The significance of the coliform group in frozen orange juice

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THE SIGNIFICANCE OF THE COLIFOBM GROUP IN FROZEN ORANGE JUICE

A Thesis Presented to the Faculty of the Department of Bacteriology The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by Julianna Martinez January 1950

UMI Number: EP55013

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T h is thesis, w ritten by

..... Jj^lA-artna..M&r.ti,jas-g.................. under the guidance of h.$X... F a c u lty Com m ittee, and app ro ved by a l l its members, has been presented to and accepted by the C o uncil on G ra duate Study and Research in p a r t ia l f u l f i l l ­ ment of the requirements f o r the degree of

...............

Faculty Committee

7

Chairman

U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA LOS ANGELES 7

Department

of

Bacteriology

January 25, 1950

Library of the University of Southern California* Bindery Departments The Master’s thesis of Julianna Martinez is acceptable to the department of Bacteriology of the University of Southern California even though it has been typed in Elite type rather than Pica*

Sincerely,

Chairman, Bacteriology Department

TABLE OF CONTENTS’ CHAPTER I*

PAGE Introduction

* • • • • • • • • • *

1

Review of Studies on Coliforms and Related Organisms inOrange Juice II*

5

Determination of Coliforms in Frozen Orange Juice

• • • • * • • • * • « *

11

Methods for Detection of Coliforms in Frozen Orange Juice

..................

Results of Coliform Detection Experiments HI*

11 *

12

*

17

Determination of Survival Time of E. Coli and S. -paratyphosa in Orange Juice

* *

Methods for Determination of Survival Time of E* coli and S_*partyphosa in Orange Juice * Results of Survival Time Experiments IV

Discussion

« •

• •

* • • « • • • • • • • • •

Summary and Conclusions

• • • • • • • •

BIBLIOGRAPHY.......... ........................

17 22 23 28 29

LIST OF TABLES TABLE I*

PAGE Numbers of Microorganisms per ml. of Frozen Orange Juice • • • • • • • . . • •

II

Presumptive Tests for E. Coli in Frozen Orange Juice

Ill

Survival

.

•

14.

ofE. Coli Stored at -8 Deg. C* in

Orange Juice of pH 3.4 IV

Survival

« * . # . . *

Survival

Survival

• •

20

of E. Coli in Orange Juice of pH 3*4

Stored at 7 Deg. C. VI

19

of Salmonella Paratyphosa Stored at

-8 Deg. C. in Orange Juice of pH 3*4

V

13

• « . . . . • •

21

of Salmonella Paratyphosa in Orange

Juice of pH 3*4 Stored at 7 Deg. C.

« .

•

21

LIST OF FIGURES1 . PAGE

FIGURE

.

1

Lactose Fermenting Yeast Producing ^Typical E* Coli11 Colonies on Eosin Methylene Blue Agar

. 16

THE SIGNIFICANCE OF THE COLIFORM GROUP IN FROZEN ORANGE JUICE CHAPTER I INTRODUCTION The initial aim of this project was to determine whether organisms belonging to the coliform group were present in frozen orange juice and if present to isolate and determine their exact classification within this group.

The

problem is important because the presence of E. coli is assumed to indicate fecal pollution whereas the presence of other members of the coliform group does not necessarily have this significance.

The problem is of particular importance

since industry is preparing unpasteurized frozen orange juice with a long storage and wide distribution potential. Certain food products can be tested for fecal pollution by using E* coli as the indicator of pollution in a manner similar to that employed in the examination of water for coliforms.

The usefulness of such an examination is based on

the premise that any water sample or like specimen which con­ tains coliforms of the type Escherichia coli affords positive evidence of fecal contamination, and may, therefore, be con­ sidered a potential source of enteric pathogens.

Testing of

potable water by this method is recognized as being fundament­ ally sound; the present problem arising is whether or not these same methods without modifications are applicable to

2 frozen orange juice with the same degree of pertinence.

The

Standard Methods for the Examination of Water and Sewage of the American Public Health Association (194^0, outlines the methods used for water analysis#

An attempt was made to apply

the procedures relating to the isolation and identification of E. coli in water to the examination of frozen orange juice. The term coliform means an organism which is a facultative anaerobic, Gram-negative, non spore-forming, lactose-fermenting rod.

