.

(viii) Typing methods This is useful for epidemiological studies. All the above mentionedtests can identify up to species level though intraspecies differentiation is not possible. Hence,

typing methods like bacteriophage typing, bacteriocin typing, antibiogram typing and serotyping are useful for intraspecies differentiation. Nowadays molecular

typing methods such as restricted fragment length

4.

Serology

Serological tests are used in the diagnosis of bacterial infections. Antigen or antibody can be detected in the serum of the patient by various methods such as precipitation, agglutination, ELISA and rapid tests. Some examples include: Widal test: Enteric fever

VDRL (Venereal disease research laboratory) test: Syphilis RPR (rapid plasma reagin) test: Syphilis

Standard agglutination test (SAT): Brucellosis

Fermention of Glucose only

Weil-Felix reaction: Rickettsial diseases

Bacterium attacks simple sugar (glucose) first and then the lactose and the sucrose. The test is read

(rii) Antibiotic sensitivity tests

polymorphism (RFLP), ribotyping and sequence based Yellow colour (acidic): Fermentation of carbohydrate methods are also available. Typing methods help to find Red colour (alkaline): No fermentation out the source of infection in epidemics or sometimes Bubbles in the butt: Gas is also produced during in outbreak of food poisoning.

for 18-24 hours.

ELISA: For various bacterial infections such

at 18 to 24

brucellosis chlamydial infections, leptospirosis

hours and not before 18 hours. During initial 8 hours, the glucose fermentation produces sufficient acid to make

II. AUTOMATED METHODS

both slant and butt yellow (acidic). At 18 to 24 hours the is depleted in the slant and the the organism begins oxidative degradation of the peptones present in the slant, resulting in alkaline by-products which change the indicator back to a red

Conventional methods take days

glucose concentration butt. Thus

colour in slant.

gas.

H.S combines with ferric ions (from ferric salts) to form

Interpretation

Composition

Negative: No turbidity

gas by sugar fermentation. This

production

of

Typhi on biochemical reactions then it can be confirmed by slide agglutination with the specific

antisera.

The

is observed by breaks/cracks is lifted up.

(PPA) test

slant and alkaline (red)

K/K.

Biochemically identifiedorganisms are furtherconfirmed

byslide agglutinationtestusing specifie antisera for the bacteria. For example if i suspected as suspected Salmonella

identify and

characterise the isolates while rapid automated methods

t time Automated methods are available for culture, takeless time. Automated methods are dvdldu utane

identification, antimicrobial susceptibility testing etc.

However fermentation (anaerobic) of

A.

Automated Blood Culture Methods

yield Conventional blood culture methods often

glucose in the butt produces a larger amount of acid, overcomingthe alkaline effect of peptone degradation,

therefore the butt remains yellow (acidic). Thus when glucose is only fermented, the medium appears alkaline

to

etc.

results and

poor

there is increased chance of contamination.

BaCT/ALERT 3D and BACTEC are two commonly used

Fig. 22.10 Triple sugar iron (1Sl) agar

blood culture systems

Unit INI

Applied Microbiology growth.

1. BacT/ALERT 3D System

(i) Principle It is based on calorimetric detection

of growth

of organisms.

(ii) Culture medium

BacT/ALERT bottles contain liquid medium containing supplemented Tryptic soy broth. A

sensor is installed at the bottom of each bottle for detection of CO,.

VITEK automated system

2. MALDI-TOF VITEK Automated System It is used for identification and antimicrobial

susceptibility testing of bacteria and

(ii)

cards are available for identification of Gram negative bacteria, Gram positive bacteria,

fastidious bacteria and yeasts. detection of fluorescence

Substrates in the

when

organisms grow in the culture medium. Culture medium The BACTEC bottle contains soybean-casein

The sensor also

the identification and is

Cards for antimicrobial also

•

agents. The minimum

inhibitory concentration (MIC) determined for antibiotic. The system uses ,

turbidimetric method for susceptibility testing.

Advantages and Disadvantages of automated

Procedure

The organism suspension is added to the wells of

Advantages

Continuous monitoring: Inoculated blood culture bottles

kept within the automated culture system and are incubated. Monitoring of microbial growth is done every 10 minutes by the system. In case of positive microbial growth, the system gives a signal (producing : beep or colour change on the screen).

Rapid 3. More sensitive than conventional methods 2.

©

For identification, the results are available within 4-6 hours.

For antimicrobial susceptibility testing the results are available within 16-18 hours. is the most widely used

automated system

Colony morphology cannot be observed as liquid medium is used.

MALDI-TOF (Matrix Assisted Laser Desorption/

lonisation-Time of flight) is used for rapid identification of bacteria, fungi and mycobacteria with

absolute

accuracy.

Automated Methods for Bacterial

Principle

Identification give

about a positive or negative microbial

for infectious

(Taq) the

methods Hepatitis C, enteroviral meningitis, herpes standard'

Pedioase

nhanacies hists aretheound oldiandinde advantageous found

which methods situations inexpensiveconventional or not available.

insensitive,

in

Diagnostic Bacteriology triggers the formation of new DNA

These three steps

are repeated again and again,

process that is automated by machine.

template for

slow,

an

the thermocycler or The products of first cycle become the

the next cycle. After 20-30 cycles

mainly of two major types,

Double stranded template DNA

Amplified methods

Single stranded DNA

acd ainylinsition lecinsusts uned MinioPolymerase ns Nudere chain

Oligonucleotide primers are added and

annealing occurs.

reaction (PCR) and

Extension

modifications

2. Transcription mediated amplification (TMA) 3. Nucleic acid sequence based amplification

One (

Ligase chain reaction (LCR)

4.

more,

exponentialincrease in the amount of DNA occurs

(Fig 22.11).

amplified methods and non-amplified methods as folows

1.

165

strand from

free nucleotides. Tag polymerase and nucleotides are added in the tube for formation of new strands of DNA.

but for diseases molecular disease diagnosis have replaced the culture as the "gold

DNA has

PCR cycle is repeated. DNA is separated into

Non-amplified method

vo single strands primers are added and

annealing occurs.

Nucleic acid probes

Amplified Methods

A.

Amplification of nucleic acid

is the

basis of all the

amplified methods. Amplified nucleic either DNA or RNA

acid

may be

1. Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) was first developed

1983 at Cetus Corporation, USA. It has made revolutionary impact in molecular biology. In 1993, Kary

in

Mullis was awarded the

Nobel prize

Two copies have become four

for this work.

MALDI-TOF identifies the microorganisms, based on the patterns of ribosomal proteins present in the organism.

It is a DNA amplification system that produces large of DNA in vitro from small amounts of starting

amount

material. It amplifies a specific DNA sequence (or gene) of interest.

Fig. 22.11 Polymerase chain reaction

(ii) Procedure It

involves four

main stages which are as follows:

Denaturation The double stranded DNA is dissociated to single stranded DNA at 94°C (denaturing temperature).

(a)

2. MALDI-TOF

1. High cost: For both instrument and culture media.

MOLECULAR METHODS

(i) Principle

The card is incubated in the automated system at

35.5 t 1°C.

in India.

Disadvantages

Automated culture methods described above,

card.

VITEK

laborious procedure

information

susceptibility testing are

available commercially.

principle microbroth dilution. The wells in the card contain doubling dilution of antimicrobial

is

blood culture methods

B.

interpreted by automated

Antimicrobial susceptibility testing is based on the

which is read by photodetector.

culture

test

system.

contains fluorescent dye.

When microorganisms grow they release CO, which reacts with fluorescent dye in sensor. LED activates this fluorescent dye. Fluorescence

2.

in

microorganism is compared with database for

(iii) Interpretation

Less

well measure various metabolic

activities of microorganism which help identification. The reaction pattern obtained from the

digest broth, yeast, amino acids, sugar, vitamins and sodium polyanethole sulfonate (liquod). Each bottle contains a chemical sensor which can detect CO, produced by microorganisms.

4.

substrate

Separate

2. BACTEC

It is based on

well contains an individual test

Each

IlI.

Molecular methods

It uses calorimetric reagent card containing 64 wells.

•

pH is decreased, this change is detected by calorimetric method.

(i) Principle

yeast.

based on biochemical characteristics metabolic properties of microorganism. It

•

pH.

The blue-green sensor becomes yellow when

help

the microbial identification is carried out by performin®

• Microorganisms growing in culture medium (positive culture) produce CO, and thus there

not

the organism grown. Conventional

various biochemical tests. Nowadays various automated methods are available for identification of microorganisms. These include:

1.

(ili) Interpretation

, decrease

However these methods do

identification

(b) Primer annealing The temperature is reduced to 50-60°C, then ougonucleotide primers attach to target DNA. This temperature is called annealing temperature and the process i known

as

annealing of primers. Thermus

aquaticus

Gel

electrophoresis.

(iii) Application of PCR The PCR provides extremely rapid analysis (one day).

versatile tool useful in infectious, genetic forensic investigations

or neoplastic diseases, in of phylogenetic relationand in the examination been applied in clinical

ships in evolution. It has

of various infectious agents laboratory for diagnosis (Table 22.1).

(c) DNA synthesis

Folymerase enzyme derived from

(d) Detection of amplified product Amplified DNA can be detected by

A specific DNA sequence of a particular

infectious agent

is amplified with the specific primers.

166

Unit III Applied Microbiology

Table 22.1 PCR

in Diagnosis of Infectious Agents Bacteria

M. tuberculosis, Legionella pneumophila, Helicobacter pylori, Chlamydia trachomatis, Mycoplasma pneumoniae Viruses

Cytomegalovirus, Herpes simplex virus, Hepatitis B virus,

Hepatitis C virus, Coxsackie virus, Measles virus, Human immunodeficiency virus (HIV-1 and HIV-2), Human

papilloma virus, Rotavirus, Rubella virus, Human herpes virus-6 (HHV-6), rhinovirus, parvovirus, adenovirus. Fungi

Candida albicans, Cryptococcus neoformans, Pneumocystis jiroveci. Protozoa

Toxoplasma gondii, Trypanosoma cruzi, Plasmodium spp.

Disadvantage of Conventional tubes with environment Contamination of reaction PCR

DNA may occur. used to amplify and

simultaneously

detect or quantify a targeted DNA molecule on real time

also detect and quantify RNA of the tes the specimen by using reverse transcriptaso real time PCR formats. Thermocycler used for real time that used in conventional PCR is different than basis. It can

organism

The amplified nucleic acid (amplicons) can be visualised simultaneously during the process of amplification and there is no requirement of gel electrophoresis to detect the amplicons as in conventional PCR.

Detection of amplicons of real

time PCR

real time PCR reaction are detected fluorogenic molecules which may be

The amplicons in

by using various

Other target amplification techniques include TMA and either non-specific or specific. Non specific methods: SYBR green fluorogenic dye used to stain any nucleic acid non-specifically. Modifications of PCR Specific methods: Fluorescent labelled oligonucleotide Besides originally described PCR, modifications of PCR include reverse-transcriptase PCR (RT-PCR), nested

PCR, multiplex PCR and real-time PCR

As originally described, PCR was a technique for DNA amplification. Reverse-transcriptase PCR (RT-PCR) was developed to amplify RNA. In this technique target is

RNA instead of DNA. After extraction of RNA, the

complementary DNA (CDNA) is first produced from

RNA with the help of enzyme reverse transcriptase and then cDNA is amplified by PCR. This technique is very useful for detection of RNA viruses or 16 S rRNA gene

probe is used. This probe binds (i.e. hydridises) only to particular region of amplicon (amplified

nucleic acid). Three types of hybridisation probes are commonly used in real-time PCR systems. 1. TaqMan Molecular beacon 3.

(ii)

Nested PCR

PCR was developed to increase the sensitivity

specificity of PCR technique. uses two pairs of amplification primers. One primer pair is used in the first round of PCR to amplify the desired sequence. The amplified product of the first round is then subjected and

second round of PCR with the second set of primers

which anneal to the sequences found only in the first round products.

Two or more primer sets designed for amplification of

different targets are included in this technique. This will

help in amplification of morethan one target sequence in 1 clinical specimen. It can detect many DNA sequences of several organisms in one reaction. For example, the

common organisms causing pyogenic meningitis, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae can be

detected by different primers in the same reaction tube.

Quantitative: Real time PCR can quantify the nucleic acid present in the specimen. Hence, it can be used

monitoring the disease progression during treatment e.g. viral load in HIV. Conventional PCR

for

of the organisms.

Nested

Fluorescence resonance energy transfer (FRET) probe

Advantages 1.

chain reaction (LCR) is a

DNA probes are their

high degree of specificity

probe amplification ability to detect minute guaneice o

cannot quantify.

Sensitivity and specificity of real time PCR are more , compared to conventional PCR. time than conventional PCR. 4. Contamination rate is extremely low. 2.

probes hybridise direct detection of microorgantisms in clinicay speeimens to one another on each of the denatured target and for identification of organismns after diphation preumnene adjacent DNA strands in such a way that a 'nick' formed. is

nick by joining the Enzyme DNA ligase then seals the

' end of one

probe and the

ligated product, as well

end

template in subsequent rounds of denaturation, annealing and ligation. It results in an exponential

as

thermal cycler. LCR based amplification has been used tuberculosis, N. gonorrhoeae :

Probes

serve are shown in Table 22.2.

increase of ligated products. Like PCR, LCR also requires for M.

Application of DNA

of the other. Each Applications of DNA probe technology in microbiology

the original target,

C. trachomatis.

B. Non-Amplified Method

Table

22.2 Applications of DNA probe technology

In clinical microbiology: - Direct detection of microbes in specimens. To detect microbes which are either difficult or

impossible to culture.

Identification of culture isolates

Nucleic Acid Probes

Nucleic acid probes are non-amplified methods. Nucleic probes are segments of DNA or RNA labelled with radioisotopes or enzymes that hybridise to

acid

complementary nucleic acid with high degree of specificity. All microorganisms contain some unique sequence:

Strain identification

To identify toxins, virulence factors Identification of resistant markers

Detection of amplified nucleic and in real

time PCR

• Detection of specific DNA fragments in Southern blotting

nucleic acid within their genome that distinguish Nucleic acid probes for direct detection of group A other organisms. This unique sequence recognised by hybridisation streptococci, Chlamydia trachomatis and Neisseria gonorrhoeae are available. Probes for identification of group A with DNA probe (Fig. 22.12). Hybridisation is the of

them from all

of nucleic acid can be

technique in

which two single-strands of nucleic acid

streptococci, group Bstreptococci, enterococci, Hemophilus

come together to form a stable double-stranded molecule. influenzae, mycobacteria, N. gonorrhoene, Staphylococcus Probes containing sequences unique to the microbe can aureus, Streptococcus pneumoniae, Campylobacter spp.,

be added to microbial cultures, body fluids or other Histoplasma capsulatum, Blastomyces dermatidis and Coccidioides immitis isolated in culture are also available. specimens suspected contain the microbe or its DNA. The DNA probe hybridises with the complementary DNA probes for detection of LT and ST toxins of Esch. coli

sequences on the microbe's DNA. The advantages

are also available.

Disadvantages The

real time PCR system is very expensive compared

conventional PCR.

Add DNA probe

2. Nucleic Acid Sequence-Based Amplification

Labelled

RNaseH and 17 bacteriophage RNA polymerase

used in NASBA, while TMA uses an RT enzyme with RNA are available.

Double stranded DNA

are

endogenous RNaseH activity and 17 RNA polymerase. NASBA based kits for detection and quantitation

Hybridise

probe

Like TMA, it is also isothermal RNA amplification method. The method is similar to TMA. RNA target is reverse transcribed into cDNA and then RNA copies are synthesised with the help of RNA polymerase It also does not require thermal cycler. AMVRI,

of HIV-1 RNA and CMV

and

methods. Probe amplification difficult or : targetamplification to culture. Nucleic acid probes method differs from target amplification method in that which bind impossible RNA can also be designed and has been only a sequence present in particularly toused contain products amplified to locate ribosomal RNA. the initial probes. A number of DNA probes have been developed for In LCR, two oligonucleotide

3. Takes less

to

167

are

(iv) Real Time PCR Real-time PCR

Diagnostic Bacteriology

Chain Reaction (LCR) 3.

