Botanical microtechnique: A course in applied microtechnique for junior college

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BOTANICAL MICROTECHNIQUE A COURSE IN APPLIED MICROTECHNIQUE FOR JUNIOR COLLEGE

A Project Presented to th e F acu lty o f th e School o f Education The U n iv ersity o f Southern C aliforn ia

In P a r tia l F u lfillm en t o f th e Requirements fo r th e Degree Master o f Science in Education

by Charles J . U ig lia z z o January 1950

UMI N um ber: E P 4 5 9 6 8

All rights re se rv e d INFORMATION TO ALL U S E R S T h e quality of this reproduction is d e p e n d e n t upon th e quality of th e copy subm itted. In th e unlikely e v e n t th a t th e a u th o r did not se n d a co m plete m anuscript a n d th e re a re m issing p a g e s, th e s e will b e noted. Also, if m aterial h ad to b e rem oved, a n o te will indicate th e deletion.

Dissertation Publishing

UMI E P 4 5 9 6 8 P ublished by P ro Q u e st LLC (2014). Copyright in th e D issertation held by th e Author. Microform Edition © P ro Q u e st LLC. All rights rese rv e d . This w ork is p ro tected a g a in st un authorized copying u n d e r Title 17, United S ta te s C o d e

P ro Q u e st LLC. 789 E a s t E isen h o w er P arkw ay P.O . Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1 3 4 6

The format and s t y l e o f type used in th is p r o jec t has my approval

June 1, 1950

£°C. Add 35 c c . o f g ly c e r in and 5 g . o f phenol.

c.

Use sta in e d or u n stain ed t i s s u e .

d.

Dehydrate by th e g ly c e r in j e l l y evaporation p r o c e ss. (1 )

Wash t is s u e i n running water*

(2)

Rinse the m aterial c a r e fu lly to remove a l l k i l l i n g flu id .

56

(3 ) Transfer the m a teria l to a 2 q t . ja r o f water and a llo w to stand undisturbed fo r 2 hours, (U) Siphon o f f most o f the water w ithou t a g ita tio n and re­ p la ce th e w ater. (5 )

Repeat th e rep lacin g o f the water a t l e a s t tw ic e .

(6 )

Transfer the m a teria l to a la r g e volume o f o f g ly c e r in in w ater.

(7)

Use a b o t t le or ja r and mark the l e v e l o f the 5>$ g ly c e r in .

(8 )

Gauge the volume so th a t a fte r th e elim in a tio n o f water th e r e sid u a l g ly c e r in w i l l more than cover the t i s s u e .

so lu tio n

(9) Evaporate th e w ater. (a )

"When rapid evaporation i s d e sir e d use an incubator oven a t 35 to l*0oC.

(b)

For ordinary evaporation u se a d e sic c a to r a t room temperature.

(c )

For rapid evaporation a t room temperature use a vacuum d e sic ca to r or vacuum oven.

(10

Evaporate u n t il th e g ly c e r in i s about 10$ concentrated.

(11

Add a fr e s h 10$ concentrated s o lu tio n o f g ly c e r in .

(12

A fter n ea rly an anhydrous co n d itio n i s a tta in e d , tr a n sfe r the t is s u e in to an anhydrous a lc o h o l.

(13

Transfer th e t is s u e to two changes o f an anhydrous a lc o h o l.

(lh

Place a p ie c e o f g ly c e r in j e l l y about as la r g e as a match head on a clean dry s l i d e and warm u n t i l m elted.

(15

Remove some t is s u e from #12 and p lace on th e warmed j e l l y .

(16

Lower a cover g la ss over the m aterial c a r e fu lly .

(17

P lace a le a d w eight on th e cover g la s s th a t w i l l squeeze out e x c e ssiv e j e l l y .

(18

Cool th e j e l l y and clean o f f th e e x ce ss w ith a lc o h o l.

57

5»

6.

(19)

S e a l the cover g la ss -with balsam or a quickdrying lacq u er.

(20)

P lace in an undisturbed p lace and l e t

(21)

Clean th e s l i d e s .