The coliforms embrace a wide and

heterogenous group of organisms, which may be divided into three more specific groups by the use of the ,,Imvic,, quartet of tests.

Imvic is a mnenomic fixing in order the four tests

which are employed in the classification of coliform bacteria: (I) indole production, (M) methyl-red reaction, (V) VogesProskauer test, and (c) the utilization of citrate as a sole source of carbon.

It has been established that IS. coli

regularly exhibits either a

or a

( - / - - ) Imvic pattern; the Aerobacter group exhibits three other combinations (-- / /)> (---- / “)> or (

/) of the

Imvic pattern while the Meoli intermediates11 exhibit the re­ maining ten possible variations between these two groups, i.e., (

), ( / / / / ) , ( / / / - ) , ( - / / / ) , ( / - - / ) , (-//-)> (/-//)> (//-/)> (/-"/)•

(Parr, 1939).

3 The significance of such classification for sanitary science is that Escherichia coli is considered essentially of fecal origin; members comprising the Aerobacter group are not primarily implicated as intestinal inhabitants, and are not as significant; the ,fcoli intermediates*1 remain a controversial group whose significance is as yet unsettled• This latter group of doubtful genesis may be isolated from excreta but it is also known that its members can be isolated from other far removed habitats. The Standard Methods for Water Analysis employs the following interrelated examinations for the detection of the coliform group in water. A. made of water.

The Presumptive Test.

This is the primary test

Graded quantities of water are inoculated

into standard lactose broth fermentation tubes.

These tubes

are incubated for 2 4 - 2 hours at 35 to 37 deg. C. Formation of gas in these lactose fermentation tests under the stip­ ulated conditions is presumptive evidence of the presence of coliforms. If no gas is formed within the given time, the in­ cubation is continued to 48 - 3 hours.

The presence of gas at

the end of the second but not the first 24 hour incubation period is considered a doubtful result.

In either case, the

presence of the coliform group should be verified by the

4 examination described below.

Complete absence of gas at the

end of the l£> hour incubation period is considered as in­ dicative of the absence of coliforms. B.

The Confirmed Test*

Lactose broth tubes showing gas

at the end of the 24.and 4& hour incubation period are streak­ ed on to Endo or eosin methylene blue agar plates, or, are inoculated into the liquid confirmatory brilliant green lactose bile broth*

The plates or the liquid media are then

incubated for another 24 hour period at 35 to 37 deg* C* Form­ ation of typical E. coli colonies on Endo or eosin methylene blue agar plates, or, formation of gas within 4# hours in the brilliant green lactose bile broth is considered a positive confirmed test* In routine work, it is permissible to submit to the Confirmed Test only the two highest dilutions (dilutions containing the smallest inocula) from all the lactose broth tubes which show gas.

ttIn such cases all remaining lactose

broth tubes showing gas that have NOT been submitted to the Confirmed Test SHALL BE CONSIDERED AS CONTAINING COLIFORM ORGANISMS, EVEN THOUGH ALL OF THE CONFIRMED TESTS MADE YIELD NEGATIVE RESULTS. 11 (American Public Health Association, 1946. Capitals by this author). C.

The Completed Test.

In case of gas formation

within the specified time in the liquid confirmatory media brilliant green lactose bile broth transfers are made into

5 standard lactose broth tubes*

If using the solid Endo or

eosin methylene blue agar, typical, well isolated Escheriehia coli colonies or in absence of typical colonies, those which are most likely to be coliforms are inoculated into lactose broth tubes and incubated at 35 to 37 deg* C* until gas form­ ation is noted*

The incubation should not exceed 1$

if 3

hours. Nutrient agar slants inoculated from the colonies picked above or from the brilliant green lactose bile broth should also be incubated at 35 to 37 deg* C. for

jf 3 hours5

Gram stained preparations from those slants corresponding to the secondary lactose broth tubes that show gas should be ex­ amined microscopically. The demonstration of Gram negative, nosporulating rods in the nutrient agar culture and the formation of gas in lactose broth is considered a satisfactory Completed Test, demonstrating the presence of a member of the coliform group. Review of Related Studies Garcia (1911), was one of the first investigators to detect the presence of coliforms in orange juice.