Fig.

22.12 Hybridisation technique

Antigen-Antibody

Reactions

Stages

reaction occurs in two stages: The antigen-antibody C.

EarE are.iaywe

23

The Primary

Stage

This reaction is reversible.

tie binding between antieen and antibody oxcurs by

Bandanna firmer covalent binding. in

PREVIEW

Types of Antigen-Antibody

Features

. Antigen-Antibody Reactions- General

Reactions

uses.

5.

Principles

and uses of immunolluorescence tests and enzyme linked f immunochromatographic test (ICT) and

Applications

by Zinsser's unitarian hypothesis (1920) which held that

antigen-antibody reactions. with

Antigen combines with its specific antibody in an observable manner and the reaction between antigen

and antibody iS specific. These antigen-antibody reactions in vitro are known as serological tests as these

2.

immunoassays. Besides serum, other specimens can also such as CSF, urine etc. for performing these be

antigens participate in the antigen-antibody reaction.

diseases.

5.

injury in

some

Antigens and antibodies can combine in varying proportions. Antibodies are generally bivalent,

although IgM may have five or more combining sites (multivalent). Antigens may have valencies upto hundreds.

Ig has a low affinity IgG but has a much higher avidity due

the Laboratory or in Vitro (i) For diagnosis of infections

2. In

Detection and quantitation of either antigens or antibodies.

than

multivalency. Hence, Ig can bind to antigen more effectively than IgG.

(ii) Helpful in epidemiological studies

B.

effects. Both of these extreme views are not true. is now an established fact that a single antibody can cause different types of antigen-antibody reactions and single antigen can stimulate duction of different classes of immunoglobulins which differ in their reaction capacities as well as in other properties (Table 23.1).

Table 23.1 Role of Immunoglobulin Classes in Serological Reactions

Serological reaction

intensity

attraction between antigen and antibody molecules. Avidity the total binding strength after the formation of antigen-antibody better indicator for complexes. Avidity IS strength of an antigen-antibody reaction.

hypersensitivity reactions and autoimmune diseases.

(iii)

as the

an antigen would induce synthesis of only one antibody which produces different types of visible (observable)

Immunoglobulin class

6.

1. Reaction is specific; an antigen combines only

Strong

Weak

Variable

Agglutination

Weak

Strong

Moderate

Complement fixation Strong

Weak

Negative

D.

Qualitative and Quantitative Tests

1. Qualitative Tests

Qualitative test detects presence or absence of antigen or antibody in the specimen and thus the result is 'positive'

false negative results. The

are

also of use when prozone

Evaluation of the performance of anydiagnostic test can

parameters.

Number of true positives Number of true positives : Number of false negatives

Specificity: It is the ability of a test

identify all those who are true negatives.

Specificity

to correctly

not having the disease i.e.

Number of true negatives Number of true negatives + Number of false positives

II. TYPES OF

ANTIGEN-ANTIBODY REACTIONS A. Precipitation Reactions

Precipitation When a soluble antigen reacts with its antibody in the

presence of electrolytes (NaCI) at an optimal temperature and pH, the antigen-antibody complexforms an insoluble precipitate and it is called precipitation. The precipitate

Precipitation may in liquid media or in

gels such as

agar, agarose or polyacrylamide.

Flocculation When instead of

sedimenting, the precipitate

iS

suspended as floccules, the reaction is called flocculation.

This is a modified form of precipitation. A serological

test (e.g. VDRL) routinely used for diagnosis of syphilis is one example of flocculation test. VDRL slide is used exact amount of antigen or antibody in the specimen perform 'Venereal disease research laboratory' (VDRL)

or

'negative'. As the undiluted specimen is used, the

cannot be detected. 2.

have stronger

When

used in IgG avidity test. 168

Precipitation

Although IgG has a low avidity initially but will avidity in later part of infection. Avidity increases with time and this property 15

Characteristics

serial dilutions of serum henomenon occurs.

usually sediments at the bottom of the tube

of

(i) It forms the basis of immunity against infectious to tissue

The reaction is firm, but reversible. The firmness of combination depends on the affinity and

avidity. Affinity is defined

antibody can be detected by

in Vivo

may lead

the fragments.

3. Only the surface 4.

A. Uses

the Body or

antigenic similarity. Entire molecules of antigen and antibody react but not

are performed in serum specimens. These are also called

1. In

its homologous antibody and vice-versa

However, cross reactions may occur due to

GENERAL FEATURES

tests. Both antigen and these immunoassays.

not

those involved in precipitation as precipitin and the corresponding antigens were called agglutinogen and precipitinogen respectively. This concept was replaced

immunosorben assay (ELISA).

flow through assay.

. ANTIGEN-ANTIBODY REACTIONS

antibody by similar method. reliable to differentiate

are more

be done by statistical analysis. Sensitivity and specificity are the two important and commonly used statistical

believed that different type antibody was involved of reaction. On this basis, the antibody for each type was called agglutinin and involved in agglutination

Prozone phenomenon.

6.

but

Quantitative tests

between true negative and

fixation, neutralisation and immobilisation of motile positives. organisms. When these reactions were discovered, was Sensitivity

Difference between precipitation and agglutination.

Types of agglutination reactions and their Principle and uses of latex agglutination test. Principles

the reaction by a number of

169

all, is followed by secondary stage which has visible Sensitivity: It is the ability of a test to correctly effects such as precipitation, agglutination, complement identify all those who are having the disease Le. true

SPECIFIC LEARNING OBJECTIVES

4.

ionic bonds, rather than This primary interaction

methods. Secondary Stage 2. The The primary stage in most of the instances,

The student should be able to learn:

3.

Antigen-Antibody Reactions

Thvleasurement of antibody is expressed as titre of units The antibody titre is the highest dilution of the sere of units, shows an observable reaction in the particular antigenantibodyreaction. Antigens may alsobe trratedinsergm

Quantitative Tests

he qualitative test is positive, the exact amount of antibody in serum can be detected by serial dilutions of serum

test (Fig. 23.1).

Prozone Phenomenon

When same amount

of

antiserum is mixed with

increasing quantities of antigen in different tubes, the

and mixing each ciution with a known quantity precipitation takes place in one of thepresent in middleintubes optimal which

of antigen.

antigen and the antibody are

Unit Ill

Applied Microbiology

Antigen-Antibody Reactions

on a slide or petridish layered with is done gel, diffusion occurs in both X and axis which is Antigen

Definition

Various Immunodiffusion Single diffusion in one dimension (Oudin procedure) one

It isan antigen-antibody reaction,in which a antigen combines withits antibody in the particulate

Tests

2.

Antibody

Antibody in Zone of equivalence (Lattice formation)

exces

equivalent proportion (Zone of equivalence). In the

preceding tubes, antibody is in excess and in later tubes,

Precipitation

(false

negative) in the presence of excess antibodies is known (a as prozone phenomenon. This is relevant clinically as

further to get the

proper results of precipitation.

Mechanism of precipitation

(1)

4

over an

two liquids. Ring tests were previously used

for detection of

Marrack (1934) proposed the lattice hypothesis which is

widely accepted. According to this concept, multivalent

antigens combine with bivalent antibodies, precipitation

This occurs occurs only when large lattice is formed.

in the zone of equivalence. In the zone of antigen excess, valencies of the antibody are fully satisfied which

the

results in failure to form a large lattice. Similarly, in the

zone of antibody excess, the valencies of the antigen are form taken up with antibody and results in failure to a large lattice (Fig. 23.3). This lattice hypothesis holds

true for agglutination also.

(ii) Clinical applications of precipitation reaction

C-reactive protein (CRP)

streptococcal grouping

It can be done in slide and Slide test:

test

and

by the Lancefield technique.

(b) Flocculation test

are two

simple and newer methods, theapplication ofthese tests is limited. Precipitation test still has a considerable value in: 1. Detection of antibody for diagnostic purposes e.g. 2.

VDRL in syphilis. Identification of bacteria e.g. Lancefield's grouping of Streptococcus.

central well and surrounding wells with different antigens.

are

(iv) Precipitation in gel

indicates oss

23.4).

which combine with the antigen but do not

inhibiting the agglutination by the completeantibody

reaction partial

©

three types:

reverse passive

In direct agglutination test, antigen directly and

in a well

tube agglutination tests are examples of direct

agglutination test.

Unrelated

TYPES OF AGGLUTINATION REACTION Fig. 23.4 A

1.

Double diffusion in two dimensions

special variety of double diffusion

in two

dimensions is the Elek's test for toxigenicity in

C.

diphtheriae.

Immunoelectrophoresis

Immunoelectrophoresis combines electrophoresis

6. Electroimmunodiffusion

Immunodiffusion can be speeded up if antigen and antibody are driven by electricity. It is combination of electrophoresis and diffusion.

23.2 Prozone phenomenon

of

and

agglutinates with the antibody. Slide agglutination

Antiserum

and immunodiffusion.

Fig.

are

These tests are performed on slide, in tube or in card. Sometimes microtitre plate may also be used.

Reaction of

identity

Antigen in well

is named one dimension. In contrast, when test

(i) Antigen excess

Agglutination reactions

•

Reaction of

When immunodiffusion is done in a test tube layered with gel, vertical diffusion occurs which

Optimal proportion antigen and antibody (zone of equivalence)

added subsequently.

Applications direct, passive (indirect) agglutination.

partial identity

cause

agglutination. They act as blocking' antibodies,

Agar gel

5.

Antibody excess

for agglutination too. The

antibodies (e.g. anti-Rh and anti-Brucella) are formed

on a slide

These are precipitation tests in gel. Immunodiffusion is generally done in 1% agar gel. Immunodiffusion tests are based on two principles: Only antigen diffuses (single diffusion) or both antigen and antibody diffuse (double diffusion).

holds good

zone phenomenon may occur when either : n antigen or an antibody is excess. Occasionally incomplete

other.

Spur formation

identity (Fig.

place better with

presentin optimal proportions. Lattice formation

hypothesis

filled

they are unrelated, the lines will cross each

examples of slide flocculation test.

Tube test: The Kahn test (tube flocculation) was done previously for diagnosis of syphilis.

Agglutination reaction takes

antibodythan with 1gGantibody.Principles governing Agglutination occurs when bothantigenand antibody

If two adjacent antigens are identical, the lines of precipitate formed by them will fuse with each

is added to ;

is more sensitivethan precipitation for Agglutination the detection of antibodies. agglutination are the same as that of precipitation.

other.

Precipitation reaction may be used either as qualitative Immunodiffusion test or quantitative test. Earlier, precipitation reactions were widely used serological tests. Now due to availability of

.

template. The antiserum (antibody) is placed in

the

drop of inactivated patient's serum on a slide and mixed by shaking, floccules appear. VDRL and RPR (Rapid Plasma Reagin) tests for detection of antibodies against syphilis

present in contrast to particulate antigen of

Advantages

Double diffusion in two dimensions procedure) Agar gel is poured on a slide. Wells are cut using

in tube.

drop of antigen solution

"tdifersfrom precipitation in whichsoluble antigen

agglutination.

(halos) are formed around the well

in liquid medium

antiserum in Antigen solution layered the junction narrow tube. A precipitate ring appears at of the

visible clumping of particles.

two dimensions (Radial

The antiscrum (antibody) is incorporated in agar gel on slide or petridish. The wells are cut on the surface of gel. Antigen is added to the wells. The antigen diffuses radially in all directions to meet the antibody and ring-shaped bands of precipitation

commonly used method.

Ring test

Single

dimension (Oakley-Fulthorpe

diffusion

immunodiffusion)

Hence, only these precipitation reactionsare described

VDRL test is the most

in which antigen is in excess, the precipitation is weak

or even absent (Fig. 23.2). Absence of precipitation

these sera should be serially diluted

Antigen i excess

Fig. 23.3 Lattice hypothesis

Fig. 23.1 VDRL slide or

3.

Double diffusion in

procedure)

171

B. Agglutination

named two dimensions.

Counterimmunoelectrophoresis

(Countercurrent-immunoelectrophoresis, CIE or CIEP)

(ii) Rocket electrophoresis

Slide Agglutination

Test

• A uniform suspension of antigen made in a drop of saline on a slide or tile. A drop of the appropriate antiserum (serum

containing appropriate antibody) is added. The agglutination reaction is facilitated by mixing the antigen and the antiserum with a wire loop or by gently rocking the slide. Clumping occurs instantly or within seconds when agglutination test is positive. Clumping after a minute may be due to drying of the fluid and should be disregarded. A control consisting of antigen suspension in saline, without adding antiserum must be included on

the same slide. It is to ensure that

antigen is not autoagglutinable.

Unit III Applied Microbiology

id) Paul-Bunnel testare

agglutinated by serum Sheep erythrocytes infectious mononucleosts. (1) It is a routine procedure to identify the bacterial strains isolated on culture from clinical specimens, MG agglutination test (titi) Streptococcus One example is to identify Salmonella species. pneumonia. ad arming and cross matching. It positive in primary atypical (ii) It is also used for blood grouping and cross mart

Uses of slide agglutination test

2. Tube Agglutination Test

This is a standard quantitative method for determination of antibodies in serum. Serum is diluted serially by doubling dilution test tubes.

An equal volume to all tubes.

agglutination

formation at the bottom of test tube and clearing of the supernatant).

Negative test indicates that agglutination has

not

occurred (i.e. button formation at the bottom of the

to settling of antigen suspension). The highest dilution of serum at which agglutination

test tube due

is antibody titre.

Uses of tube agglutination test (1)

agglutination

precipitation reaction can be converted into agglutination test by attaching soluble antigens the surface of carrier particles such as latex particles cells. Such bentonite and red blood tests are called passive agglutination tests and are used for detection A

antibodies. This conversion IS done because agglutination tests are more sensitive than precipitation tests for detection of antibodies. Passive agglutination tests are very sensitive. of

(for antigen detection) When instead of antigen, the antibody is adsorbed on the carrier particles for estimation of antigen, it is

(Streptococcus MG agglutination

test)

Two main complications may (1) Prozone phenomenon

as reverse passive agglutination.

(i) Latex

agglutination test

widely employed to adsorb several types of antigens.

be

Latex particles can also be coated with antibody for

detection of antigen (Fig. 23.5). These tests are very convenient, rapid and specific. These are used for detection of hepatitis B antigen, ASO, CRP, RA factor,

Blocking antibody

(i) Prozone phenomenon Serum

known

Polystyrene latex particles (0.8 - 1 pm in diameter) are

Complications related to tube agglutination

with high concentration of antibody may not be HCG, bacterial typing (N. meningitidis) and many other

able to react with antigen and may give a false negative result. Several dilutions of the serum should be tested prevent such false negative results due to prozone.

Blocking antibody Blocking or incomplete antibodies may be detected by performing the test in hypertonic (5%) saline or albumin saline. Antiglobulin (Coombs) test is more reliable for detecting these antibodies.

Heterophile antibodies have a property to react with due to

Toxigenicity Test

is diphtheriatoxin-antitoxin neutralisation test

in animals. 2

Latex particles

Antibody

Itis diphtheria toxin-antitoxin neutralisafion in

1.

Fig.

..

23.6 Latex agglutination tile

humans.

antibodies

sensitised with Erythrocytes of antibodies. detection

detection of RA factor

Antistreptolysin 'O' (ASO) Test

ASO testwas used fordetection of antistreptolysin 'O'

uaemagglutination test antigen are used

test.

It is no longer used..

(it) Coagglutination

their

named as

virus

3.

Specific IgG immunoglobulin is coated on these Cowan

of Staphylococcus aureus. Fc portion of IgG

test.