(22)

Label and s t o r e .

RESINOUS MOUNTING:

dry.

(See Figure 5*)

How t o make resin o u s mounts.

a.

Use f o r a l l t i s s u e s in g e n e ra l.

b.

P lace sta in e d or un stained t is s u e in x y l o l, chloroform , or tu rp en tin e, depending what so lv e n t was used in l a s t s t a in .

c.

Place a sm all drop o f balsam on th e specimen which has already been placed on th e s l i d e , (u s e fu l in the p a r a ffin m ethod.)

d.

Warm the balsam s l i g h t l y and cover the specimen thoroughly.

e.

P lace a c o v e r -s lip upon th e specimen.

f#

Permit the mounted specimen to remain f l a t

g.

Clean th e s lid e s w ith x y lo l or tu rp en tin e.

h.

Label th e s l i d e s and s t o r e .

VENETIAN TURPENTINE:

f o r a few days.

(See F igure £ .)

How t o make V enetian tu rp en tin e mounts.

a.

Use when the dangerous x y lo l sta g e i s d esir ed to be elim in a ted and f o r whole mounts.

b.

Proceed from th e s ta in in g .

c.

P lace in 10$ g ly c e r in u n t i l concen trated .

d.

Wash the g ly c e r in out thoroughly in 95$ a lc o h o l.

e.

Complete the dehydration in 100$ alcohol*

f.

Transfer to a 10$ Venetian turpentine in absolute a lc o h o l. (Make tr a n sfe r q u ic k ly .)

g.

Allow the turpentine to th ick en as the alcoh ol evap orates. ( I f too th ic k add a few drops o f ab solu te a lc o h o l.) (1 )

(See Chapter 9 & 1 0 .)

Use an e x sic c a to r f o r evaporation.

58

(a )

7.

E xsiccator c o n s is ts o f a saucer f u l l o f soda lim e (sodium hydroxide w ith lim e ) on a p la te o f g la s s and cover w ith a b e l l j a r . (Use care in n o t l e t t i n g any o f th e soda lim e or other d r ie r g e t in to th e tu r p e n tin e .)

h.

Mount the m a teria l in a few drops o f th e Venetian tu r­ pentine by p la c in g the thickened medium on th e m a te r ia l.

i.

Add the cover g la s s c a r e fu lly .

j.

P lace in a dry p la ce u n t il hardened.

k.

Glean th e ex cess m aterial w ith ab solu te a lc o h o l.

1.

Store and la b e l fo r use and exam ination.

HYGROBUTOL:

(See F igure $ .)

How to make hygrobutol-balsam mounts.

a.

Suggested fo r permanent mounts o f d is s o c ia te d s e c tio n s .

b.

S ta in and wash t i s s u e . (Unstained t is s u e can be mounted a l s o . ) (See Chapter 9 & 1 0 .)

c.

Transfer a sm all amount o f th e m a teria l to a watch g la s s o f $0% a lco h o l and observe w ith th e m icroscope.

d.

I f th ere i s to o much d is to r t io n , tr y 20% a lco h o l on another b ath . (W ell-hardened m aterial w i l l stand $0%.)

e.

A fter a s ta r tin g p o in t fo r dehydration i s e s ta b lis h e d , carry the m aterial in step s of 20 to approxim ately 70% a lc o h o l.

f.

Add to the 70% a few drops o f sto ck so lu tio n of counter­ s t a in , i . e . , e o s in Y, ery th ro sin B, or f a s t green. (See Chapter 1 2 .)

g.

S atu rate the s o lu tio n in ab solu te a lco h o l or methyl c e l lo s o lv e .

h.

Leave in th e counter s ta in u n t i l s l i g h t l y overs ta in e d . (This r e q u ire s about ii-12 h o u rs.)

29

i.

Rinse in 70$ a lco h o l and tr a n sfe r through the fo llo w in g s e r ie s fo r o n e -h a lf to one hour in te r v a ls : (1 ) (2 ) (3) (U)

3 parts a lco h o l to 1 p a rt hygrobutol. 2 p a rts a lco h o l to 2 p arts hygrobutol. 1 p art a lco h o l to 3 p arts hygrobutol* Pure hygrobutol; change tw ice a t 15> minute in t e r v a ls .

j.