Ifolford

and Berry (194&a) determined the number of coliforms in orange juice and called attention to the previous work of Stone (1929), who reported coliforms in orange juice in a

6 personal communication with these authors*

Nolte and

Van Loesecke (1940) found coliforms in numbers ranging from 0 to 1 ,0 0 0 per cc in raw orange juice*

Beard and Cleary (1932), studying the survival time of various organisms in orange juice at -4- deg* C. at pH 3*5, found that the survival time of two strains of E* typosa and one strain of Shigella dysenteriae was limited to 170 hours, whereas Salmonella partyphosa survived 96 hours and Salmonella schottmuelleri survived 72 hours* Schrader and Johnson (1934) > working on frozen orange juice, studied the behavior of E. coli and other organisms in this medium*

They stated that E. coli was unable to multiply

in orange juice at any temperature and that its death rate was most rapid at 37 deg* C*, slower at 25 deg* C., and still slower at -12 deg. C*

They concluded from their work the

E* coli failed to survive longer than two weeks in orange juice. McFarlane (1942), studied the behavior of E* coli and other microorganisms in fruit juices and fruit juice-sucrose solutions stored at -17.8 deg. C. He stated that in un­ sweetened juice stored at -17*8 deg* C. 9 % of the E. coli were destroyed within IS hours*

He inoculated 8,400,000

E* coli in an orange juice sample and, by the end of one week of storage at -17*8 deg* C., was able to recover only three E* coli* Testing at 2-, 4“, and 6 - week intervals he obtained

7 only doubtful positive reactions for this organism, Wolford and Berry (1948a) worked on two types of frozen orange juices

One prepared from oranges that showed no

visible spoilage, the other from oranges which were soft and showed varying degrees of decay but which, however, contained no visible mold growth.

They noted that the total microbial

counts of the orange juice, prepared from oranges with soft rot, were approximately 2,500 times greater than that ex­ hibited by orange juice from the sound fruit,and that the coliforms were still viable after eight months in the juice from the soft rot oranges, while none could be recovered after one month in the lower count juice. The coliforms isolated resembled the Aerobacter group* Attempts to discover the source of these coliforms led to the examination of the slime and debris from the equipment in the juicing plant.

These workers (1948b) learned that the slime

harbors an. extensive bacterial flora including many eoliforms. They isolated organisms from this slime exhibiting an Imvic pattern similar to that displayed by the Aerobacter and the "intermediate types". Wolford (1949), studied frozen orange juice stored from two to forty-three weeks at -10 deg. F., (-23.3 deg. C.) and reported that during the first six weeks of storage the plate counts drop rapidly, later decreasing more slowly and at a logarithmic rate.

The initial plate counts varying from

a 28,000 to 225,000 organisms per ml. reflected plant sani­ tation.

The initial coliform index varied from 11 to IS

organisms per 100 ml., and from here, decreased in an irregular manner as the storage period increased.

Seventy of seventy-

nine samples examined were found to contain coliform organisms, £. coli being present in thirty of these samples.

Seventeen

of the samples contained E. coli at a two per 100 ml. level while only three samples contained a level greater than ten per 100 ml.

Of the 236 coliform cultures isolated seventy-

nine resembled E. coli and 170 were Aerobacter species. Furthermore, from their previous studies these workers ob­ served that of over half of the samples which contained coliform organisms, only those coliforms considered normal to plant material (Aerobacter type) were present. Although the Genus Erwinia has not been studied in this problem the possibility of confusing members of this genus with E* coli has been suggested by various workers.

Con­

sequently future studies on frozen orange juice should attempt to determine whether Erwinia members occur in the juice and if so, the significance of their presence when test­ ing for coliforms.

According to Elrod (1942), the Genus

Erwinia is closely related to the Escherichia-Aerobacter group.

These organisms are Gram negative, do not produce

spores, are motile, and ferment a variety of carbohydrates including lactose with the production of gas.

The members of

9 this genus are of particular interest in the citrus industry. They possess many other characteristics which are common to the coliforms, e.g., they are antigenically heterogenous; they have various biochemical characteristics and fermentative relationships in common, i.e., aerobic to anaerobic; they vary in their reaction to the methyl red, Voges-Proskauer, and citrate tests. As a matter of fact, Elrod states that the main criterion of differentiation between these two groups lies in the ability of the Erwinia organisms to attack plant tissues producing soft rot.

A particular enzyme,

proto-

pectinase, elaborated by these organisms is said to be responsible for this characteristic.