Virus Neutralisation Tests Viruses may also be neutralised by

antibodies and these

neutralisation tests.

It is based on the presence of protein A on the surface of some strains of Staph. aureus (especially Cowan strain).

neutralisation

these antibodies.

Principle

1 strains

in serum by

Nowlatexagglutination testis done for detecting

for

Rose-Waaler test employed for of rheumatoid arthritis

was a haemagglutination

Nagler Reaction

It is useful for detection of a-toxin of Clostridium E.

Immunofluorescence

Fluorescence is

the

property of certain dyes

which

molecule binds to protein A whereas antigen combining absorb rays of one particular wavelength (ultraviolet Fab terminal

remains free. When the corresponding

light) and emit rays with a different wavelength

antigen is mixed with these coated cells, Fab terminal (visible light). Coons and his colleagues (1942) showed binds to antigen resulting in agglutination. This test

commonly used for detection of bacterial antigens longer used. in blood, urine and CSF. It is

was

C.

Complement Fixation Test (CFT) Principle

that fluorescent dyes can be conjugated to antibodies and these 'labelled' antibodies can be used to detect antigens in tissues. The commonly used fluorescent dyes are fluorescin isothiocyanate (FITC) and lissamine rhodamine, exhibiting blue green and orange red fluorescence respectively. Immunofluorescence test

The antigen-antibody complexes have ability to 'fix' complement. This reaction has no visible effect. To

of two types:

Direct immunofluorescence test 2.

1.

Indirect immunofluorescence test

Direct

Immunofluorescence

Test

Principle

2. Applications

CFT was widely used in diagnosis of Mycoplasma, Chlamydia, Rickettsia and some viral infections. Complement fixing antibodies were detected by this method.

The specific antibodies tagged with fluorescent dye (i.e. labelled antibodies) are used for detection of unknown

antigen in a specimen. antigen is present, t reacts with labelled antibodies and fluorescence can be observed under

ultraviolet light of fluorescent microscope (Fig

Other complement dependent serological testsinclude: 23.7). This is depicted in flow diagram as follows: Antibody coated latex particles

1.

Antigen

OXK) strains are agglutinated

by sera of patients with rickettsial infections. This is due to antigenic sharing between these Proteus strains and Rickettsial species.

Shick Test

In vitro tests

reaction

Some Proteus (OX19, OX2

are as follows:

173 and 'in

In vivo tests

common antigenic sharing. (1) Weil-Felix

Antigen-Antibody Reactions examples of neutralisation test in

almost not in use now.

Agglutination Test

microorganisms or cells of unrelated species

perform Some

detect the fixation of complement, an indicator system is used. Earlier it was commonly used method but is

(iz)

3. Heterophile

latex

to

test (MAD) for demonstration of spechire

(b) typhus fever (Weil-Felix reaction) (c) infectious mononucleosis (Paul-Bunnel test) (ii) For diagnosis of primary atypical pneumonia

used

The test is performed microtitre plate and the resul is read under microscope The example i microscopic

Reverse passive agglutination test

(d) brucellosis

agglutination tile

Test 4. Microscopic Agglutination in a

Serological diagnosis of

(a) enteric fever (Widal test)

(i1)

of

antibodies in leptospirosis. of a particulate antigen is added Agglutination Test 5. Passive (indirect) (for antibody detection) (i.e. clump

Positive test indicates

occurs

Latex

antigens. agglutination test (Fig. 23.6)

2

Agglutination

Fig. 23.5 Latex agglutination fest

D.

Treponema pallidum immobilisation test for syphilis When live motile Treponema pallidum is mixed with patient's serum in the presence of complement, the organism becomes non-motile. Sabin-Feldman dye test for Toxoplasma gondii.

Neutralisation

Test

Neutralisation tests are less commonly used nowadays.

Specimen (Positive for antigen) Labelled antibodies

Fluorescence observed

Antigen is present in specimen

Unit

I1l Applied Microbiology

anorescence, the

y

Test

General I (positive test)

specific antibody

Antigen in testserum getsattached to the solid phase

features

of the well by passiveadsorption. Washing step is done. After washing, the enzyme-labelled antibodies

ELISA) are . It is done on solid phase (absorbing material). The test can be done in polystyrene tubes (macro-ELISA) polyvinyl microtitre plate containing 23.8)

micro-ELISA) (Fig

Indirect immunofluorescence

(antibody to antigen in testserum) are added. Washing step is done. After

washing, a substrate is added. serum contains the antigen (positive colour is produced.

96 wells

The intensity

Known Antigen

Patient serum containing antibody

Example:

labelled

(containing anti-treponemal antibodies which is globulin in nature) Fig. 23.7 Direct

and

Fluorescii

Fluorescence

(Positive)

Antibody

Immunofluorescence

The indirect method is employed for detection of antibodies in serum or other body fluids. It is also used for detection of autoantibodies (e.g. antinuclear

Antigen

antibody) in autoimmune diseases.

on

slide. The

competition

for

For detection of this antigen-

al

Antibody

antibody reaction, fluorescin-tagged antibody to human

globulin is added. In positive test, fluorescence occurs under ultraviolet light (Fig. 23.7). One specific example

detecting antibodies in serum of syphilis patient is

also included.

Advantages

A single antihuman globulin fluorescent conjugate can

be employed for detection of antibody t any antigen. All

antibodies are globulin in nature, therefore, antihumar

ELISA has been applied widely for detection of variety of antibodies and antigens. It is simple and nearly as sensitive as radioimmunoassay. It requires only microlitre quantities test reagents. The principle of ELISA is almost same as that of

Sera to be tested are added in these coated wells.

• If antibody IS present in the specimen, it binds to coated antigen. To detect this antigen-antibody reaction, antihuman

enzyme

immunoglobulin (secondary antibody) conjugated

with an enzyme is added.

Enzyme conjugated antihuman immunoglobulin binds to antibody (immunoglobulin in nature) to be

Enzyme acts on substrate to produce colour

detected.

Colour detected by spectrophotometer

•

their principles

23.9).

The intensity

of

measured

by

spectrophotometer or ELISA reader. Positive and negative controls are always

DIRECT ELISA

put up

alongwith test sera.' Incubation and washing is done

antigen detection)

Direct ELISA is used

substrate is added and

This procedure is also named as indirect ELISA (Fg

There are several types of ELISA with some variation

(for

this binding,

acts on substrate to produce colour

positive reaction.

Types of ELISA in

To detect

enzyme

(ELISA reader)

curve.

Immunosorbent Assay

•

Substrate

their radioactivity is measured. The concentration test (unlabelled) antigen is calculated from the ratio the bound and total antigen labels, using : reference

G. Enzyme Linked (ELISA)

precoated with the antigen.

contain

Antigen

fixed amounts of

and of

INDIRECT ELISA

Indirect ELISA is used for detection of antibody or less

For antibody detection, the wells of microtitre plate

ELISA kits are available commercially, which the reagents for performing the test.

specific antibody between a known radiolabelled antigen and unknown unlabelled (test) antigen. This competition

of

Colour detection

(ELISA reader).

Radioimmunoassay (RIA)

RIA is based on

Substrate

rise to a colour change by adding specific substrate (o-phenyl-diamine dihydrochloride for peroxidase,

the reacting system. After antigen-antibody reaction,the unknown antigen is separated into the'free' and bound' fractions

antibody (serum) is applied to the slide. If antibody (globulin) is present in the serum, it attaches to known

of

and immunosorbent (absorbing material that absorbs antigen or antibody). Substrates are specific for each enzyme. The enzyme (horseradish peroxidase, alkaline phosphatase) gives

is determined by the level of the test antigen presentin

Principle

antigen on the slide.

F.

or

labelled with enzyme

ELISA name is derived from enzyme linked (enzyme is linked either to antigen or antibody)

Labelled antibody

Test

antibody

p-nitrophenyl phosphate for alkaline phosphatase). commonly antigen. Alkaline phosphatase with this substrate produces a yellow colour which is detected by spectrophotometer For antibody detection

Disadvantage

prepared against each antigen to be tested.

enzyme labelled

Primary antibody (antibody to antigen)

Fig. 23.8 ELISA test

1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, Sandwich' Technique of Immunofluorescence faeces, tissues and other specimens. Antigen beingin the middle with labelled and unlabelled 2. It is a sensitive method to diagnose rabies, by antibody on eitherside,forms a sandwich. This is used detection of the rabies virus antigens in brain for detection of antibodies.

Separate specific fluorescent labelled antibody has to be

by

Antigen (test serum)

globulin attaches to all antibodies. This has overcome the disadvantage of direct immunofluorescence test.

known antigen is fixed

test), the

measured

substrate.

labelled

smears.

antibody,

antigen,

UV light (positive test)

indirect immunofluorescence tests

Uses

A

is

These are basic steps of all types of ELISA. Washing is donein between each step to wash off the unbound

Flourescein Fluorescence under

antigen

antibody

syphilis patient

Treponema pallidum

Indirect

of

spectrophotometer.

Slide

2.

detected) is precoated with

antigen or antibody) of microtiter plate.

FL.IS A can be used for detection of antigen or antibody. rests for specific immunoglobulin classes (e.g. Ig also available.

Fluorescence under UV

Fluorescein labelled

serum (containing antigen to be

added into the empty wells (not

test.

Slide

175

serum (Fig, 23.9).

positive

on

acts

Unknown Antigen

Antigen-Antibody Reactions

only difference being, an

Direct immunofluorescence test

for detection of

antigen in

the

every step to wash

off unbound reagents.

Unit III Applied Microbiology Indirect ELISA antigen detection)

Indirect ELISA

Direct ELISA (For antigen detection)

(For antibody detection)

antigen detectionempty and

Antigen-Antibody Reactions immunoassay.

are

not

well. The antigen

Step

the primary antibody (antibody to antigen) is added. This antibody binds to antigen in the well conjugated antihuman immunoglobulin Enzyme (secondary antibody) is added. is added.in positive test. The substrate detected

Patient serum (antigen)

Patient Serum

177

This type of ELISA is routinely used for

wells Microplate antibody. diagnosis of dengue, chikungunya and japanese precoated with the or encephalitis. antigen to be detected) is added to antigen (containing The wells of microtitre plate are pre-coated with antiSerum gets absorbed onto the well.

For

(antigen)

•

Step

The

The test serum is added in the coated wells. If IgM antibody (primary antibody) is

To detect this reaction (anti-human-IgM antibody

Ig

antibody detection

For antigen detection

Antigen coated

Antigen (test serum

Primary antibody

with enzyme

the reaction.

•

Secondary antibody Substrate

Colour detection

Colour detection

A substrate is added to know the binding of secondary

antibody. If binding occurs (positive result), an enzyme acts substrate to produce colour, intensity of which

on

be read by spectrophotometer or ELISA reader. The use of avidin-biotin helps in amplifying the signal generated by this binding complex, thus increases the

labelled with enzyme

Substrate

Step

This secondary antibody binds to the antigen present

or

Secondary antibody labelled

antibody + antigen), antibody (specific for

the antigen) conjugated with an enzyme (secondary antibody) is added.

sample)

Serum (antibody to be detected)

in

in the wells.

indirect ELISA is shown in box below.

well

present

serum, it binds to anti-human-lgM antibody. • Recombinant antigen (e.g. dengue antigen) is added

colour is

Principle of

Step 3

human-IgM antibody (capture antibody).

•

sensitivity of the assay. Colour (Positive)

Ig Antibody Capture

Antibody coated

Anti-human IgM antibody

(MAC) ELISA

SANDWICH ELISA Sandwich ELISA

(For antigen detection)

Sandwich ELISA

Colour (Positive)

Colour (Positive)

Sandwich ELISA detects

Competitive ELISA

(For antibody detection)

(capture antibody)

Serum (Antigen)

of

Recombinant antigen

(conjugated)

against the antigen to be detected.

Secondary antibody-

Substrate

wells. Specimens to be tested are added in coated If antigen is present in specimen, it binds to coated antibody.

Step:

Serum (Primary antibody)

Primary antibody

For antigen detection in a specimen, the wells coated with specific antibody microtitre plate

Step 1

coated well

well

the antigen in serum. It is so

named because the antigen gets sandwiched between two antibodies.

avidin-enzyme (conjugated)

biotin

Colour detection

Substrate

To detect this antigen-antibody reaction, antiserum

Conjugate is washed out as antigen not free to bind the

Step

conjugate

(antibody) conjugated with an enzyme (primary antibody) is added.

antigen This conjugated antiserum binds to already attached to coated antibody. substrate is added to know the binding of

• A

conjugated antiserum to antigen-antibody complex.

If the binding occurs (positive result), an enzyme acts

Step 4

Substrate Enzyme (tagged to conjuga

Colour (Positive) Antigen

Antibody

not there to act on

No Colour (Positive)

Secondary antibody (conjugated) [Antibody tc antibody ()1

Primary antibody (not conjugated)

substrate

substrate to

Colour detection can also be seen This

Primary antibody (conjugated) [antibody to antigen (4)1

Positive and

detection.

Antibody detection It has been used

done to wash off

of •

gM ANTIBODY CAPTURE (MAC) ELISA This is an enzymatically amplified sandwich-type of

detection of HIV

antibodies.

•

colour indicates a negative test.

There

two

specific antibodies, one conjugated (if serum

with enzyme and other present in serum IS

washing

unbound reagents.

for

Positive result shows no colour whereas appearance

negative controls should always be

included in the test. At every step of ELISA test, incubation and IS

COMPETITIVE ELISA

Competitive ELISA can be used for antibody or antigen

by naked eye.

type of ELISA test is known as sandwich ELISA

(Fig. 23.9).

Colour product Fig. 23.9 ELISA

produce colour, intensity of which can be read by spectrophotometer or ELISA reader.

on

Colour detection

positive for antibodies).

Competition oxcurs between two antibodies tor same antigen. The microtitre plate wells arethese withand coatedwells Fllvancgen incubated Sera to be testedis added

then washed. at 37°C and

Unit III Applied Microbiology

treated with

If antibodies are present, antigen-antibody reaction

parallel wells of microtitre

occurs.

To detect this reaction, enzyme labelled specific HIV antibodies are added. There is no antigen left for these antibodies to act. These antibodies remain free and washed off during washing.

Substrate is added but there is no enzyme to act on it.

Therefore, positive results show no colour (Fig. 23.9). If serum tested is negative for antibodies,

antigen is there to combine with enzyme conjugated and enzyme acts on substrate to produce

•

antibody complexes there is no

such effect

Avidity(%)

Absorbance of the well with urea Absorbance of the well without urea * 10)

Low avidity =

colour.

Inconclusive (test to be repeated)

this type of competitive ELISA, competition is between the two antigens to bind the primary

Uses

Primary antibody is incubated in

Clinical application

a

solution with a

This

mixture is then added to the microtitre well

precoated with the antigen (it is same type of antigen which is to be detected in patient's serum). If antigen is present in the test serum, this

mixture

will contain antigen-primary antibody complex, free primary antibodies and free antigen.

Only free primary antibodies in the mixture will bind to antigen coated on the well and the rest of the components in mixture (antigen-primary antibody complex and remaining free antigen) are removed after washing. Now enzyme conjugated secondary antibody added. This will bind to the primary antibody which

has already bound to the antigen coated on well. • After washing, the substrate is added. In a positive test, the colour is detected. This

is indirect type of competitive ELISA.

The principle of IgG avidity ELISA is similar to that

IgG avidity

test is available

serum used is treated with urea.

Principle

Avidity of IgG indicates the firmness with which the IgG antibody binds with its antigen. Low avidity antibodies: These are produced during primary infection or in individuals with

munosuppresion.

High avidity antibodies: These

are

synthesised

secondary infection, or reactivation, or vaccinated individuals. Theseantibodies are produced

during

by memory cells.