T ransfer to a la r g e volume o f $% s o lu tio n o f balsam in hygrobutol in a sh o rt wide-mouthed b o t t l e .

k.

Allow th e hygrobutol to evaporate slo w ly a t a temperature o f about 3£>°C.

1.

"When th e balsam i s s l i g h t l y more f l u i d than used f o r covering s e c t io n s , mount th e m a te r ia l.

m. Remove a s u ita b le q u an tity of th e p la n t t is s u e w ith i t s enveloping balsam, p la ce on a dry, clean s l i d e andlower a dry cover g la s s c a r e fu lly .

8.

n.

Dry the fin is h e d s lid e s in a h o rizo n ta l p o s itio n .

o.

Store and l a b e l.

DIOXAN-BALSAM:

(See Figure £ .)

How t o make dioxan-balsam mounts.

a.

Suggested fo r whole mounts o f filam en tou s and d e lic a te m a te r ia l.

b.

S ta in and wash t i s s u e . (Unstained tis s u e can be mounted a l s o .) (See Chapter 9 & 1 0 .)

c.

Pass through a s e r ie s o f dioxan, con tain in g 20$, h0%, 80$, 90$, and 100$ dioxan. (Use a lco h o l as th e s o lv e n t. Use 1 to 2 hour in t e r v a ls .)

d.

Pass through two changes o f new pure dioxan fo r an in te r v a l o f 1 to 2 hours.

e.

Examine a few fila m en ts under a m icroscope mounted in the la s t flu id .

f.

I f t is s u e s in good c o n d itio n , tra n sfe r to a 10$ so lu tio n o f balsam in dioxan.

g.

Use a wide-mouthed b o t t le and gauge th e volume of the liq u id so th a t the m aterial does not become exposed a s th e dioxan evap orates. ( I t takes 2 to 8 hours t o ev ap ora te.)

60

h.

P lace the uncovered container in to an oven or a d u st-fr e e p lace a t a temperature o f about 35°C.

i.

Leave th e m a teria l in th ic k balsam in which i t i s mounted. 2?

j.

Continue th e p rocess under m and n in th e proceeding method. (Hygrobutol-balsam Method.) SOURCES

1*

2.

C.

BOOK SHELF:

Help y o u r s e lf to a d d itio n a l a id s .

a.

Chamberlain, pages (100-111) (2 3 -2 6 .)

b.

Johansen, pages (1 1 0 -1 2 0 .)

c.

McClung, pages (198-20U.)

d.

S a ss, pages (1 0 9 -1 1 0 .)

SPECIAL REFERENCES:

Added a id s fo r you.

a.

Stew art, A. A .: "The Mounting of C e llo id in S e c tio n s in S e r ie s" . S c ie n c e , 2j.2s 872. 19U>.

b.

McWhorter, F. P ., and E. W eiser: " P ossib le Uses o f Dioxan in B otan ical M icrotechnique". S ta in Technology, 11: 107-119. 1936.

ACTIVITIES: Laboratory performances th a t may help you in p r e P ^ in g good mounts fo r your s l i d e s . 1*

TRYING: Try th e variou s methods of mounting on your t is s u e s and p e r fe c t your technique on one or more o f th e methods. Compare your r e s u lt s w ith the samples prepared by th e in ­ str u c to r .

2.

ASSISTING: Look over your fe llo w stu dents s lid e s and a s s i s t him in s e le c t in g an appropriate mounting medium. Compare re­ s u lt s and reach c o n c lu sio n s. P e r fe c t the use o f t h i s mounting medium. Submit your r e s u lt s to the in s tr u c to r fo r c r it ic is m .

3.

EXAMINING: Look over some o ld s lid e s and se e how w e ll th e mounting mediums have held up. Make use o f t h i s knowledge in your mounting.

61

D.