Elrod has devised a

simple test employing carrot or turnip tissue to determine the presence of this enzyme.

The ability of an organism to

macerate either of these vegetable tissues is considered as evidence of the existence of this enzyme and therefore proof that the organism under study is a member of the Genus Erwinia. The continuous subculturing of these organisms will lead to the loss of this property and make their differ­ entiation from the l,coli-aerogenesn group highly difficult. Isolates of this genus when classified by the Imvic tests are said to approach the characteristics of Escherichia freundii and those of Aerobacter cloacae with greatest frequency. Standard Methods for Mater Analysis does not provide a differential test for these organisms, because they are not

10 indigenous in water and are not present with sufficient frequency to warrant the inclusion of such a test for water analysis* Orange juice on the other hand, might possibly harbor such organisms, for organisms of this genus are known pathogens of citrus fruit.

The brown spots commonly seen in oranges are

due to members of this Erwinia group.

Their occurence is not

one which can be considered infrequent; thus the probability of such organismsbeing present in orange juice should be considered*

CHAPTER II

DETERMINATION OF COLIFORMS IN FROZEN ORANGE JUICE METHODS' The cans of frozen orange juice which were to be ex­ amined were placed in cold running water to allow the juice to melt prior to the initiation of the examination.

The

melted samples were shaken vigorously and opened aseptically. Plate counts were made to obtain an index of the microbial population for each orange juice sample tested. Saboraud1s glucose agar was employed for mold and yeast counts while tryptone glucose agar was utilized for total counts (Difco products)* A portion of the orange juice was neutralized with sterile NaOH*

One ml. samples of.Is10, 1:100, and Is1000

dilutions were plated out.

The plates were poured and in­

cubated at room temperature for IS hours# In the qualitative examination for coliforms, the inocula used were 10 ml., 1 ml., and 0 .1 ml., quantities, placed in standard lactose, lauiyl sulfate-tryptose, and brilliant-green lactose bile broths. were employed with the 10 ml. inocula.

Double strength broths Parallel inoculations

were made into all three media and these were incubated under the conditions set forth for the Presumptive Test in Standard

12 Methods for water analysis* Transfers were made by streaking on Bacto-eosin methy­ lene blue agar plates from tubes showing gas* In absence of typical Escherichia coli colonies, others which most nearly resembled their description were picked and inoculated into lactose broth tubes as recommended in the Completed Test, concomitant with transfers to agar slants from these same colonies. RESULTS a* Counts of total microbial population in frozen orange juice. The counts obtained on Saboraud1s dextrose agar and tryptone glucose agar are given in Table I.

The counts are

divided into three groups* Group Is

Counts obtained from orange juice within one

week after receipt from plant*

Group includes 23 samples

stored at -8 deg. C. Group IIs

Counts obtained from orange juice stored

between one to two weeks after receipt from plant*

Group in­

cludes 32 samples stored at -8 deg. C. Group Ills

Counts obtained from orange juice stored

between two to three weeks after receipt from plant. includes 30 samples stored at -8 deg. C*

Group

13 TABLE I TOTAL MICROBIAL COURTS OF 85 SAMPLES OF FROZEN ORANGE JUICE AS OBTAINED WITH TWO DIFFERENT MEDIA

ORGANISMS PER ML. OF FROZEN ORANGE JUICE GROUP

MEDIUM

HIGHEST COUNT

MEAN COUNT

LOWEST COUNT

1

16,000

10,000

6,000

2

2A.000

11.000

6.000

1

11,000

7,000

5,000

2

9.000

8.000

5.000

1

7,000

5,500

3,000

2

5.000

.3.000

2.000

I

II

III

1 Saboraud *s dextrose agar. 2 Tryptone glucose agar.

14 Saboraud1s dextrose agar -was employed for demonstrating the yeast and mold population, while tryptone glucose agar was utilized for the determination of bacterial numbers#

The two

media appear to give approximately the same count, since mold and yeast which are the predominant flora seemed to be able to grow in tryptone glucose or SaboraudTs dextrose agar plates with equal facility#

As can be seen from the data in Table I, the

numbers appear to decrease with storage*

Whether this drop in

count actually occurs as it appears or is modified by an elongated lag phase induced by continued storage at below freez­ ing temperatures cannot be ascertained from this evidence, b. Results on the determination of coliforms in frozen orange juice* Sixty-two samples examined for coliforms using 10 ml*, 1 ml#, and 0#1 ml# inocula gave the following results: TABLE II RESULTS OF PRESUMPTIVE TESTS FOR COLIFORMS IN FROZEN ORANGE JUICE (Sixty-two samples tested) MEDIA (BROTH) Size of inocula 10 ml*

Standard lactose 62 pos# *

Lauryl sulfate tryptose

Brilliant-green lactose-Bile

54 pos.