Urea has ability to dissociate low avidity antibodies. Thus, urea is used for IgG avidity test. Method

Untreated patient's serum

and

and used for rubell,

toxoplasmosis, cytomegalovirus infections etc.

micro-ELISA. The result is read visually.

detection of HIV and

antigens

the nitrocellulose membrane in the cassette. Test serum is added on the membrane. In positive serum, antibody will bind to the appropriate antign After washing to remove unbound antibody, conjugate (enzyme labelled antihuman immunoglobulin antibody) is added. It washed again to remove unbound conjugate substrate is added. A positive

result shows a

a spot on the membrane serves as

the serum specimen

can

used

for

an automated system

principle of ELISA with the following

All the steps are performed by the instrument

Detection method is fluorescence based and not by colour production as in ELISA.

The solid phase receptacle present in reagent strip with serves as solid phase, which is either coated

antigen (for antibody detection) or antibody (for antigen detection). This is equivalent to well in microtitre plate in in ELISA. The principle of ELFA has been shown flow chart.

These automated systems based ELFA technology are commercially available e.g. VIDAS.

Principle The reagent strip (solid phase coated with antigen or antibody)

¥ Enzyme acts

Uses of ELISA

It has been used for detection of antigens and antibodies of examples

as

substrate

Fluorescence (Positive test)

Takes more time (2-3 hours) as compared to rapid tests (10-20 minutes). Expensive equipments such as ELISA washer and ELISA reader are required.

The intensity of the fluorescence is related to the

amount of antigen or antibody present in the specimen

(ICT).

Flow

through assay e.g. TRI-DOT

assay.

flow or lateral immunoassay is one of the most popular form rapid mmunoassays.

Immunochromatography

It can detect both antigens and antibodies. It has advantage of being a step test. It can be completed within 30 minutes. It contains nitrocellulose membrane. It is I strip based test. The strip contains a chromatographic pad with three zones: sample pad, conjugate pad and capture line (Fig 23.10).

The conjugate pad may be having colloidal gold, dye, latex beads as conjugate which produces signal. The specimen is applied to the sample pad and flows

or

laterally by capillary

action.

Upon reaching the conjugate pad, it may bind to

conjugate antigen or antibody is present in the specimen and forms antigen antibody complex.

This complex then flow laterally to reach capture line. Here it is captured by antigen or second antibody present in the test line.

further and binds to gold labelled antibody) moves anti-human immunoglobulin t form colour control line.

Immunochromatographic test (ICT) is also available in a cassette format. 2. Flow

Through Assay

Flow through assay is another type of rapid test which

differs from ICT as follows:

Uses 1.

2.

test There is a positive control line also to check i properly performed. The e conjugate (colloidal

Substrate

High sensitivity and specificity Disadvantages of ELISA

Two principles of rapid tests are available. 1. Lateral flow assay e.g immunochromatagraphic

The presence of colour line is a positive test.

Enzyme labelled conjugate

•

follows:

the

(automated system). 2.

Principles

. Immunochromatography

(ELFA)

1.

bed side (point of care) and without any laboratory

equipment.

test

ENZYME-LINKED FLOURESCENT ASSAY

be tested using the

various microorganisms. Some

in IGRA (interferon gamma release

measuring sensitised T lymphocytes

for diagnosis of latent tuberculosis.

Economical

•

quantitative

producing the interferon gamma (IFN-1). IGRA is used

that site.

Advantages of ELISA Large number of specimens 96 well microtitre plate.

control, as shown

used for

of cells producing antibodies or cytokines.

differences:

Specific type

training is required. These tests are also called Pointof-care tests (POCT) as these can be performed at

modification of ELISA. It is

It is a simple modification of ELISA for testing one or few samples of sera at a time. The test is rapid (about

Cassette ELISA is being used for

Rapid tests are very simple to perform, rapid (takes 1020 minutes), do not need any instrument and minimal

HIV

Dengue Toxoplasma

based on

type and 2 antibodies. are immobilised on

Procalcitonin

Rapid Tests

H.

Enzyme-linked flourescent assay

hours taken for

TORCH infection (antibody)

Rotavirus (antigen) 2. Biomarkers

Hepatitis Hepatitis C

CASSETTE OR CYLINDER ELISA

10 minutes) as compared with the 2-4

(antigen and antibody) Measles, mumps

Antibody detection B

assay)

179

HIV

Rotavirus in

detection

ELISA mentioned above for antibody detection. Only by development of colour at the test

•

It

coloured spot. Human immunoglobulin immobilised at

of

Antigen-Antibody Reactions

Antigen detection surface antigen (HBsAg) • Hepatitis for dengue NS1 antigen faeces

ELISPOT Test

between recent and past infection. To diagnose congenital infection.

and

IgG Avidity ELISA

avidity < 40% avidity > 60% avidity 40-60%

To differentiate

antibody.

test serum.

antibodies

which remain bound to antigens.

High avidity

In

high avidity

Interpretation

antibodies

Antigen detection

used for ELISA test plate. Intwe

urea

Infectious diseases

Hepatitis markers

(i)

Protein A is used to label antibody instead

colloidal gold.

of

Antigen-Antibody Reactions

Unit III Applied Microbiology 3. Uses It is available for detection of antigens or antibodies such as hepatitis, HIV, TORCH against infections

Sample

POSITIVE SAMPLE

infections.

Testline positive Capture line zone

Conjugate pa

Sample

For antibody

Control

For detection of biomarkers such as procalcitonin.

J.

Conjugate pad

Sample

negative

reaction can be

line

zone

Antibody to antigen (4)

Antigen 1 Conjugate [antibody to antigen

Control

1 Antibody to antibody (?)

Fie, 23.10 Immunochromatography strip for antigen detection in sample purple andproton.one.themom.mier.c.og control region to

the

The specimen flows vertically through nitrocellulose membrane instead of lateral flow

The test is present in a cassette format. It consists of nitrocellulose membrane and absorbent pad. nitrocellulose membrane contains two test regions (coated with HIV-1 and HIV-2 antigens) and a third control region (coated with antihuman

• The

immunoglobulin). The specimen (serum) and buffer reagents are added sequentially from the top. •

As the serum passes through the nitrocellulose membrane, HIV antibodies, if present bind to the coated HIV antigens. Test dots: Protein-A conjugate (present in buffer)

binds to the Fc portion of the HIV antibodies give pinkish purple DOT(s) for HIV

to

DOT. For

irrespective of whether HIV

ICT.

Like ICT, flow through assay can also be used detect antigen or antibody. HIV-TRI DOT is one example of flow through assay to detect antibodies to HIV-1 and 2 separately patient's serum.

give a pinkish

not.

present

I.

Chemiluminescence-Linked

Immunoassay (CLIA)

Chemiluminescence refers to a chemicalreactioninwhich

is emitted in the form of light. Luminescence mens energy emission of light. Chemiluminescence immunoassy is a combinationof chemiluminescencetechniau. (CLIA) with immunochemical reactions. 1.

The principle of CLIA is similar to that of other immunoassays such as ELISA; however chromogenic (colour producing) substance is replaced by chemiluminescent compounds(light producing)suchas luminol and acridinium ester. These chemiluminescent

compounds emit light during chemical reaction. The light

can be

and HIV

detected by a luminometer.

Antigen-Antibody complex

separately (Fig. 23.11).

Control dot: Control region is coated with antihuman

immunoglobulin. Protein A can bind to any IgG present in serum and this IgG-protein A complex can bind to antihuman immunoglobulin present in

Chemiluminescent compound Produce light

under electron microscope.

2. Immunoelectronmicroscopy

Viral particles

• Protein A conjugate

Control

Positive

HIV antigen

HIV-

antibody-

(b)

Nitrocellulose membrane

Fig 2111(@ HIV-TRI DOTtest(b)Diagramaticrepresentation

2.

Advantages

Highly sensitive (more sensitive than ELISA)

Individual specimen can be tested as compard to ELISA which is preferred for testing multiple

specimens. Disadvantage It

has limited

application in diagnostic

microbiology in contrast to ELISA technique

Syphilis

Double diffusion (Elek's

Diphtheria

toxigenicity test) 3.

Slide agglutination

4.

Tube agglutination (widal test)

5.

In immunoblots, antibodies can detect proteins (antigens) mixtures. The mixture of

electrophoretically separated

Tube agglutination (serum agglutination test)

Enteric fever Brucellosis

Hetrophile agglutination (Weil- Typhus fever Felix reaction)

Western Blot in

Salmonella

infections, Cholera

Immunoblotting

in

oteins (antigens) is gel. The separated

proteins are then transferred from gel to a nitrocellulose

paper. These nitrocellulose paper strips are reacted with test sera and subsequently with enzyme-conjugated anti-human immunoglobulin. A suitable substrate added, colour produced by enzyme where specific in test sera has reacted

7. Hetrophile agglutination (PaulBunnel test) 8.

Hetrophile agglutination

mononucleosis

Primary atypical

test) 9.

Latex agglutination

10.

Latex agglutination (RA factor)

with separated proteins

ELISA positive HIV antibody cases. This is known as Western Blot test. It detects antibodies against various

Infectious

(Streptococcus MG agglutination pneumonia

on strip. This test has been widely used to confirm the 12.

Meningitis Rheumatoid arthritis

Latex agglutination (ASO test)

Rheumatic fever

Latex agglutination (CRP test)

Inflammatory diseases

protein (antigen) fractions in test sera. Southern Blot

13. Direct immunofluorescence

Rabies

The above procedure may also be applied to analyse DNA or RNA. When DNA is transferred on nitrocellulose

14. Indirect immunofluorescence

Syphilis

strips from gel, this test is referred to as Southern Blot test.

Northern Blot

When RNA is transferred, it is named as Northern Blot test.

Applications of Antigen-antibody Reactions The following antigen-antibody reactions are used for

L.

Light detection by luminometer

Slide flocculation (VDRL test)

2.

and

as clumps. This method is applied in some viruses such as hepatitis A virus and rotavirus causing diarrhoea.

K.

Disease

used) 1.

infections, Shigella

are mixed with specific antisera observed under the electron microscope. These are seen

antibody

Principle

visualised

Antigen-antibody reaction (lest

Tests

Test

Ferritin (electron dense substance) conjugated antibody is used to react with an antigen. This antigen-antibody

SAMPLE

(ii)

Immunoferritin

1.

NEGATIVE

Test line Capture line

so many other diseases.

23.2 Applications of antigen-antibody reactionsin Table diseases

detection in COVID-19

immunoelectronmicroscopic

Sample

diagnosis

of

181

The list includes very few diseases. However, antigenantibody reactions may also be applied in diagnosis of

diseases mentioned below (Table 23.2).

(FTA-ABS test) 15. ELISA

HIV infection

16.

ELISA

Hepatitis

17.

ELISA

Rotavirus

18.

Immunochromatographic test

Meningitis

infection

(ICT)

19. Immunochromatographic test (ICT)

HIV infection

Antimicrobial Susceptibility Testing

24

Antimicrobial Susceptibility Tests

IlI.

Molecular Methods

of

1.

test bacterium is inoculated on the central and lower thirds one third and control on upper The

tests.

of

3. Kirby-Bauer disc diffusion method. 4. Epsilometer E test.

•

the plate.

However, in modified Stokes disc diffusion method, the test bacterium is inoculated over the

upper and lower thirds of the plate and control

Minimum inhibitory concentration (MIC).

on central one third. An uninoculated gap

Minimum bactericidal concentration (MBC).

of 2-3 mm wide should be

kept between the standard and test inocula where

Pathogenic

antibiotic discs are applied (Fig. 24.1). bacteria exhibit great strain variations in

susceptibility to antibiotics. It is, therefore, essential to determine the susceptibility of isolates to antibiotics that are

likely to be used in the treatment. Antimicrobial

susceptibility test (AST) is performed only for pathogenic bacteria isolated from specimens and not for the

I.

commensals.

ANTIMICROBIAL

is the best

medium to use for

testing aerobes and facultative anaerobes. Nutrient agar is another alternative medium used. For certain fastidious organisms such as Streptococcus pneumonine, MHA with 5% sheep blood is used.

• Kirby-Bauer disc diffusion method

A.

antibiotic discs are applied. Sensitivity to the drug determined from the inhibition of bacterial growth around the disc.

Hinton agar (MHA)

Diffusion tests Stokes disc diffusion method Dilution tests Broth dilution method Agar

charged with appropriateconcentration of the drugs

test bacterium is inoculated on the medium and The these

The medium should support good overnight growth of both the test and the control organisms. Mueller-

Antimicrobial susceptibility tests are of two types:

B

The 'disc diffusion' method uses filter paper discs

Medium

SUSCEPTIBILITY TESTS A.

be unsuitable for slow growing microbes.

dilution method

• A maximum of six antibiotic discs can be applied 100 mm diameter plate (petridish). The plates are

control, the difference between the two be more than

Intermediate sensitive The

zone size of the test bacterium should be at least difference between the

mm and the

The zone size of the test bacterium is smaller than 2 mm. 2. Kirby-Bauer Disc Diffusion Method Dip a cotton swab into the inoculum.

Inoculate the

Mueller-Hinton agar

streaking the swab three times over

before applying the antibiotic discs, using either sterile forceps or multidisc dispenser. On a plate of 100 mm diameter, six discs may be applied, one in the centre and fivein the periphery (Fig. 24.2). Antibiotic discs should not be placed closer than 24 mm (center to center).

The plates are then incubated at 37°C for 16-18 hours. The zones of complete growth inhibition around each of the discs or

vernier caliper

measured using

then incubated at 37°C for 16-18 hours.

Zone of inhibition

The reporting of results is done by comparing the zones

of inhibition of control and test bacterium. Measure the

zone size i.e. the distance in mm from edge of the disc to the

zone edge. It is interpreted as

Antibiotic disc

follows:

Sensitive The zone of test

bacterium is equal to, or larger than

that of control strain.

Here,theantibioticisallowedtodiffuse through a solid mediumsothat the concentration is highest near thesite of application of the antibiotic disc and decreases with the distance. These tests have, however, been found to

182

are inoculated

in

Fig. 24.2 Kirby-Bauer disc diffusion method

Test strain

Antibiotic disc

The density of the organisms in broth isadjusted to approximately 1.5 10% cfu/mL by comparing

turinditywith that ofd5, on rial baycon amne.th tube.

Control strain

suitable broth

medium (peptone water) and incubated at 35-37°C for 4-6 hours.

Zone of inhibition

Test strain

Control strain Stokes disc diffusion method Fig. 24.1 Stokes

ruler

The diameter of the disc IS

included in this measurement.

medium.

Isolated colonies

plate by the entire

agar surface. Allow 3-5 minutes for the surface of agar to dry

REPORTING

The organisms are isolated in pure culture on a solid

Diffusion Tests

should not

mm.

Inoculum

Disc diffusion tests are most widely used to determine the susceptibility of isolates of pathogenic bacteria to antibiotics which are likely to be used in the treatment.

183

size of test bacterium i smaller than that of

Resistant

Stokes Disc Diffusion Method

: diffusion method.

Antimicrobial Susceptibility Testing If zone

control strain should be at least 3 mm.

those clinically relevant should be tested.

student should be able to learn:

antimicrobial susceptibility

control strain should also be

Similarly, inoculum the case of test strain. prepared as in Discs Antibiotic be prepared in the laboratory or Antibiotic discs may Filter paper discs of

Only

SPECIFIC LEARNING OBJECTIVES

Stokes

1inoculum reducesthesizeofinhibition

for

Automated Systems for Antimicrobial Susceptibility

5.

Too heavy zones.

and not those used Antibiotics should be chosen with discrimination.

Testing

The

even

pirchased commercially. For preparation of discs in the in diameter are used. agents should be used iboratory, pure antimicrobial clinical practice.