EVALUATION? Samples of -ways in -which your m astery o f good mounting techniques may be checked. 1.

2.

COMPLETION:

Supply the m issin g word in th e statem en ts,

a,

Nygrobutol-balsam should be used f o r

s e c t io n s .

b,

Amann* s Lactophenol should be added to make temporary s lid e s l a s t lo n g e r,

c,

Venetian turpentine medium should be used on t is s u e which cannot stand th e dangerous s ta g e ,

d,

Algae and fu n g i should be mounted i n __________ ,

e,

P a ra ffin s e c tio n s should be mounted in

f,

__________ should be used as a so lv e n t fo r resin o u s m a te r ia l,

g.

An incubator should be used fo r

medium to

.

evaporation,

BEST ANSWER: Place an X in each space f o r -which your mounting technique q u a l if i e s . Yes

No )

Your aqueous mounts should l a s t lo n g e r i f you add a 10$ g ly c e r in s o lu tio n ,

)

Your aqueous mounts should be durable f o r a lg a e, fu n g i, and fe r n s .

)

Non-drying o i l s should be used fo r mounting t is s u e s over a lo n g p eriod o f tim e.

d.

)

You should se a l cover g la s s e s -with glue or g ly c e r in j e l l y .

e.

)

Resinous mounts should be made of alm ost a l l tis s u e s •

f.

)

Dioxan-balsam and V enetian turpentine should be used as mounting mediums fo r whole mounts*

b.

62

PART V. STAINING HOW TO SELECT THE PROPER STAINS With so many s ta in s to s e l e c t from i t becomes q u ite a problem to make a v i s e c h o ic e .

The fou r chapters t o fo llo w are so arranged th a t

su g g estio n s are given and then by p r a c tic e , c o n s is te n t r e s u lt s can be ob tain ed .

The only way to in su re su cc ess i s to become fa m ilia r w ith

the a c tio n of each s t a in upon th e variou s str u c tu r e s . *

be measured by th e in t e n s it y o f your enthusiam*

Your su ccess w i l l

63

CHAPTER 9 .

UNEMBEDDED TISSUE

HOW TO STAIN UNINFILTRATED TISSUE A.

B.

MOTIVATION; Advantages t o be gained by properly knowing how to s ta in u n in filtr a te d tis s u e * 1.

WIDER RANGE OF EXPERIENCE: To know a s t a in i s n o t enough* You must know how i t works and what can be expected . This tr a in in g comes on ly w ith experience*

2.

GREATER POSSIBILITIES: R e a liz in g th e e f f e c t s o f each s ta in g iv e s you v a s ts f i e l d s o f new p o s s i b i l i t i e s .

3*

SELF CONFIDENCE: To know th a t a s ta in w i l l do a c e r ta in job under a s e t s itu a t io n g iv e s grea t freedom o f a c t i v i t y .

PRESENTATION: H elpfu l h in ts to g e t you s ta r te d on the r ig h t road f o r sta in in g u n in filtr a t e d t i s s u e . DIRECTIONS 1.

GRINDING: How t o s t a in f o s s i l s e c tio n s prepared by the grin ding method. a.

2.

PEELING: How t o s ta in f o s s i l s e c tio n s prepared by the p e e lin g method. a.

3.

G enerally n ot sta in e d .

G enerally n o t sta in e d .

FREEHAND:

How to s t a in freehand s e c t io n s .

a.

I f an aqueous s t a in i s to be used , pass down the whole s e r ie s o f a lc o h o ls {9$%, 85%, 50%, 35%), u sin g f i v e m inutes in each b efo re proceeding to s t a i n .

b.

I f th e s ta in i s in a strong a lco h o l (85-95%), tr a n sfe r d ir e c t ly to the s t a in .

c.

I f th e s t a in i s in a 70/6 a lc o h o l, pass th e t is s u e through 95% and 85% a lc o h o ls w ith 5 minutes in each b efore s ta in in g .

d.

Make a s e le c t io n o f s ta in s and methods in Chapter 12.

64

e.