55 pos*

1 ml*

62 pos#

3 pos*

0 pos*

0.1 ml*

62 pos*

0 pos*

0 pos*

#(The appearance of any amount of gas within 4# hours is indicated by the word positive.)

15 Twenty-three other samples were later run, using only the 10 ml. inocula, with the following results: Standard lactose broth

23 pos.

Lauryl sulfate tryptose broth

21 pos.

Brilliant-green lactose bile broth

19 pos.

In most cases where positive tests were obtained gas appeared in lauryl sulfate tryptose and brilliant green lactose bile broths only at the IS hour reading.

Some gas

could usually be detected in the standard lactose broth tubes containing the largest inocula after 2J+ hours. Streaking from the positive presumptives on eosin methylene blue agar produced a variety of colonies.

Yeasts

and large Gram-positive nonsporulating rods resembling Lactobacilli were the organisms which were most frequently encountered; the yeasts definitely predominated. On occasions small, discrete, dark-centered colonies with a green metallic sheen were observed on plates of eosin methylene blue agar streaked from standard lactose and lauryl sulfate tryptose broths showing gas.

These colonies which

were typical of E. coli in appearance were found to be yeasts. None of the colonies isolated from the eighty-five samples examined proved to be Gram-negative, non-sporulating lactose fermenting bacilli*

16 FIGURE I

LACTOSE FERMENTTNG YEAST PRODUCING "TYPICAL E. COLI" COLONIES ON EOSIN METHYLENE BLUE AGAR

17 CHAPTER I H DETERMINATION OF SURVIVAL TIME OF E. COLI AM) SALMONELLA PARATYPHI IN FROZEN ORANGE JUICE. An attempt was made to determine the survival time of E. coli and of one of the enteric pathogens in orange juice in order to clarify the conflicting data reported in the literature. A typical strain of E. coli and one of Salmonella paratyphi were used* The organisms were grown on nutrient agar slants for 24 hours*

Suspensions of the organisms were made in sterile

distilled water and seven dilutions differing by a factor of ten and ranging from Is 10 through 1:10,000,000 were prepared* Total numbers of the organisms in suspension were determined by plate counts on tryptone glucose agar* Ten ml* portions of orange juice shown to be coliform negative by previous examination were inoculated with 1 ml* quantities of each dilution.

Several lots of orange juice

containing a full dilutions series were quick-frozen with dry ice and stored at -8 deg. C.

Other samples inoculated with

the same numbers of organisms were stored at 7 deg. C*

Para­

llel series with Salmonella paratyphi and E. coli were pre­ pared in every case.

Samples containing a mixture of the two

strains were also prepared and handled in the same manner. Each frozen sample,once melted,was not frozen again, obviating the

18 errors which would be incurred by repeated freezing and thawing* E. coli was recovered by inoculating brilliant green lactose bile broth tubes with 10 ml. portions of orange juice and incubating these at 37 deg* G* for twenty-four hours. Eosin methylene blue agar plates were subsequently streaked from those brilliant green lactose bile broth tubes showing gas after 24 hours.

Brilliant green lactose bile broth tubes

which were negative for gas at the end of this period were incubated for another 24 hour period and those showing gas at the end of this time were streaked on eosin methylene blue agar plates.

Typical E. coli colonies were picked and trans­

ferred to agar slants and again to brilliant green lactose bile broth.

If Gram stained preparations from the growth on

the agar slants demonstrated Gram-negative nonsporeforming rods, and the corresponding brilliant green lactose bile broth tube showed gas within IS hours from a typical E. coli colony picked on eosin methylene blue agar plates, it was assumed that some of the E. coli organisms inoculated were still viable. The presence of Salmonella paratyphi was detected by inoculating 10 ml* of orange juice into tubes of Difco Tetrathionate broth enrichment medium and incubating for 12 to 18 hours at 37 deg. C., subsequently streaking from this medium to S-S agar.