PREVIEW

I.

inoculated on the medium by spreading This broth isswabs. with sterile inoculum after overnight incubation gives Theideal semi-confluent growth,

Modified Stokes disc n method

Affusion method and modified Stokes disc diffusion method

Unit III Applied

Microbiology

Kirby-Bauer Disc Diffusion Method Table 24.1 Interpretation Chart used in Diameter of zone of inhibition (in mm) Disc concentration

Antibiotic"

For

Enterobacteriaceae

Intermediate sensitive

Resistant

20-22

$ 19

30 #g

$ 17

15-16 20-22

19

10 pg

2 21

22-25

≤ 14

30 ug

2 23

18-20

$ 21

5 ng

Sensitive

2 23

(1)

•

$ 21

Tetracycline 2.

2 22

11-15 15-18

$ 10

1.25/23.75 pg

≥ 16 2 19

16-18

$ 15

2 19

Enterococcus

Ampicillin Vancomycin

The interpretation of zone size

21-22

$ 20

30 pg

2 17 2 17 : 23

15-16

§ 14

30 ng

The antibiotic concentration at which ellipse edge intersects the E-test strip is taken as MIC value of

into sensitive,

intermediate or resistant is based on interpretation

that antibiotic.

chart of Clinical and Laboratory Standards

Institute (CLSI) guidelines (Table 24.1).

disc diffusion methods, as described above,

done after the the

pathogenic bacteria are isolated

clinical specimens. When results

are

the specimen, the 'primary disc diffusion

Zone of Inhibition

test' may

MIC point

The results of the primary test should be verified by testing the isolates

subsequently. This direct test is of no use when mixed growth of different bacteria is suspected in the specimen e.g. sputum, stool etc.

•

modification sensitivity test. It uses an absorbent strip with a known gradient of is a recent

nutrient agar plate without any antimicrobial agent. at 37°C for 16-18 hours and plates these Incubate examine them for growth, if any.

susceptibility. The commercially available panels containing antibiotic solutions in serial dilutions used for determining the antimicrobial susceptibility of

which The minimum

of the agar diffusion

antibiotic concentration along its length. The strip is placed on the agar plate inoculated with the test organism. The antibiotic diffuses into the medium.

MIC = 2 mg/l (a)

subculture, show growth, drug that fails for that test strain (Fig. 24.4). MBC of the drug while MBC growth bacterial MIC inhibits the

of slow

of

standardised. small degrees of resistance are to be

demonstrated. (v)

When the therapeutic dose accurately as endocarditis.

in

the

Antibiotic Concentration (/mL)

methods and are based on microbroth dilution. Most

systems are computer assisted and contain softwares to analyse the growth rate and determine the antimicrobial

the organism. Some of the automated systems available include:

VITEK system (biomerieux): Besides

1.

For testing antimicrobial susceptibilities bacteria for which disc diffusion tests

(iv) When

TESTING

antimicrobial susceptibility testing, it also

kills

the bacterium. The uses of determination of MIC are:

testing antimicrobial sensitivities

ANTIMICROBIAL SUSCEPTIBILITY

to be regulated

treatment of bacterial

2.

identifies the organism. Details of VITEK system have been described in Chapter 22. Pheonix system (Becton Dickinson)

I11. MOLECULAR METHODS

Polymerase chain reaction (PCR) is the most commonly used molecular method to amplify the specific drug resistance gene. For MRSA detection, mecA gene can

be amplified and identified. Van gene can be detected

for vancomycin resistant Staph. aureus (VRSA) vancomycin resistant Enterococcus (VRE) by PCR. Resistant to rifampicin can be detected by CBNAAT (commercially available as Gene Xpert). CBNAAT is

cartridge based nucleic acid amplification test.

0.25 pg

64 pg

Fig. 24.3 (a) Epsilometer or E-test on the agar plate b)

Details of E-test strip

Mueller Hinton broth

B.

Dilution Tests

Antibiotics are serially diluted and each dilution i tested with

Turbidity growth)

No growth

the organism for antimicrobial test. The

minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) may be determined

by these tests.

Depending upon whether the dilutions of antibiotics

The minimum inhibitory concentration (MIC) 1S e made in broth or agar, these are two types of dilution recorded as the lowest concentration of the gradient tests i.e. broth dilution method and agar dilution which inhibits the growth of the organism.

bactericidal concentration (MBC) IS

ofisthe The tube containing the lowest concentration the on

growth

the antibiotic discs are applied.

It

•

Bacterial

be performed. Here, the clinical specimen IS directly inoculated uniformly on the surface of plate and

plate containing an antibiotic dilution. The

Several automated systems are available. Theseare rapid

E-test strip

required

4. Epsilometer or E-Test Epsilometer or E-test is antimicrobial sensitivity test detect minimum inhibitory concentration (MIC) of antibiotic (Fig. 24.3).

on

lowest concentration of the drug at there is no visible growth(Fig. 24.4).

growing bacteria such as tubercle bacilli.

are

urgently and single pathogenic bacterium is suspected in

into plates. Many strains can be inoculated and poured each

Incubate

tests.

from

method.

185

Dilution Method

BErtray.e

(ii) For

Primary Disc Diffusion Test

3.

Agar

Serialdilutionsofthe drug are preparedin molten agar

plate)

() For confirmation of the results of antimicrobial susceptibility tests obtained by disc diffusion

Control strains Staph. aureus, Esch. coli, Ps. aeruginosa etc. should be tested each time when a new batch of discs or agar is used.

The

Microbroth dilution (in

2.

of the drug in Mueller-Hinton broth test strains are spot inoculated This method is more Serialdilutions in tubes and a standardised suspension of convenient when several strains are to be tested at the taken are time. same known An organism bacterium inoculated. the test also be titrated. II. AUTOMATED SYSTEMS FOR sensitivityatshould 37°C for 16-18 hours.

For Gram positive bacteria Staph. aureus

Antimicrobial Susceptibility Testing

Method

Broth Dilution is of two types dilution method in tubes) and BrothMacrobroth dilution(performed microtitre

MBC = 32 pg/mL subculture

No growth

Growth

Fig. 24.4 Broth dilution methods

showing MIC and MBC

Sterilisation and Disinfection (iii)

for killing insects by using rogBuchniqueinused form of aerosol. 11 is '

25

Sterilisation and Disinfection

fine

spray

fungi and viruses are killed

pore sterilisation/Disinfection

by moist heat.

of materials, instruments used in

(iv) Type of

surilish and diagnostic procedures. For media and reagents

used

laboratory

I. Introduction: Some Definitions

IV.

and

Practice

Spaulding Classification System

to ensure safety

are some of the organic substances.

(a) Dry heat e.g. red heat, flaming,

Inoculating wires or loops, tips of forceps and needles are held in the flame of a Bunsen burner till they become

incineration,

red hot

hot-air oven

() Moist heat e.g. pasteurisation, tyndallisation,

SPECIFIC LEARNING OBJECTIVES

5. Types of radiations and their Uses of gases as chemical method of sterilisation and role of ethylene oxide in sterilisation of disposable items.

to become red hot.

Chemical methods

5.

INTRODUCTION: SOME DEFINITIONS The agents which achieve criteria of Microorganisms are responsible for contamination are called disinfectants. Depending upon

disinfection

the efficacy of disinfectant, they are classified as high-level and infection. They are present all around. The aim of sterilisation and disinfection is remove or destroy disinfectants, intermediate-level disinfectants and lowthem from materials or level disinfectants. These are described later from surfaces. Thus the in this sterilisation and disinfection procedures are of prime chapter.

associated infections.

Antiseptics

Sterilisation

Antiseptics are chemical disinfectants which can be

both in the vegetative

Sterilisation process has microbicidal action against

The technique by which, the occurrence of

infection

bacteria (including bacterial spores and tubercle into an uninfected tissue is prevented. bacilli), all viruses and fungi. The agents which can Decontamination achieve this criteria are called sterilants. It is the process of neutralisation or removal of germs, Disinfection I means the destruction of all pathogens or organisms

capable of producing infections spores. All organisms may not be killed but the number is reduced to a level that is no longer harmful

dangerous substances

object or person.

or

radioactivity from an area,

is the process

of disinfecting

of certain chemicals.

for

acid, plasma sterilisation

(iv) Hot Air Oven

It is the most widely used method of sterilisation by dry is electrically heated and is fitted with

heat. The

ensure adequate and even distribution of hot

a

air

the most reliable

and commonly employed

the method of method of sterilisation. It should be

in the chamber

(Figs. 25.1 and 25.2).

is also fitted

with a thermostat that maintains the chamber air at a chosen temperature.

choice unless contraindicated. Two types of heat used Temperature and are, dry heat and

with this

disposal of biomedical waste.

Physical Methods

1. Heat

animal carcasses

method. It is also one of the methods for treatment and

compound oxide (ETO) 7. Gases e.g. ethylene A.

this purpose. Soiled dressings,

bedding and pathological materials are dealt

peroxide,

Surface active agents e.g. quaternary ammonium

time

is required for 160°C for two hours (holding time) hour). was 160°C for

moist heat.

sterilisation (previously

Principle

organisms by denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated levels of electrolytes.

(1) Dry heat kills the

time However, alternative temperatures and holding 30 minutes. one

include 170°C

hour and 180°C

for

However, the possibility of DNA damage also incriminated as one of the mechanisms of 1s

Shelf

inactivations of microbes. (ii) Moist heat kills the microorganisms by

Door

Factors influencing area with the fumes

(1) Nature of

(11)

heat: Dry heat or moist heat

Temperature and duration: for

186

by burning. Instrument named incinerator may be used

denaturation and coagulation of proteins.

but not necessarily Fumigation

to health.

b.

By this method, infective material is reduced to ashes

hypochlorite, povidone

Oxidising agents e.g. hydrogen

Heat

applied safely to living tissues and are used to prevent infection byinhibiting the growth of microorganisms.

(iii) Incineration

Alcohols e.g. ethyl alcohol, isopropyl alcohol 2. Aldehydes e.g. glutaraldehyde 3. Phenols e.g. lysol, chlorhexidine iodine

It is a process by which an article, surface or medium is

Glass slides, scalpels and mouths of culture tubes are passed through bunsen flame without allowing them

radiation Radiation e.g. gamma

4. Halogens e.g. sodium

7. Methods used for testing of disinfectants.

importance in preventing the transmission of health care

(ii) Flaming

autoclave

Filtration e.g. HEPA filters, ULPA filters

1. Difference between sterilisation and disinfection. 2. List of physical methods and chemical methods of sterilisation. 3. Principle, temperature, time period and uses of hot-air oven, autoclave, pasteurisation, inspisation and tyndallisat. 4. Uses of filtration a types of filters.

all

DRY HEAT STERILISATION (i) Red heat

1. Heat

The student should be able to learn:

made free of all microorganisms or spore form.

STERILISATION

Physical

VI.

Surgical

OF

of organic

lethal action of heat. Materials containing organic substances require more time for sterilisation. Proteins, sugars, fats and starch

methods

V. Testing of Disinfectants Central Sterile Supply Department

Methods of Sterilisation III. Sterilisation Methods in Clinical

METHODS

material: A high content

spores against the

from

PREVIEW

30 minutes at 65°C

substances tendto protect the vegetative form and

in the microbiology

and drug manufacturing , in food contaminating organisms.

187

Characteristic of microorganisms and spores present: Bacterial spores are killed by moist heat at 121°C for 15 minutes. Most vegetative bacteria,

sterilisation

iS

The time required

inversely proportional to

temperature to which organisms are exposed.

Thermostat

Fig, 25.1 Hotair oven-Diagramnatic

Unit III Applied Microbiology

holder method (63°C

for 30

hash method (PC for 20

minutes)and

followed is cooling quickly to 13°C orseconds lower)are nonsporing pathogens such as used. Af brucellae and

salmonellae

are

mycobacteria killed

Coxiella burnetii which being relativelyexcept sistant may survive in holder method hea (b)

Inspissation

Inspissation

(fractional

is

sterilisation);

used for some serum

egg based media whichmay be destroyed atorhigher temperature. The article sterilised by heating for half at 80-85°C temperature an hour daily

consecutive days. This process sterilisation is called inspissation. The instrument

Fig. 25.2 Hot air oven

on

Uses

three

used is called inspissator.

It is used for

sterilisation of (i) Glasswares like glass syringes,

petridishes, flasks,

pipettes and test tubes.

(11)

Surgical instruments like scalpels, scissors, forceps etc.

(ili)

Chemicals such

as

The first exposure

sulphonamide powders etc.

the intervalsinto the remaining sports between the heatings forms which

It should not be overloaded. material should be arranged in a which allows free circulation of air.

(i)

should

be

wrapped

the oven.

Egg based media: Lowenstein-Jensen medium and

in special vaccine baths at 60°C for one hour

Serum or body fluids can be sterilised by heating

for one hour at 56°C in a water bath on several

successive days.

(vii) The oven must be allowed to cool for two hours

be used for glass syringes and rubber stoppers. is

spores of Bacillus

(b) Tyndallisation or Intermittent sterilisation:

kept inside the oven. These spores should

be destroyed if the sterilisation is proper. Thermocouples may also be used. (iii) Browne's tube with green spot is available. After proper sterilisation a green colour is produced

Steam at 100°C for 20 minutes on three successive days is used.

This is known as tyndallisation or intermittent sterilisation.

(11)

(after two hours at 160°C).

MOIST HEAT STERILISATION This method of sterilisation may be temperatures as follows. (1) At

into vegetative forms which subsequent heating.

are killed on

Uses

temperature below 100°C

Temperature below

kills all the vegetative forms, and in

the intervals between the heatings the remaining spores

(ii) At a temperature of 100°C At a temperature above 100°C

It

is used for sterilisation of egg, serum or

containing media, which

100° C

(a) Pasteurisation of milk: Two

The first exposure

types of method,

Temperature above 100°€

(under pressure) vapour pressure boils when its

equals that of

Water

100°C but pressure water raised. At normal inside a closed vessel increases, the

when pressure

applied in autoclave and pressure

principle has cooker.

Fig. 25.3 Vertical autoclave

AUTOCLAVE

Safety valve

Discharge tap

Principle killing or saturated steam has a better Steamabove 100°C dry heat. Bacteria are more susceptible than power bacterialprotein coagulates rapidly. moist heat as easily. can penetrate porous material Saturated steam steam comes into contact with a cooler surface

Chamber

its latent heat to

condenses to water and liberates 1600 mL of steam that surface, for example,

ondince tns and or tines coun preain, volume

tray

Water

at 100°C

Heating

mL of

calories of heat. The 100°C and releases 518 the in sucks more steam to

Fig. 25.4 Autoclave (inner view)

reduction large

hame site and the process continues till theThetemperature to that of raised of the article is water produces

steam.

The lid is closed with the discharge tap

condensed

•

Safety valve is adjusted to the required pressure. mixture is After boiling of water, the steam and air allowed to escape till all the air has been displaced.

cooker or boiler. lid and

electric

Chamber

I consists of a vertical or horizontal cylinder of gunmetal of stainless steel in a supporting iron case. On this basis, and autoclaves are of two types: vertical autoclave horizontal autoclave. Lid

and rendered air The lid is fastened by screw clamps lid bears a discharge tight by an asbestos washer. The

temperature of autoclave.

are

The autoclave can be used

damaged

at

sugar

higher

valve.

Heating is generally done

to generate free steam.

The valves are left open so that pressure does not

rise

The

been

by

electricity to produce

and is

and the The cylinder is filled with sufficient water material to be sterilised is placed in the chamber.

displaced.

steam pressure rises inside and when

The

reaches

the desired set level 115 pounds (lbs) per square inch),

the excess steam escapes. the safety valve opens and holding period (15 minutes) is

From this point, the counted.

is stopped and pher the holding period, the heating the pressure gauge autoclave allowed to cool till

is

equal to the

Beas insertierysihanaeiesed indicates that the

pressure inside

atmospheric pressure.

now opened and

to enter

steam. The steam circulates within the jacket inner chamber supplied under high pressure to the where materials are kept for sterilisation (Figs. 25.3 and 25.4).