Suggested s ta in s : (Safranin and D e la fie ld * s hem atoxylin; sa fra n in and a n i l i n b lu e; sa fr a n in and lig h t green; m alachite green and Congo red; sa fr a n in and gentian v i o l e t ; methyl green and a c id fu ch sin *) (See Chapter 1 2 ,)

f.

Use a sm all brush to tra n sfe r t i s s u e .

g.

Label and sto re fo r u se .

4*

HAND MICROTOME: How to s ta in hand microtome s e c t io n s . (See in s tr u c tio n under freehand s e c t io n s .)

5*

SLIDING MICROTOME: How to s t a in s lid in g microtome s e c tio n s . (See in s tr u c tio n under freehand s e c t io n s .)

6.

TEASING:

7*

8,

How to s ta in se c tio n s th a t have been tea sed a p a r t.

a*

Follow su ggestion s under th e freehand method.

b.

Hematoxylin s ta in s are su ggested .

DISSOCIATION:

(See Chapter 1 0 .)

How to s ta in se c tio n s th a t have been m acerated.

a.

Follow su ggestion s under th e freehand method.

b*

An aqueous safran in s t a in i s recommended.

SMEARS: a,

(See Chapter 1 2 .)

How to s t a in sm ears.

Johansen*s Methyl V io le t Method. (1)

Transfer smears from th e water to 1% aqueous so lu tio n of m ethyl v i o le t 2B. S ta in for 15 minutes or l e s s . (Use fr e sh s t a i n .)

(2)

Transfer th e s lid e s to water fo r o n e-h a lf an hour.

(3)

D iffe r e n tia te in 70% and 95% a lc o h o l to which has been added 0,5% p ic r ic a c id . Give 10 seconds in each.

(4)

Immerse the s lid e s fo r 15 seconds in a 95% a lc o h o l to which 4 drops of ammonia have been added to each 100 c c . a lc o h o l.

(5)

Complete dehydration in a b so lu te a lc o h o l fo r about 10 to 12 seconds.

(6)

Complete d iffe r e n ta tio n i n pure c lo v e o i l for 8 to 15 seconds.

65

b.

e.

(7)

Wash in x y lo l con tain in g a tra ce o f ab solu te a lc o h o l.

(8)

Leave th e s l i d e s in pure x y lo l fo r about 2 hours and mount in balsam. (See Chapter 8 .)

Tuan's M odified Hematoxylin Method. (1 )

S ta in in 0 .5 $ hematoxylin f o r 20 m inutes.

(2 )

Wash out e x cess s ta in -with w ater.

(3)

D iffe r e n tia t e in a satu rated aqueous s o lu tio n o f p ic r ic a cid fo r 20 to 70 m inutes, depending on the sta g e of d iv is io n .

(it)

Examine o c c a sio n a lly under th e microscope to note th e progress of the d e sta in in g .

(5)

Wash in running water fo r 30 m inutes, then proceed to the dehydration and mount in balsam. (See Chapter 8 .)

C apinpin's B r a z ilin Method. (1)

S ta in in a r ip e 0 .5 $ so lu tio n o f b r a z ilin in 70$ a lco h o l fo r 2 to 6 hours. (Ripen s t a in fo r a t l e a s t a w eek.)

(2)

Wash b r i e f l y in 70$ a lc o h o l.

(3)

D iffe r e n tia te in 1$ ammonium sulphate in 70$ alcoh ol fo r 5 to 10 m inutes.

(U)

Examine th e smear sta in in g which show a chromosomes brownish-black or b la c k , cytoplasm pink and c e l l w a lls c o lo r le s s •

(5)

Wash in two changes o f 70$ and one in 95$ a lco h o l fo r 10 minutes in each.

(6)

Dehydrate in ab solu te a lco h o l and pass through the fo llo w in g m ixtures: (a) Equal p a rts o f absolute a lc o h o l and cedar o i l . (b) Equal p a rts o f x y lo l and th in cedar o i l . ( c ) 9 p a rts x y lo l and 1 part cedar o i l .