The plates of this latter medium were

19 Incubated at 37 deg. C. for 24 to 1$ hours*

Colorless

colonies surrounded by yellow zones of discolored media were picked off and the organisms grown on agar slants and in­ cubated at 37 deg. C. for 24- to 4& hours.

If the growth on

the agar slants proved to be Gram-negative nonsporulating rods it was assumed that some of the Salmonella partyphi organisms inoculated were still viable. TABLE III SURVIVAL OF E* C P U AT -S DEG. C. IN FROZEN ORANGE JUICE pH 3*4 Initial inoculum

Organisms per 10 ml. of orange

(Storage time in days) 1

2

3

4

5

6

12,000,000

/

/

/

/

/

-

1,200,000

/

/

/

-

-

-

120,000

/

.iuice

_

-

12,000 1,200 120

mm

12

/ indicates re-isolation of E. coli from stored sample

20 TABLE 17 SURVIVAL OF SALMONELLA PARTYPHI STORED AT -8 DEG. C. .IN FROZEN ORANGE JUICE OF pH 3*4

Initial inoculum Organisms per 10 ml.

Q (Storage time m days)

-

300,000,000

/

30,000,000

/

3,000,000

/

300,000

/

-

-

30,000

-

-

-

-

-

3,000

-

-

-

-

-

300

-

-

—

-

-

30

-

-

-

-

-

/

/

-

-

-

-

-

•

-

,

-

Tables I U through IV show the results for typical experiments.

No data is presented for the survival of

mixtures of E. coli and S. paratyphi since they exhibited the same viability in mixtures as when each occurred alone.

21 TABLE V SURVIVAL OF E* COLI IN ORANGE JUICE OF pH 3*4 STORED AT 7 DEG. C. Initial inoculum Organisms per 10 ml. of orange juice

Storage Time A

5

Weeks 1 2

/

/

/

/

/

/

/

/

/

/

/

/

/

/' /

/

/

/

-

12,000

/

/

/

/

/

/

-

-

1,200

/

/

/

/

/

-

-

-

120

/

/

/

/

-

-

-

-

12

/

/

-

-

-

-

-

-

1

Days 2 ?

12,000,000

/

/

1,200,000

/

120,000

? /

TABLE VI SURVIVAL OF SALMONELLA PARATYPHI IN ORANGE JUICE OF pH 3.4 STORED AT 7 DEG. C. Initial inoculum Organisms per 10 ml. of orange juice

1

Storage Time ... Weeks Days'.. 2 A 5 1 2 3 3

300,000,000

/

/

/

/

/

/

/

/

30,000,000

/

/

/

/

/

/

/

/

3,000,000

/

/

/

/

/

/

/

/

/

/

/ -

-

300,000

/ /

-

30,000

/

/

/

/

/

-

-

3,000

/

/ -

-

-

-

30

/

/

/ / -

—

/

/ / -

-

300

/ /

/ -

-

-

-

•

22 As can be noticed by the data in Tables III through VI, E. coli seems to exhibit a longer survival time even though a lower inoeula was used. Another interesting and im­ portant point that seems to be brought out by these tables is the apparent longer survival of both E. coli and Sj. paratyphi at 7 deg. C. than at -8 deg. C.

CHAPTER IV DISCUSSION The determination of coliform organisms in frozen orange juice by procedures employed in water analysis affords a rather difficult task*

From the work presented previously

it may be observed that many false positive results are ob­ tained*

The results might be due primarily to yeasts which

seem to occupy a dominant role in the microbial flora of this juice*

In the Presumptive Test when larger inocula are used,

by virtue of the character of the orange juice, other sugars such as dextrose and sucrose are introduced into the test medium which allow orangisms that are non-fermenters of lactose to produce acid and gas*

The fact that standard

lactose broth does not seem to exert any selective action for E* coli is a significant point and is due to the presence of large numbers of yeasts in frozen orange juice which are capable of fermenting lactose.

From the data in .Table II it

can also be noticed that lauryl sulfate tryptose and brilliant green bile broths through their apparent selective action seem to eliminate fermenters in the lower dilutions* The overall result emphasized by the data in Table II is the apparent inadequacy of Standard lactose broth for the testing of frozen orange juice for coliforms. The attempt to obtain quantitative information on the

24 numbers of E. coli surviving under different conditions by plating samples of the orange juice on eosin methylene blue agar and counting the Atypical11 E. coli colonies was un­ successful.