Procedure

This can be tested by passing the escaping steam-air mixture into pail of water through rubber tubing, pail it When the air bubbles stop coming in the indicates that all the air has closed. discharge tap is now

heater.

Heating heater

open.

The electric heater is turned

moist conditions for killing the microbes

present. Autoclave is a modified pressure It comprises of three parts: chamber,

Pressure gauge

Perforated

and a safety tap for air and steam, a pressure gauge

Principle

germinate used at different

(iii)

(i) At a

not recommended for the sterilisation of

instruments used for surgical procedures.

subtilis subsp. niger(NCTC 10075 or ATCC 9372) are

Boiling: Boiling for 10 to 30 minutes may kill the vegetative forms but many spores withstand boiling for considerable time. When better methods are not available, boiling may most of

before opening the doors, since the glasswares may crack by sudden cooling.

inside above torise At (ii) a

Sterilisation and Disinfection

reventsthetemperature

Components of autoclave

(ii) At a Temperature of 100°C (a)

and thus

When

(ii) Serum based media: Loeffler's serum slope (c) Vaccine bath: Bacterial vaccines are sterilised

in

(vi) Rubber materials (except silicone rubber) or any inflammable material should not be kept inside

Sterilisation control (i) Biological indicator: The

killed on

Dorset's egg medium

manner

plugs.

Petridishes and pipettes

are

Inspissation is used for sterilisation of:

(ii) Material to be sterilised should be perfectly dry. (iv) Test tubes, flasks etc. should be fitted with cotton craft paper.

vegetative

germinate Uses

(ii) The

(v)

kills all the vegetative forms, and in

liquid paraffin, fats, subsequent heating.

Precautions (i)

Principle

the instrument 100°C.

removed. the sterilised material on the pressure cooker works Note: The domestic

same principle and may be used

for sterilisation of smal

articles in clinics.

Sterilisation conditions 121°C Temperature

Chamber pressure Holding time

: 15 lb per square 15 minutes

inch

190

Unit III Applied Microbiology

These conditions are generally used, however, sterilisation can also be done higher temperatures, at 126°C (20 lbs/square inch) for 10 minutes or 133°C (30 lbs/square inch) for 3 minutes.

pass

through filter, therefore, filtered preparations are

safe

(1)

surgical instruments,

dental instruments, anaesthetic equipments rubber material, gowns, dressing, gloves etc.

Membrane filters: Membrane lters are made up of cellulose esters These are routinely used in water analysis.

sterility testing,

(ili)

hot air

must be

allowed to

than that of

(ii) High

(11)

chamber.

(ili)

or larger. This is also used to clean air I laminar

(i) Biological indicator: Spores of Geobacillus

flow system and biological safety cabinets.

are used as the test organism. This organism has an optimum

(iv) Surgical

growth temperature of 55-60°C and its spores

are killed in 12 minutes at 121°C. An envelope

and incubated at 55*C for five days. Spores are

sterilising conditions of autoclave are proper. This is the bestindicator for testing

destroyed

effectiveness of sterilisation.

Chemical indicators: Browne's

tube contains

red solution which turns green, when exposed

to temperature of 121°C for 15 minutes

autoclave.

in

(1V) Autoclave tapes 2.

and

for protection from air-borne infections.

Sterilisation conditions Serratia marcescens and Brevundimonas diminuta

two bacteria used as sterilisation control to test pore size of a filter.

3.

are

the

sterilisation:

cosmic rays.

to be found.

IS

The following factors influence the potency of a (i) concentration (11) time of action

used as

antiseptics

antiseptic or disinfectant

should

(i) have wide spectrum of activity and be effective

against all microorganisms including bacteria (both vegetative protozoa and fungi,

spore forms), viruses,

act in the presence of organic matters, have high penetration power and quick action,

(iv) be stable and effective

in acidic

as well as in

(iv) temperature

(v) nature of organism and (vi) presence of organic matter. Disinfectants can be divided into three groups

here is no appreciable increase in temperature.

Uses

High level disinfectants. 2. Intermediate level disinfectants. 3. Low level disinfectants.

High level disinfectants The effectiveness of high level disinfectants may be

equivalent to that of sterilisation. These disinfectants are used for certain

types of endoscopes, cystoscopes

and surgical instruments with plastic components which

cannot withstand the sterilisation procedures such as

autoclaving. The high level

disinfectants include

glutaraldehyde, hydrogen peroxide, peracetic acid and ethylene oxide.

Table 25.1 Disinfectants Used in Hospitals

Chemical methods

Activity against

Level of

bacteria bacterial

viruses

fungi

Aldehydes e

High level disinfectants

Advantages

glutaraldehyde, ortho-

phthalaldehyde Oxidising agents e.g.

hydrogen peroxide, peracetic

acid, plasma sterilisation*

3. Ethylene oxide*

Sodium hypochlorite"

Alcohols e.g, ethyl alcohol, isopropyl alcohol

cresols, lysol 2. Phenols 3. Povidine-iodine

Intermediate level disinfectants

. Quaternary ammonium

Low level disinfectants

(except

(except n

tubercle

enveloped

bacilli)

viruses)

These chemical methods act as sterilants. Effective

etc.

(i) Rapid method

(Table

25.1):

They have very

all cells including vegetative bacteria and spores. They damage DNA by various mechanisms This method is also known as cold sterilisation because

The pore size of filter is around 0.75 pm in diameter which allows viruses and mycoplasma to

(x) be easily available and cheap. The disinfectant which possesses all the above criteria

high penetrating power. They are highly lethalto

Limitation retains bacteria but

disinfectants. An ideal

(ii)

(ix) be safe and easy to use, and

spores

are used for

Gamma radiations from a Cobalt 60 source are commercially used for sterilisation of disposable items such as plastic syringes, swabs, culture plates, cannulas, catheters

hospitals, especially dialysis water.

variety of chemical agents

(ili)

(vi) be compatible with other disinfectants, (vii) not cause local irritation, (vii) not be toxic if absorbed into circulation,

alkaline conditions,

ionising and non-ionising. (1) lonising radiations: Ionising radiations include

Uses of filtration

in

A

Radiations

This method of sterilisation is useful for substances which get damaged by heat process e.g. sera, sugars, antibiotic solutions etc.

(in) For bacteriological examination of water

B. Chemical Methods

disinfection

Two types of radiations

gamma rays and

marked

theatres. Most vegetative bacteria are susceptible to UV radiation but spores are highly resistant. Susceptibility of viruses is variable.

N-95

microorganisms depending upon their poresize. Theseare used bymedical personnel, especially

Filtration

(1) To sterilise sera, sugars and antibiotic solutions.

layered)

minutes

enclosed areas such as bacteriological laboratory, inoculation hoods, laminar flow and operation

and

191

It

replication. UV radiation is used for disinfecting

These are used to purify inhaled air. They remove

sterilisation. After sterilisation is over the strip

suitable recovery medium

mask (three

respirator:

containing a filter paper strip impregnated with 10° sporesis placed inside the autoclave during

(111)

Ultra-low particulate air (ULPA) filter is another filter which can remove particles of 0.12 pm

Sterilisation and Disinfection (v) not corrode metals.

sterilisation of syringes and disinfectant

acts by denaturation of DNA bacterial protein and interference with

(HEPA) filters:

all

directly by potentiometer.

the

air

rapid mass

nm of 240 to 280 bactericidal activity.

to deliver clean bacteria.

larger.

stearothermophilus

for

with wavelength catheters. Ultravioletfor radiation 30 has

HEPA filters can remove particles of 0.3 um or

Thermocouple: It is to record the temperature

is inoculated into

efficiency particulate

These include infrared Non-ionising radiations: (UV) radiations. Infraredis used and ultraviolet

filtrationin laminarair flow system and biological safety cabinets in microbiology laboratories.

the

Sterilisation control (1)

(11)

used in air

Materials should be arranged in such a manner free circulation of steam inside

Nitrocellulose

air to a cubicle or a room. High efficiency free particulate air (HEPA) filters

than that of pure steam.

as to ensure

preparation of

bacteria.

These filters are used

escape from the

chamber as temperature of air-steam mixture lower

use.

sterilisation Control has been used for testingthe efficacy Bacillus pumilis radiations. ofionising

0.015 to 12 um. The 0.22 um filter is the most commonly used because the pore size is smaller

Treatment of biomedical waste. The air

the

(Millipore) membrane filters are widely used Membrane filters e available in pore sizes of

Precautions (1)

and for

solutions for parenteral

(1i) It is particularly useful for materials which cannot

withstand the higher temperature of

(ini)

Types of filters

Uses

(i) To sterilise culture media,

for clinical use.

power (it can sterilise materials, ) High penetrating through outer packages and wrappings) is Absence of heat (temperature not raised).

Not effective may

bacteria

not be effective.

Include all vegetative bacteria including tubercle bacili.

viruses : Include both enveloped and non-enveloped viruses.

compound

192

Unit IlI Applied Microbiology

(iv) For killing of bacterial cultures and suspensions

Intermediate level disinfectants Intermediate level disinfectants may not be effective against bacterial spores, hence these disinfectants are for instruments (e.g. laryngoscopes, fiberoptic endoscopes) where contamination with spores and other

used

highly resistant organisms is unlikely. The intermediate

level disinfectants include alcohols, iodophores

phenolic compounds.

(v)

For destroying anthrax

spores in hair and

described Formalin gas has been

is effective against bacteria (including M tuberculosis), fungi and viruses (includinghuman It

Many organisms can survive on exposure to low level viruses, hepatitis used for items immunodeficiency disinfectants. These disinfectants virus and enteroviruses). It also kills spores. less toxic and which con in contact with the patients but they do not to the eyes and skin than ormaldehyde. It is irritant penetrate into tissue. Stethoscopes, electrocardiogram

electrodes etc. are examples of such items. The low level rapid, broad spectrum and high level disinfectant It can act in the presence of organic matter and disinfectants include quaternary ammonium compound does not damage plastics.

Alcohols

1.

It is used as 2% buffered solution. It disinfects articles within 20 minutes but may

•

Ethyl alcohol and isopropyl alcohol are the most

•

require longer time to kill spores. It is available commercially as 'cidex'.

frequently used. They act by denaturing bacterial proteins. They rapidly kill bacteria including tubercle bacilli but they have sporicidal or virucidal activity. However, human immunodeficiency virus (HIV) is

It can be used for

lenses.

Glutaraldehyde is available in inactive form and thus has to be activated by alkalization before use

susceptible to 70% ethyl alcohol and 70% isopropyl alcohol in the absence of organic matter. To be effective, they should be used at centration f 60-70 per cent in water.

a con-

After activation, it remains active for

sterilisation of semicritical items such cystoscopes, endoscopes and bronchoscope.

(i) For

(ii)

volatile.

as

metal

instruments.

Alcohol based handrub.

Disinfection of non-critical instruments e.g. thermometers, stethoscopes. Disinfection of rubber stoppers medication vials or vaccine bottles.

•

Disinfection of laboratory bench

•

Used as skin antiseptics.

of multi-dose

area.

Methyl alcohol is effective against fungal spores and is used for treating cabinets affected by them. Methyl alcohol vapour is toxic and inflammable.

Ortho-phthalaldehyde(OPA) is a high level disinfectant. more stable during storage

and more

rapidly

mycobactericidal than glutaraldehyde. 0.5% OPA slowly sporicidal. Unlike glutaraldehyde, it does not require activation. OPA vapours may irritate the respiratory tract and eyes, therefore, it must be handled with appropriate safety precautions. It can be used for materials where glutaraldehyde is used such as endoscopes. This new disinfectant may be useful for

glutaraldehyde resistant mycobacteria.

2. Aldehydes

FORMALDEHYDE

3. Phenols

markedly bactericidal, sporicidal and virucidal. iS used both as aqueous solution and in gaseous form.

Lister, the

It is

(i)

Preservation of tissue for histological

examination. (li) To sterilise

bacterial vaccines.

prepare toxoid from toxin.

(ii) Llorhexidine and cetrimide) is widely used in Savlon taure-operative disinfection of skin, as bladder wounds, They are

father of antiseptic surgery, first introduced

The lethal effect of phenols is due to cell membrane

damage, thus releasing cell contents

and

causing

lysis.

Phenol (1%) has bactericidal action.

is readily absorbed by skin and causes toxicity. Certain phenol It

all the chlorinecompounds is

elc. It is bactericidal at a high dilution.

The activity of chlorineis markedly influenced by

the presence of organic matter Sodium hypochlorite (1%) solution is the

most

more

widely used for human

active against Gram positive than Gram negative immunodeficiency virus (HIV) infected material The with no action against tubercle bacilli or spores hypochlorites have a bactericidal, fungicidal, virucidal

irrigant

hacterla

have very little activity against viruses. They have

and

rood fungicidal activity.

and rapidly sporicidal action. Hypochlorite solution decays rapidly and should be prepared daily

Itshould not beused in the presence of formaldehyde

the most

used biguanide disinfectant. It is bactericidal, sporostatic.

It is used in

as one of the reaction products is found carcinogenic. Chloramines are used as antiseptics for

as skin disinfectant

dressing wounds.

ind for hand hygiene. Hand rub (0.5%) is used for hand

Uses

1. Chlorine gas

hygiene.

IS

used in

swimming pools.

Uses

2.

before surgery.

water supplies

and

Sodium hypochlorite (1%) is used as disinfectant in laboratory for disinfecting blood and other specimens. It is also used for disinfecting blood

spillage area.

Advantages 1.

2.

Broad spectrum efficacy.

lodine in alcoholic and aqueous solutions is used as a skin disinfectant. It is actively bactericidal, with

Longer action than alcohol based handrub.

Disadvantages Its activity is reduced in the

moderate action against spores. It is active against M. tuberculosis and viruses. Like chlorine, it is also presence of organic inactivated by organic matter. Compounds of iodine

Surface active agents (povidone) known

with

matter.

claimed to be more active than the iodophores, aqueous or alcoholic solutions of iodine. lodophores It is less dettol. are toxic and water stable, non-staining and release iodine less soluble, active ingredient slowly. They are used as preoperative antiseptics as irritant. It is readily inactivated by presence of organic

(iii) Chloroxylenol

well as disinfectants. Betadine (povidone-iodine) is one example of commonly used iodophores.

matter. It is inactive against Pseudomonas.

(iv) Hexachlorophane

It is more active against Gram positive than Gram

negative bacteria. It is applied on skin as prophylaxis

use of phenol (carbolic acid) in surgery (1867). Phenols are produced by distillation of coal tar between The formalin (40% aqueous solution of formaldehyde) temperatures of 170°C and 270°C. is commercially available and the most widely used. It is active against amino group in the protein molecule. Mechanism of Action Uses

against

active

and chloramine are also

dueto release offree chlorine. When hypochforinds1s added to water, the chlorinereactswith waterto Sore acid which is a strong oxidising agent hypochlorous and effective disinfectant.

3. Low irritation.

ORTHO-PHTHALALDEHYDE It is

They are

Mechanism of Action The disinfection action

this used in handwash solutions, mouth wash, body wash solutions and as skin disinfectant. It is also used

To sterilise plastic endotracheal tubes, face masks,

corrugated rubber anaesthetic tubes and

Uses

©

14 days.

Uses

Isopropyl alcohol is preferred to ethyl alcohol

it is a better fat solvent, more bactericidal and less

of organic matter. fooT ated by thewide presence range of organisms.

widely

delicate instruments having

form)

used.

is a solution of cresols in soap It is most commonly Lysol 15 " sterilisation of infected glasswares, cleaning used for sinfection of excreta. They are not readily

GLUTARALDEHYDE

193

theform of bleaching powder(powder form),sndiun hypochlorite (liquid

(i)

phase disinfectants"

and chlorhexidine.