(7 )

Complete th e c le a r in g in two changes o f x y lo l, and mount in balsam . (See Chapter 8 .)

66

d.

Other methods th a t can be used.

(See Johansen.)

(1)

B e llin g * s Iron-acetocarm in method.

(2)

McCallum’s Iron-propionocarmin method.

(3)

M cClintock’s Permanent Acetocarmin method.

(4)

Z irk le*s method.

(5)

Warmke *s method.

(6)

H illary*s method. SOURCES

1.

2.

BOOK SHELF: H elpful r e fer e n c es which w i l l g iv e you something t o aim a t i n p e r fe c tin g your own techn iqu e. a.

Chamberlain, pages (1 4 0 -1 5 8 .)

b.

Guyer, pages (2 1 8 -2 3 6 .)

c.

Johansen, pages (155-169.)

d.

HcClung, pages (165-173*)

e.

S a s s , pages (94-97*)

f.

T obias, pages (5 0 -5 2 .)

SPECIAL REFERENCES: a. bi

A d ditional a id s fo r you.

Conn, H. J .: B io lo g ic a l S ta in s . 3rd Ed. Commission on Stan dardization o f B io lo g ic a l S ta in s . Geneva, New York.

1936.

Conn, H. J .: H istory o f S ta in in g . Commission on Stan d ard ization o f B io lo g ic a l S ta in s . Geneva, New York.

1933.

c.

Conn, H. J .: "Haematin and Acid Fuchsin". 4: 9 7 . 1929.

S ta in Technology.

d.

Dickson, D. T .: "The D if f e r e n t ia l S ta in in g of P lant Pathogen Host". S cien ce NS. . 52: 63- 64 . 1920.

e.

Gourley, J . H .: "Basic Fuchsin S ta in in g fo r Vascular Bundles". S ta in Technology. 5: 99-100. 1930.

f.

Stoughton, R. H.: "Thionin and Orange f o r the D if f e r e n t ia l S tain in g o f B acteria and Fungi in P lan t T issu e s" . Annals o f A pplied B io lo g y . 17: 162-164. 1930.

67

C.

D.

ACTIVITIES: s ta in in g .

Pro.jects t o help you p e r fe c t your technique o f

1*

TRYING: S e le c t a number o f your u n in f iltr a te d t i s s u e s and proceed t o t i y your experience with th e su ggested s t a in s . Compare the r e s u lt s with the prepared samples su p p lied by the in s tr u c to r . Get su g g estio n s from th e in s tr u c to r .

2.

CRITICIZING: Compare your r e s u lt s with th a t of your c la s s m ates. Share su g g estio n s and make u se o f th e s e su g g estio n s in your tech n iq u e.

3.

READING: Look over the new s e r ie s o f S ta in Technology magazine and u se t h i s inform ation in p e r fe c tin g your own tech n iq u e.

EVALUATION: Some check-ups th a t w i l l be an a s s is ta n c e to your m astery o f sta in in g u n in filtr a te d t i s s u e s . 1.

TRUE-FALSE: T

2.

P lace an X in th e c o rrect space fo r tru e car f a l s e .

F

a.

( ) ( ) F o s s il s e c tio n s should not be sta in e d .

b.

( ) ( ) You should always consid er whether you are going to use an aqueous or a lco h o l so lv e n t s t a in .

c.

( ) { )

d.

( ) ( ) You should properly la b e l your t i s s u e s .

e.

( ) ( ) You should use th e same s ta in in g tech n iq u es for hand microtome and s lid in g microtome s e c t io n s .

Safranin and D e la fie ld , s hematoxylin should be used to s ta in freehand s e c t io n s .

RATING SCALE: Rate th e follow in g statem ents in the order o f a p p lic a tio n to th e s ta in in g of u n in filtr a te d t i s s u e . Use th e numbers 1 , 2, 3> 4 , and 5 . Use number 5 for the h ig h est r a tin g . a.

P lace the fo llo w in g s ta in in g techniques in the order o f t h e ir d iffic u lty . Johansen's Methyl V io le t smear method. Tuan m odified hematoxylin smear method. S ta in in g te a s in g s e c t io n s . S ta in in g freehand s e c t io n s . S ta in in g f o s s i l t i s s u e s .