It was assumed that this method would give

reasonably accurate results since Howard and Thompson (1925) found that 89.6% of all colonies having typical*1 (colonies 2-3 mm. in diameter, dark centers, purple to black, flat or slightly concave surfaces, distinct metallic sheen) appearance proved to be E* coli. However, as previously mentioned, a yeast was present in most of the samples which gave a colony indistinguishable from the E* coli type thus making the attempted procedure impossible to develop.

The presence of the yeast In the

orange juice did not interfere with the qualitative results reported above since it does not grow in brilliant green lactose bile broth. The data in Tables III and VI point out the interesting and perhaps paradoxical result of E. coli and S. paratyphi surviving longer at a higher temperature (7 deg. G.) than at a lower temperature (-8 deg. C.) in orange juice.

A longer

lag phase might have occurred at the lower temperature, re­ sulting in a failure to recover the organisms with the techniques and times of incubation employed.

Another factor

which bears consideration may be found in the report of McFarlane (1942).

He noted that the freezing of a solution

25 resulted in differences in concentrations of solutes in the various layers of a frozen specimen.

This phenomenon would

imply variations in pH as well as osmotic pressure throughout a specimen of frozen orange juice, creating diverse environ­ mental conditions for microorganisms within the same sample. Thus, lack of uniformity in the quantities of the samples to be frozen could conceivably lead to inconsistencies in results of viability experiments unless continuous shaking or stirr­ ing were carried out in all samples during freezing. The fact that E. coli seems to survive longer than S. paratyphi in orange juice even when present in a smaller inocula might be the result of a strain difference. If one recalls the report of ¥olford and Berry (1948b)$ in which coliforms were found extant in the slime and debris of the equipment in juicing plants in numbers as high as three billion per gram, it might be considered reasonable to recover a few coliform organisms from orange juice.

The

coliform organisms isolated by these workers seem to be of the Aerobacter and wintermediate types”. If such a condition were ascertained to be normal in plants operating with reasonably adequate sanitary measures, the advisability of establishing a certain standard for numbers of coliforms in orange juice as is commonly employed with certified milk today should be considered.

26 The lack of agreement as to the viability of E, coli in orange juice seems to be a controversial issue.

The dis­

crepancies reported might perhaps be explained on the basis of variations in resistance between strains of E, coli. The pH afforded by orange juice is too low to allow normal development of coliforms isolated from other sources.

If

coliforms, however, are present in the slime and debris of the juicing plants in as large numbers as are reported,' it would appear that these organisms are multiplying to some ex­ tent in an environment which probably contains some residual juice.

This would then necessitate the probability of the

existence of special strains which are capable of multiplying at a low pH, which in turn would explain certain reports claiming the isolation of coliforms in orange juice after as long a period as eight months, Coliforms have been established as being variable organisms occurring in many unsuspected and remote places, Beckwith (1931) found them present in paper pulp slime, Tonney and Noble (1932) reported them as occurring in wooden tanksj cheese cloth bags, pump nozzles, fiber gaskets, and graphited string packers; Spaulding (1933) isolated them from jute packing in water mains, etc.

Thus it might be feasible

that the strains which have withstood- a month or more of storage in frozen orange juice are special strains which have, through unknown circumstances, become adapted and are

27 resistant to conditions normally inhibitory to the more common coliform strains*

Further investigation of those

coliforms isolated under such circumstances should be con­ sidered* The determination of total microbial flora might assume a more important position, since it can serve as an excellent index for reflecting plant sanitation and quality of the juice*

It might be deemed advisable to set standards

on total microbial counts and reject specimens which have obviously not been produced under adequate sanitary con­ ditions*

This in itself would tend to minimize the possi­

bility of an unwholesome and potentially dangerous specimen reaching the market* A certain period of cold storage might also be recommended to lessen the probability of survival of enteric pathogens* The major problem which must be resolved in the ex­ amination for coliforms seems to be the suppression of yeasts* Whether this is accomplished through the use of more select­ ive media, i.e*, the use of penicillin lactose broth (Silliker 194$), the incorporation of other antibiotics or drugs known to inhibit yeast at levels not inhibitory to coliforms, or through the use of other tests employing different incubation temperatures, such as the Eijkman Test

28

(Stuart et. al. 1942), is a matter that must still be settled. The problem would appear to merit further research. SUMMARY AND CONCLUSIONS Eighty-five commercially packed orange juice samples were examined for coliforms.