Sterilisation and Disinfection

chloroxylenol and

Wool

under

and Glutaraldehyde i the most commonly used high-lever disinfectant in hospitals. It is accepted as sterilant

Low level disinfectants

,cresol, chlorhexidine,

like

derivatives phane are commonly used as antiseptics

Uses

(i) As antiseptics

Wound cleaning: 5% topical

against staphylococcal infections. It is bacteriostatic at is potentially toxic and should very high dilutions. be used

Skin preparation during surgery: 10% is used. Mouth wash: 1% is used as antiseptic.

with care.

4. Halogens Chlorine and

iodine

two commonly

used

disinfectants. These are bactericidal and are effective against sporing bacteria and viruses. Chlorine

Chlorine is used in water supplies, swimming pools, in food and dairy industries. Chlorine compounds

solution or

ointment.

(in) As disinfectant

For medical instruments e.g. thermometers

5.

Oxidising Agents

(i) Hydrogen peroxide against • Hydrogen peroxide H.O,) is effective 3-6%, while most organisms at concentration

Sterilisation and Disinfection Unit III it kills all

Applied Microbiology

organisms including spores at higher

concentration (10-25%). Mode of action

is by

(d) Biological indicator spores Bacillus atrophaeuscontrol

decomposition c H,O,, These free radicals are the active ingredient in the disinfection process

(e) Uses

are used to Plasma sterilisers

H.O, is used to disinfect contact lenses ventilators, •

surgical prostheses and plastic implants. It

is

used for high level disinfection and

sterilisation.

Vaporized H.O, is used for plasma sterilisation. oxidising agent. It is one of the high level disinfectants. It is sporicidal at the concentration of less

materiale

1. Non-hollow items Electrocautery instruments

2.

Hollow items

the presence of organic matter.

Laryngoscopes

•

The end products (acetic acid and oxygen) of this

3.

agent are non-toxic.

also used

in plasma sterilisation procedure.

It is also used for sterilising endoscopes.

sterilisation as there

is

no

non-toxic and

Thus it is

4. Low

minutes for one

temperature: Useful for heatlabile items.

(g) Disadvantages

Liquids, powders and strong absorbers can

unstable state of matter. Plasma steriliser is & special instrument used to create the plasma state.

be sterilised.

2. It

not

requires specific synthetic packaging of the

Sterilisation chamber is relatively smaller

process called oxidation. The

turns the molecules of hydrogen peroxide

6.

Surface Active Agents

Into Thei radicals. awhich are highly unstable. These which alter energy relationships at interiocs Substances stable producing return to a

state, come in contact with the microorganisms in

1 reduction of surface tension, are known as

the surface active agents or surfactants.

load and effectively destroy the components of these microorganisms, such as enzymes and nucleic acids.

Therefore it distrupts the metabolism of microorganisms Quaternary ammonium compounds and eventually kills them.

are the most

surface active agents. Although these

species are more susceptible. The idliquid hvd rnren into the sterilizer. organisms, arns tit more suct They They have noGram aicthiopositive n on spore inserted is heatedisup H,O,perside in order to turn into

H,O, gas is now heated to an even higher to turn it into plasma. The plasma dispersed inside the steriliser chamber so that all gas. The

temperature

microorganisms are oxidised and killed.

are

Glasswares: syringes,

Hot-air oven

petridishes, test tubes, flasks, universal container, oily fluids (Paraffin)

Disposable syringes and other disposable items

3.

more active against viruses with lipid

envelopes

e.g. rabies virus) and much less against non-enveloped viruses (e.g. enteroviruses).

The

Uses

o Itis specially used for sterilising plastic and rubber

articles, respirators, heart-lung machines, sutures, dental equipments and clothing.

(ii)

commercially used to

It is

plastic

Toxin, serum, sugar and antibiotic solutions

sterilise disposable

Infective material

hence

(iv)

to human beings, including (1) It is potentially toxic

mutagenicity and carcinogenicity. It is highly inflammable.

(in) It forms explosive mixture when its concentration is greater than 3 per cent.

common compounds

are acetyl trimethyl ammonium bromide (cetavalon o cetrimide) and benzalkonium chloride The activity of

its explosive tendency can be eliminated.

(iv) It is irritant, and personnel working with it have to take precautions. (v) It takes 12 to 14 hours

of

more for sterilisation

process.

(vi)

10.

Rubber, plastic and

Tincture lodine, ethanol, savion

Glutaraldehyde

polythene tubes

Table 25.3 Recommended Concentrations of

Disadvantages

(ii)

Incineration

animal carcasses Skin

used

for commercial purposes. ETO is non corrosive.

like

soiled dressings, beddings,

sterilise

withstand high temperature of autoclave. be hence (iii) Capacity of chamber is large

Filtration

Pasteurisation

Milk 8.

(i) High penetrating power prepackaged materials.

Tyndallisation

serum, egg 6.

syringes, petridishes etc.

Advantages

Ethylene oxide Gamma radiations

Autoclaving

Culture media

5. Culture media containing

By mixing it with inert gases such as carbon dioxide,

compounds are bactericidal for a wide range of

(c) Procedure

2.

Red heat

reacts with

in

Quaternary ammonium compounds

important

microorganismsincluding viruses

Methods

Inoculating wires or loops

(i) Useful for sterilisation of articles which cannot

load.

(b) Principle

Materials

the widely used

Mechanism of Action and the amino, carboxyl, hydoxyl t acts by alkylating

safely

also safe for

sterilisation while ethylene oxide can take 14 hours. cycle

It is an

Plasma sterilises by

are

environment.

3. It takes 3545

one of

Disinfection

Some Important Materials

in sulphydryl groups DNA and RNA.

In

evaporate into the air.

increase in temperature

during the process Hydrogen peroxide is used for sterilisation. Plasma is the fourth state of matter (solid, liquid, gas and plasma). In nature, plasma is widespread in outer space. Plasma is created when : gas is heated sufficiently or exposed to a strong electromagnetic field.

Table 25.2 Methods of Sterilisation

protein molecules. In addition,

and oxygen which

Plasma sterilisation is an example of low temperature

Recommended concentrations of some commonly used disinfectants are given in Table 25.3.

penetrates

plasma

contrast ethylene oxide and formaldehyde, both are toxic chemicals At the end, the plasma is converted into water

(a) Introduction

Methods of sterilisation and disinfection of some important materials are shown in Table 25.2.

aralias eighty trai.neee is

ammonium

compound are now used for operation theaters.

intimicrobial the instrument or thesurfaceisfree rettef actionwhen disinfectants are applied. Detergentscan hip beforethe cleaning.

of

commercially

and is thus safe.

copper, bronze and brass.

methods. Glutaraldehyde, quaternary

strong detergent but weak Most sinfectants have a

properties.

It is a highly penetrating gas and it readily andspores.some plastics.

1.

Plasma sterilisation

Common

This was widely employed for fumigation of operation

theatres, wards laboratories etc. However, this method is no longer preferred and is being replaced by other

Pare

(f) Advantages

Disadvantage is that it may corrode steel, iron,

Cleaningagents soaps, have

195

Formaldehyde gas

activitisreducedinthe thesecompounds Antibacterial matter thern organic incivates presence of

(ETO) Ethylene oxide (ETO) oxide Ethylene

and their blades

Fiber optic light cables Endoscopes: rigids and flexible endoscopes. Sterrad 100 available steriliser.

alkaline pH. Acid

7. Gases

Surgical power drills

in

at an

beusedfor

Laser probes

than

It is a more potent germicidal agent than hydrogen peroxide.

high heat

sterilise the

Defibrilator paddles Dopplers

•

(iti)

biological

follows:

1%.

•

as

humidity of

Peracetic acid is

It is effective

used

liberation of free hydroxyl indicator for quality

radical on

•

is greatest

High cost of instrument and consumables

Sterilisation control

biological control for

as Bacillus atrophaeus is testing of ethylene oxide sterilisers,

Some

Disinfectants

Concentration

Disinfectant

Ethyl alcohol

Glutaraldehyde Lysol

Savion (chlorhexidine and Dettol (chloroxylenol)

2% 2.5%

2%, 5%

4%

gm in one Bleaching powder (calcium 14 water hypochlorite)

Sodium hypochlorite

1%, 0.1%

Betadine (lodophore)

2%

196

Unit INI

Sterilisation and Disinfection

Applied Microbiology

CLINICAL AND SURGICAL PRACTICE Common sterilisation methods and

intact skin

chemical

are

disinfectants used in clinical and surgical practices

3.

shown in Table 25.4.

but do not penetrate them.

Non-critical: These

equipments come in comb.nd Examples

skin

IV. SPAULDING CLASSIFICATION SYSTEM

or blood pressure cuffs.

The Spaulding classification, originally proposed in 1957,

The procedure of sterilisation iS used according to category

Spaulding classification system divides

the

medical equipments/instruments into three categories as follows:

1. Critical: These equipments/instruments come in contact with sterile areas f the body including

of

disinfection

Equipment/Item

in clinical

and surgical practices in the

Method of Sterilisation/Disinfection Ethyl alcohol (70%), chlorhexidine (0.5%)

gluconate

Glutaraldehyde (2%) or Ortho-Phthalaldehyde (0.5%)

Endoscopes Cystoscopes

Glutaraldehyde + quaternary ammonium compound (Bacillocid)

Operation theatre

- Surface cleaning Fogging

Surface disinfectants in hospitals

Phenol/cresol/lysol/chloroxylenol Povidone iodine (Betadine)

Skin antiseptic for Wounds

Pre-operatively Before venepuncture

comes

High level disinfection (minimum) contact with mucous • Sterilisation is preferred. skin

Non-critical

Equipment/instrument which • Low level disinfection comes in contact with only Intermediate level disinfection intact skin.

In Rideal Walker test, suspensions containing similar quantities of organisms are submitted to the action of varying concentrations of phenol and of the disinfectant

to be tested. The dilution of the test disinfectant which

sterilises the suspension of Salmonella typhi in

given

time is divided by the corresponding dilution of phenol. This gives the phenol coefficient.

Interpretation the test

disinfectant has been as effective as phenol. Higher the phenol coefficient, more effective is the disinfectant.

Disadvantage

2.

Typhi i

suspension in a given time

The highest dilution of phenol Salmonella Typhi

only very rarely yields a positive culture. The efficiency of new disinfectant is determined by its ability to

STERILISATION OF PRIONS Prions are infectious proteins without any detectable

Kelsey-Sykes Test (Capacity measure

of

They are highly resistant to physical and

chemical agents. They produce slow infections with long incubation period.

Heat: Prions are extremely resistant to dry heat. A hour has not been temperature of 360°C for found to be effective completely. They are more resistant to moist heat than bacteria, spores, fungi

and viruses. Steam at a temperature of 134-138°C for

test)

2.

of

used

aeruginosa) is added to the disinfectant in three successive

5% cresol

of disinfectant solutions in actual 1 in hospital practice is examined quantitatively for viable

organisms. A use-dilution is then determined which

nucleic acid.

microbiologically. It is also named as capacity test. The standard organism (Staphylococcus aureus, Esch. coli, Ps.

Gloves Catheters

In-Use Tests

1.

the capacity disinfectant to retain its activity when repeatedly

Surgical instruments except sharp instruments

4.

and its modifications.

Chick Martin Test

This test gives

Aprons

carried out under both 'clean' and 'dirty' conditions. Thus it also measures the effectiveness of a disinfectant in the presence of an organic matter.

Chick Martin test is a modification of Rideal Walker test. In this test, disinfectant acts in the presence of organic matter to simulate natural conditions. Organic matter, in the form of dried yeast or faeces, is included. 3.

Respirators

its

inactivate known number of a standard strain of

disinfectant which kills Salmonella

Catheters Sutures

18 and 28 minutes respectively to

recovery medium. The disinfectant is judged by

ability kill bacteria (growth or no growth on recovery medium) and not by comparison with phenol. The test

phenol coefficient does not, however, give any pathogenic staphylococcus on a given surface within certain time. The results of such tests are generally indication of how the test disinfectant will function more useful than those of the phenol coefficient test practically in the presence of organic matters.

suspension in a given time

Plasma sterilisation (vapourised H,O,)

transferred at 8,

The liquid phase

The phenol coefficient of 1.0 means that

specimens before disposal

Disposable syringes Disposable plastic petri dishes Heart-lung machine components

Blood pressure cuffs

contact with disinfectantfor eight minutes and samples

are

which kills

Ethylene oxide

Stethoscope

ECG machine

Phenol coefficient

For blood spillage

Heat sensitive items

Flexible endoscopes

Laryngoscop

Thermometers

The highest dilution of the test

In discarding jar (laboratory)

Scalpels

Endotracheal tubes

but does not penetrate them.

Infected needle or syringe

Sharp instruments

Implants Needles

Equipment/instrument which membrane or non-intac

Sodium hypochlorite (1%) (Freshly prepared)

Disinfectant

Ventilator

Surgical instruments

sterile

of body including blood.

1. Rideal Walker Test (Phenol coefficient test)

Isopropyl alcohol

• Clinical thermometers

9.

contact

Examples

Sterilisation

Bedpans

In-Use tests

chemical disinfectants used

Hand rubs/antiseptics

Disinfection of blood and other

comes

Minimal Inactivation Level

which

3. Kelsey-Sykes test (Capacity fest)

hospital

7.

Semi-critical

TESTING OF DISINFECTANTS 1. Rideal Walker test (Phenol coefficient test) 2. Chick Martin test

syringes.

1.

Equipment/instrument area

V.

blood. Examples are surgical instruments, needle,

Table 25.4 Common sterilisation methods

Category 0

Critical

equipment/

This has been

Definition of Category

Equipment/Instrument

are stethoscop.

non-cititu critical,insemi-critical, instrument i.e.shown Table 25.5.

this classification. The

I non.

flexible endoscopes, laryngoscopes."Pley

with only intact

is a widely used system for disinfection or sterilisation of inanimate objects or surfaces based on the degree of H. Spaulding introduced risk involved in their use.

197

Table 25.5 Spaulding Classification System of Medical Equipments/Instruments

2.

lots at 0, 10 and 20 minutes. These three lots are

in

8 minutes is found to be effective.

Chemicals: Prions

are

inactivated

by sodium

hypochlorite (25% available chlorine) if treated

for one hour. They are also sensitive to household bleach, phenol (90%) and iodine disinfectants. Chemicals such as aldehydes, hydrogen peroxide,

ethylene oxide, ethanol and ionising radiations are found to be ineffective.

198

Unit

I11 Applied Microbiology

VI. CENTRAL STERILE SUPPLY

removal

DEPARTMENT

It s

reusable equipments, medicaldevicesand consumanof for subsequent use in operation theatre (OT) and also other aseptic procedures such as catheterisation. mound stitching etc. in medical and surgical practices in the hospital.

3.

processing in CSSD

mimian unsterile amea to sterile area separatating

plusical barriers. CSSD is divided into Rive matted bs sterilisation area, sterilestorageares,andGistributrion

decontamination area, assembly and processing area, as

(Fig. 25.5). The unidirectional processing occurs

from and processing area are sterilis tn

sterilisation methods

oxide steriliser

Decontamination area:

received from

The reusable

Healthcare Associated Infection

area and stored hera

Distribution area: Distribution countor

supplies

the sterilised items to operation theatres andOther

areas of

PREVIEW

the hospital.

II. Hospital Infection Control Policy

I. Healthcare Associated Infection

Decontamination area

items are

the consumer departments

initial cleaning, disinfection by various mathohds

Cleaning/disinfection

method

of item to be sterilisen

Sterileare areatto this After sterilisation the stenied storage items shifted

follows:

1.

by such as autoclave plasma steriliser. Theethylene

used depends on the type 4.

unidirectional

26

Sterilisation area: The packed items received

assembly

for

The

of sterilant during sterilisation

also protect the item from the contaminat should sterilisation till the time it is used. afte.

The

SPECIFIC LEARNING OBJECTIVES

for

may be done by manual

Distribution

washing or by automated machines

2. Assembly and area: All pre-cleaned reusable items processing

processing area flow

Sterile storage area

Fig.