CHAPTER 1 0 .

EMBEDDED TISSUE

HOW TO STAIN INFILTRATED TISSUES A.

B.

MOTIVATION: Outcomes th a t m i l be yours i f you p e r fe c t your technique o f sta in in g on i n f i l t r a t e d t i s s u e s . 1.

PROFESSIONAL EXPERIENCE: To gain p r o fe s sio n a l sta tu s in s ta in in g methods a wide range o f experien ce i s n ecessa ry .

2.

OUTSTANDING SLIDES: Experience b rings wide range of p o s s i b i l i t i e s which means ex cep tio n a l s l i d e s o f superior q u a lit y .

3.

FINANCIAL ASSETS: S lid e s th a t f i t in to the category of pro­ fe s s io n a l men always demand and r e c e iv e b ig d ivid en d s.

PRESENTATION: Key ste p s which in su re th e b e st p o ssib le sta in in g o f in f i l t r a t e d t i s s u e .. DIRECTIONS 1.

2.

3*

SOAP:

How to s ta in t is s u e s embedded in soap.

a.

Use the p a r a ffin method o f s ta in in g .

b.

Make other s e le c t io n from Chapter 1 2 .

FREEZING:

(S ta rt a t # 4 -d .)

How to s ta in se c tio n s prepared by th e fre e z in g method.

a.

Use the p a r a ffin method o f s ta in in g .

b.

The freehand method o f sta in in g i s a ls o recommended. Chapter 9 .)

c.

Make oth er s e le c t io n s from Chapter 12.

CELLOIDIN:

(S ta r t a t # 4 -d .) (See

How to s ta in c e llo id in s e c t io n s .

a*

T ransfer t is s u e s from th e 95$ a lc o h o l bath by means sm all brush.

b.

G radually run dowi th e s e r ie s o f a lc o h o ls u n t il the t is s u e i s in a water s o lu tio n . Allow 3-5 m inutes i n each s t e p . (See # 4 -d .)

c.

Send through th e d esir ed s t a in .

(See Chapter 1 2 .)

of a

69

d.

Follow ing s t a in in g , tr a n sfe r by means o f a sm all brush to r in s e s i n f i v e changes o f an anhydrous a lc o h o l or acetone for 2-5 m inu tes. (Take care not to dry th e t i s s u e .)

e.

I f a b so lu te a lc o h o l i s u sed , make two changes o f e th e r a lco h o l to d is s o lv e th e c e l lo i d in out o f the t i s s u e .

f*

Flood th e t is s u e w ith c a r b o l-x y lo l fo r 5-10 m inutes.

g*

Rihse in 5 changes o f x y lo l and mount.

_h.

i.

4.

(See Chapter 7 & 8 .)

Transfer o f t is s u e can be made to th e microscope a t any tim e as lo n g as th e t is s u e i s not dried o u t. (This view in g i s recommended very h ig h ly .) Recommended s t a in s fo r c e l lo i d in . '•'1- i

-

(1)

Haidenhain’s iron -haem atoxylin, E r lic h 's haem atoxylin, haem atoxylin and sa fr a n in , and sa fra n in and f a s t green.

PARAFFIN:

How t o s t a in p a r a ffin s e c t io n s .

a.

Remove the p a r a ffin by p la cin g th e s lid e s i n x y lo l fo r a t l e a s t 5 m inu tes. (Never attempt to h asten the p rocess by applying heat by means o f a lamp or oven .) (See Figure 6 , S ta in in g -d ish arrangem ent.)

b«

Send the s l i d e s through two ehanges o f x y l o l .

c.

Remove the x y lo l by f i r s t p la cin g th e t i s s u e in equal parts of a b so lu te a lc o h o l and x y l o l fo r 5 m inutes. (1)

d.

Transfer t o two changes o f ab so lu te a lc o h o l for about f i v e m inutes i n each bath.