No coliforms were recovered

from any sample. A lactose fermenting yeast, producing colonies very similar to those of E. coli on eosin methylene blue agar was isolated.

This yeast could conceivably be a source of false

positive (Presumptive and Confirmed) results in the exam­ ination of orange juice for coliforms. Lactose broth did not appear to be sufficiently selective for its use in the Presumptive Test in the exam­ ination of orange juice for the detection of coliforms. A more specific method appears to be needed for the examination of coliforms in orange juice.

29 BIBLIOGRAPHY

American Public Health Association 1946 Standard Methods for the Examination of Water and Sewage pp. 193-201 9th Ed. Amer. Pub. Health Assoc., N.Y. BEARD, P. J. and CLEARY, J* P* 1932 - The importance of temperature on Survival time of bacteria in acid foods. J. Prevent. Med. 6:141-144. BECKWITH, T. D. 1931 - The bacteriology of pulp slime J. Bact. 22: 15-22. BERGEY, D. H., BREED, R. S., and MURRAY, E. G. C. 1948 Manual of Determinative Bacteriology 6th Ed. William & Wilkins Co., Baltimore CALDWELL, E. L. and PARR, L. W. 1933 Pump infection in normal conditions in controlled experimental fields. J. Am. Water Works Assoc. 25: 1107-1117 CITED BY: Wolford, E. R. and Berry J.A. 1948 Bacteriology of slime in citrus processing plant with special reference to coliforms Food Research 12s(4) 340-346 ELROD, R. P. 1942 - The ETwinia-coliform relationship J. Bact. 44s433—440 GARCIA 1911 Annual Rept. Comm. Health Penn. Pt. 2^ 102-1024 CITED BYs Tanner, F. W. 1932 Microbiology of Foods pp. 310. 1st. Ed. Twin City Printing Co. Champaign, Illinois. HOWARD, N. J. and THOMPSON, R. E. 1925 - The isolation of the colon group in water. The Canadian Engineer 48:413-417. McFARLANE, V. H. 1942 - The behavior of microorganisms in fruit juices and in fruit juice-sucrose solutions stored at -17.8 deg. C. Food Research 7s 509-518 NOLTE,

A. J. and VAN LOESECKE, H. W. 1940 - Types of organisms surviving commercially pasteurized citrus juices in Florida. Food Research 73-81.

PARR, L. W. 1939 - Coliform bacteria. Bact. Revs. 2k 1-48

30 SCHRADER, J* H. and JOHNSON, A. H. 1934 - Freezing orange juice. Insutrial and Engineering Chem. 26:369-874* SILLIKER, J. H. 1948 - The effect of penicillin and tyrothricin on organisms of significance in the Presumptive Test for water analysis. Thesis presented in partial fulfillment for M* S. degree, Bacteriology Dept., University of So. Calif. SPAULDING, C. H. 1931 - Contamination of mains by jute packing. Am* J. Pub. Health 21: 1330-1384 STUART, C. A., ZIMMERMAN, A., BAKER, M., RUSTIGIAN, R. 1942 Eijkman Relationships of the coliforms and related bacteria J. Bact. 43: 557-572 TONNEI, F. 0. and NOBLE, R. E. 1932 - The interpretation of direct counts of Colon-Aerogenes in well waters. J. Bact. 2£: 473-479 WOLFORD, E. R. and BERRY, J. A. 1948a - Conditions of oranges affecting bacterial content of frozen juice with special emphasis to coliform organisms. Food Research 12 (2): 172-177 WOLFORD, E. R. and BERRY, J. A. 1948b - The bacteriology of slime in citrus processing plant with special re­ ference to coliforms. Food Research 1^(4): 340-346 WOLFORD, E. R. 1949 - Bacteriological studies on commercially

prepared orange juice stored at -10 deg. F. Ninth Annual Convention and Exhibition, Institute of Food technologists. Food Ind. 21(7): W 53 Abstract #35 University of Southern C alifornia Ulw&rjr