The student should be able to learn: Definition of healthcare associated infectio

1.

2. Modes of transmission of healthcare associated infections. 3. Common healthcare associated infections and causative organisms responsible for these conditions.

Unidirectional

from decontamination area are

in thisarea forassemblyand processing transferred drying of pre-cleaned items, these are arclosed in a material or a container (packagings. The packaging should allowthe penetration hia

Assembly and

area

4.

Diagnosis, control and prevention of healthcare associated infections. Universal precautions and standard precautions

Sterilisation area

25.5 Central Sterile Supply Department(CSSD)

I. HEALTHCARE ASSOCIATED INFECTION A.

Definition

The terms health care associated infection (HAI) is defined

mechanisms diabetes,

due

to

pre-existing diseases, such

immunosuppression

and

as

patients with

prosthetic implants. They are, therefore, more susceptible to infection.

as infection developing i patients after admission to

2. Hospital Environment The hospital environment is heavily laden with a wide variety of pathogens. These pathogens may be present the hospital or, sometimes, after their discharge. The in air, dust, water, food or antiseptic lotions. Equipment infection is acquired after 48 hours of admission and may be contaminated. Bedding, linen and utensils may

the hospital, which was neither present nor in the incubation period at the time of hospitalisation. Such

infections may become evident during their stay in

t admitted in hospital for management act as fomites. Patients shed the organisms from their bodies while hospital personnel spread these organisms of this infection. HAls also include the following:

patient was

Occuptational infections among healthcare personnel

(e.g. infections transmitted through needle stick injury). Infections in neonates acquired during birth.

Hospital acquired infections and nosocomial infections are

terms used in the past. Since these infectious may be

acquired from any facility providing healthcare and not just the hospital, hence the change in name to HAL

B. Factors Influencing Infection Several factors treatment of infecting organisms.

to the occurrence and severity

infections. These include: 1.

Impaired Defence Mechanisms

Many patients in

hospitals have

3.

Diagnostic or Therapeutic Procedures

Diagnostic or therapeutic procedures such as insertion of urethral intravenous catheters may introduce infection in susceptible patients. Most of these infections are

4.

Healthcare associated infection is probably a great hygiene and problem today despite advances contribute

through their hands and clothes.

of these

due to endogenous flora of patient.

Drug Resistance

Hospital treatment i

infections

are generally refractory

to

the infecting agents are usually multidrug

resistant. 5.

Translusion

Blood, blood products and intravenous fluids

used for

transfusion, if not properly screened, can transmit many

impaired defence infections,

199

Unit III Applied Microbiology There of spread of infection.

C. Sources of Infection

Hospital-infection is

adequate

winninga.chene parinmeheditern.cesattendants

transmission of microorganisms from one scopeof person

1. Exogenous

another Staph. aureus and Str. pyogenes patient or member of the staff or from the environment pathogens spread by hand contact. mostly exogenous

from

important

in the hospital Patients and hospital personnelsuffering

asymptomatic carriers are the most (iñ) Inanimate objects sources Environmental sources include Certain instruments (endoscope,

from infection,

important

or

inanimate objects, air, water and

properlydisinfected,bronchoscope,

may transmit Inanimate objects in the hospital are medical equipments pathogenic organisms (e.g Pseudomonas aeruginosa). food in the hospital. cystoscope), if not

(endoscopes, cystoscopes, catheters etc.), bed pans,

surfaces contaminated by patient's secretions, excretions,

blood and body fluids 2.

2.

Airborne

(i) Droplets

Endogenous

Droplets of respiratory

infection are

inhalation.

Patients own flora may invade the patient's tissue during some surgical operation or instrumental manipulations. (ii) Dust D.

Microorganisms

Dust from bedding,

Almost any microorganism can, on occasion, cause hospital acquired infection but those that survive in

the hospital environment for long periods and develop resistance to antibiotics and disinfectants are particularly important

of

The ESCAPE Pathogens: ESCAPE contains a number

organisms which responsible for majority multidrug resistant nosocomical infections. Enterococcus faccium

of

Styphylococcus aureus

wound

floors, exudates

transmitted by dispersed from

sheddingof skinscales(measles,staphylococcal natural heycontiouteinsprcad ofinfectionseg. seps1s)

Poeudomons

aeruginosa, Staph. aureus. (ini) Aerosols

Aerosols produced by nebulizers, humidifiers and air

conditioning apparatus

transmit certain pathogens

respiratory tract. Occurrence of legionellae~ hospital watersupply has ledto outbreaks ofinfections to the

mainly with Legionella pneumophila.

Klebsiella pneumoniae

Acinetobacter baumannii

Oral Route

Pseudomonas aeruginosa

Hospital food may contain antibiotic-resistant bactera

Enterobacter species

Staph. epidermidis and Group D streptococci also are

(Pseudomonas aeruginosa, Esch. coli, Klebsiella spp. and

others),which may colonise the intestine and later cause sometimes responsible for health-associated infections. infection in susceptible patients. Other bacteria include, Esch. coli, Proteus and Serratia which have become important hospital pathogens,

particularly because of dissemination among them of R factor conferring multiple drug resistance.

Other HAIs include antibiotic associated diarrhoea (Clostridium difficile), COVID-19 (SARS-CoV-2) and

Candida albicans infections.

With the

introduction of disposable syringes

blood donors has reduced the risk

period.

large extent.

HIV escapes detection during the window

Modes of Transmission There are four main routes of transmission of infection: E.

1.

Contact

transmitted by blood transfusion

(i) Hands or clothing The

hands of hospital staff

F. Types of Infection

tissue

are

an important vehicle

Healthcare Associated (HAI) (Table 26.1)

Catheter Associated Urinary Infection (CAUTI

Tract

3.

Ventilator Associated Pneumonia (VAP) Central

Line Associated

associated

in case of indwelling urinary catheter

with catheterisation infection associated Staph epidermidis tract infection Urinary tractcatheter urinary associated as by i named Infection is caused Esch. coli, Klebsiella, in patiends rate increases

(CAUTI). and enterococci Each coli in can Proteus, Pseudomonas organism implicated. Infection the most common by strict asepsis during catheterisation. prevented be should be used only when its tise

patients with

Indwelling catheter

is unavoidable.

Predisposing risk

hospital acquired UTI

factors

Burrs on bed sone

include: 6) extreme of age (ii) (iv) 2.

Presencn o& cestral le

females

Poot itbection conned peectiven &

catheterisation for > 2 days severe underlying

disease

Ventilator Associated Pneumonia (VAP)

It is

of

the second common

4. Surgical Kite felection

HAls after UTI

Aspiration in unconscious patients and pulmonary to nosocomial pneumonia. It ventilation may lead called ventilator

associated pneumonia (VAP)

The

major pathogens include Staph. aureus, Klebsiella spp., Enterobacter, Serratia, Proteus, Esch. coli, Pseudomonas aerugnosa, Acinetobacter, Legionella pneumophil and respiratory viruses. Risk factors includes: Device related: ndotracheal intubation

Infection that develops I surgery (witian

surgies) is called The incidence in

Surgical clean-contamit

multidrug resistant organisms. Semiconscious state: Aspiration of oropharyngeal

wound. This

Poor infection control practices e.g. poor hand

hygiene 3.

as Device

Blood Stream Infection (CLABSI) associated infections (DAIs) 4. Surgical Site Infection (SSI)

from

clean wound

wounds,

pathogens Blood

Central Line Associated Stream Infection (CLABSI)

Other HAls

is the fourth common cause of HAls. It may

to consequences of infection

due

site but is generally

Gastrointestinal Infections Food poisoning and necratal sepocaes in

Table 26.1 Common Healthcare Associated Infections

Causative organisa

Healthcare associated infection

These are associated with devices used hence named

elderly patienns

pathogen, followed by Ps

Esch. coli, Protrus, staphylococd.

Prolonged ICU stay: More risk of hospital acquired wound

and

Major healthcare associated infections (HAIs) include 1.

Tract Infection caused by ada

flora.

been infrequent. However, certain infections

2.

It is the principal route of transmission of nosocomial pathogens.

usually

needles, transmission of infection by parenteral route has may be

Associated Urinary

(CAUTI)

Parenteral Route

4.

donation, contaminated blood products(factor VIII) and HIV and hepatitis B and C viruses are transmitted contaminated infusion fluids. Hepatitis B and HIVare by contaminated blood or blood products. Screening two viruses which may betransmitted in this way. of

However,

Catheter

intetionait for the majrity accounts (Als) (UTThinfections This i0 puttunpre lidartoms urinarytractinfection toilet heteas associated with catheterisation. Infection of healthcare 1.

Klebsiella, Proteus,

CAUTI

Esch. coli,

Ventilator associated pneumonia (VAP)

Staph. aureus, Klebsiella, Enterobacter, Serratis, Proteus,I Acinetobacter, Legionella pneumopiila and respiratery curuse

Surgical site infections (SSI) Gastrointestinal infection Burns

Central line

associated blood

stream infections (CLABSI)

Staph. aureus, Pseudomnas aeruginass, Esch. all,

negative staphylococci

Salmonella, Shigella sonnei and viruses Staph.

aureus, Pseudomonas acruginesa, Acinetobacter and St7, pip80

Gram

negative bacilli, Staph, epidermidis

202

Healthcare Associated Infection

Unit III Applied Microbiology

have been reported. Salmonella and Shigella sonnel mainly associated with these infections.

are

(i)

and

be MRSA),

Pseudomonas aeruginosa,

pyogenes are responsible

hospital associated infections in cases of burns. G.

for

whereroutinenot alleprcsi HIVm ons/ p ati e ntsars rotomuanly infectious for

Universal Precautions

pathogens.

Diagnosis and Control

Healthcareassociatedinfection may, sporadically or as outbreaks. Diagnosisis by the routine bacteriological methods such

direct smear examination, culture testing. This requires the samples sources of infection such as hospital

2.

bome

All bloodspecimens or body fluids should beplaced in a leak-proof impervious bags for transportation to the laboratory Use gloves while handling blood and

and sensitivity

3.

personnel, inanimate objects, water,air or food Typing of isolate(phage typing, bacteriocin typing, biotyping antibiogram) may indicate a causal connection.

use face masks with glasses or goggles. 4. Wear laboratory coats or gowns while

from possible

Control of hospital infection should be a permanent

ongoing activity. Examples

of

sources of hospital

outbreaks are nasal carriage of staphylococci in hosptal therefore,sterilisation techniques haveto be tested. Unfortunately, in many hospitals, infection control is attempted by use of more and more antibiotics. This

5.

6.

HBV)and human immunodeficiency virus (HIV)led to the introduction of 'universal precautions , to minimise the infections in medical laboratoryworkers and health

care personnel. H.

completed. 7.

Prevention

Transmission of infection

8.

Limit use of needles and syringes situationsfor which there are no other alternatives. work.

can be controlled by

agents.

3. Propersterilisation and disinfection of the inanimate objects should be done. This helps to source of infection.

4.

5.

IS

necessary to control the exit point of infection. The use of sterile dressings, surgical gloves, face-

masks and I/V fluids further of infection.

6.

control the

Disinfection of excreta and infected material

contribute in control

Preoperative disinfection of the patient's skin.

Rational antibiotic prophylaxis. 8. Properinvestigation of hospital-associated infection and the treatment of such cases.

for laboratory

potentially contaminated materials the laboratory should be decontaminated beforf disposal or

9. All the

10.

reprocessing.

Always wash hands after

completing laboratory

remove all protective clothings before

leaving the laboratory. Most common agents which

Spillage

16) of see chapter up any spill

Clean

disinfect promptly.

associated with

aboratoryacquired infections include hepatitis B virus

Coccidioides immitis, Bacillus anthracis, Brucella species,

infection Now it is replaced by

tularensiecns.

in the past for the

control while handling patient's bodyfluids

"standard precautions" which

include a set of infection control practices to prevent

of diseaseswhichcan be acquired by contact transmission with blood, body fluids, non-intact skin (including membranes. Unlike Universal

rashes) and mucous

precautions',

"Standard Precautions

are

not restricted

body fluids only. These standard precautions apply to

all patient care regardless of patient status ie. whether

infective or not.

The components of standard precautions include: 1.

2.

Hand hygiene (For details see chapter

16)

Personal protective equipment (PPE) (For details see chapter 16)

infectiv

and

material and

infection. The

Patient care

/ehat.cencadpustedl raiten e maternal should sither be discarded practices

Safe injection should Aseptic techniques preparing

and

be

or

7. Environmental cleaning

• Surface and floor should be cleaned especially in operation theaters, ICUs etc. B. Sharp handling

All sharps including needles should be handled with extreme care.

9.

Respiratory hygiene (cough etiquette) when coughing Covering

the

nose/mouth

or

HOSPITAL INFECTION CONTROL POLICY have an effective hospital infection

control committee (HICC) which should be responsible for the control of health associated infection

(HAI).

related to hospital

Committee will review infection control

of different antimicrobial agents, sterilisation and

and types of infections and antimicrobial sensitivity patterns of the prevalent pathogens.

The HICC supervises the implementation of hospital infection control programme. The various functions include:

HAI surveillance Investigating and controlling hospital acquired

infections.

Antimicrobial stewardship programme: Develops antibiotic policies, monitor the antibiotic usage. Conduction of teaching sessions for various healthcare workers.

and update the hospital infection control

guidelines from time to time. Vaccination of staff and also monitor the

and heads

of

all

clinical

matters

of needle stick injury.

Outbreak management

Communicates with other departments hospital such as:

Central sterile supplies department Laundry department

The committee should be chaired by medical superintendent and should have a microbiologist hospital infection control officer

use

Review

sneezing

Every hospital must

matters

disinfection procedures, hospital environment,incidence

used while

administering medicines

on

manage outbreaks of health associated

activities of the hospital, emergence of drug resistance,

Disinfectionequipment, linen contaminated with

b.

update policy

infection and to

HICC Constitution

proper washing of hands, disinfection Francisella Mycobacterium speciestuberculosis, shigella equipments and change of working clothes. 2. Administration of antibiotic therapy to the carrier Standard Precautions staff or source patient to destroy the pathogenic Universal precautions were used regular

procedures are

Biological safety hoods should e used

sister as members. Chief of all the supportive services (O.T., dietetics, laundry, house keeping etc.) should be

fluids.

thelaboratory worksurfaceswithan Droplaminate after the spillage o blood appropriate disinfectant

work and

The healthcare associatedinfections can be prevented by following means: 1.

Never pipette by mouth. Mechanical devices should be used.

or other body fluids and when the

may lead

to selective colonisation by multiresistant pathogens and has a harmful effect. Concerns about transmission of the hepatitis B virus

working Wrap-around gowns should These should not be taken outside'

in the laboratory

preferred.

officer, chief of nursing services andinfection control

goggles, waste management (For details see includedas invited members. Biomedical Functions of HICC chapter 16) cleaning (Fordetailsofspillmanagement ICC must meet at least once every month to formulate

spattering,

staff or

Pseudomonas growing in lotions. Carriers should be suitably treated. The cause of infection may be a defective autoclave.

specimens and other objects exposed body fluid them there a likelihood of

4.

203

body departments,bloodbank,microbiologist,medical record

Wear gloves or fluids. PPEssuchas gown,mask, glasses Wear other shoes etc. whenever indicated.

Standard precautions

(ii) Transmitted based precautions

Staph aureus (may

Acinetobacter

while handling blood

Broadly prevention can be done by

Burns

2

and

Pharmacy

Blood transfusion committee Antimicrobial usage committee

of the

(CSSD)

27

Hand Hygiene

U

t II U apter 1

U

Biomedical

Waste Management Refer to Unit II Chapter 16.

28

sus e zo

의

i o

음

임으▇

▇

a

i

홈

i

출를

パ9コs:

o

品o5る

1aる

oi o マ

を2

i cり

守oLr

区

6 oi ooo

⼀

ci coマ wr o

曲

ci oマ

ci ooマ