Transfer to s t a in . (1)

I f an aqueous s t a in i s t o be used, pass down the whole s e r ie s o f a lc o h o ls (95$, 50$, 35$) using f iv e minutes in each before proceeding to s t a i n .

(2)

I f th e s ta in i s in a stron g a lc o h o l (85-10056) tra n sfe r d ir e c t ly to the s t a in .

(3)

I f th e s t a in i s in a 70$ a lc o h o l, pass through 95$, and 85$ a lc o h o l w ith 5 m inutes in each b efore s ta in in g .

70

e.

Follow th e s ta in in g procedure below. (1)

I f s e c tio n s have been sta in e d w ith a strong a lc o h o l, tr a n s fe r to 95/6, and th en to 100$ a lc o h o l far 2 m inutes in each .

(2)

I f s e c tio n s have been sta in e d i n an aqueous s o lu tio n , p ass through the s e r ie s of 5$, 10$, 20$, 35$, 50$, 70$, 65$, 95$, and 100$ a lc o h o l fo r 2 minutes in each .

f.

Clear t i s s u e s through a s e r ie s o f two or th r e e changes o f x y l o l , c lo v e o i l , or cedar o i l w ith about 2-5 m inutes in ea ch .

g.

Mount s e c tio n s in balsam, l a b e l, and sto r e for u s e . (See Chapter 8 .)

h.

Suggested s ta in s fo r p a r a ffin : (l)

5.

Safranin gen tian v i o l e t , a l l haemataxlyns, sa fr a n in f a s t green, Flemming1s T rip le s t a in , alm ost any s t a i n , depending on th e e f f e e t d e sir e d .

CELLULOSE ACETATE:

How t o s ta in c e llu lo s e a c e ta te s e c t io n s .

a.

Transfer s e c tio n by means o f sm all brush to pure acetone fo r 1-2 minutes to remove c e llu lo s e a c e ta te .

b.

Make two rapid changes in a ceton e.

c.

Wash i n a s e r ie s o f a lc o h o ls u n t il the concentration corresponds to th e so lv en t o f the s t a in . (See # 4 -d .)

d.

Transfer t o th e s t a in .

e.

Dehydrate as i n the p a r a ffin s e c t io n s .

f.

Clear a s in p a r a ffin s e c t io n s .

g.

Mount as i n th e p a r a ffin s e c t io n s .

h.

Su ggestion s fo r c e llu lo s e a c e ta te s t a i n s . (1)

(See Chapter 12 fo r s e l e c t io n s .) (See # 4 - e .)

(See # 4 - f .) (See # 4 -g .)

A n ilin c h lo r id e , methylene b lu e , Congo red , ammoniacal fu c h sin , and the variou s haexnatoxylins.

f 3$% ALCOHOL

IL O L

XYL& 100%

100#

■COHOL

ALCOHQ

XYLOL

FIGURE 6 . SUGGESTED STAINING-DISH ARRANGEMENT

72

SOURCES 1.

2.

BOOK SHELF: Added sou rces which w i l l a s s i s t you in per­ f e c t in g your s ta in in g technique* a.

Chamberlain, pages (Ij.2-79*)

b*

Johansen, pages (60-78*)

c*

McClung, pages (l8i*-195>0

d.

S a s s , pages (6 0 -7 8 .)

SPECIAL REFERENCES: a.

C.

P.

Added a s s is ta n c e ,

See Chapter 9*

ACTIVITIES: Laboratory work to help you m aster th e s ta in in g o f in f i l t r a t e d t i s s u e s * 1*

TRYING: Use th e suggested sta in in g procedures and s t a in some o f your i n f i l t r a t e d t i s s u e s . Check your r e s u lt s w ith th o se o f your neighbor and exchange c o n str u c tiv e c r it ic is m s which can be used in p e r fe c tin g your method.

2*

PERFECTING: P e r fe c t one or two o f th e sta in in g techniques by rep eatin g th e method u n t il a smooth running p attern i s a tta in e d .

3*

READING: Consult the magazine, S ta in Technology, f o r new methods and incorp orate them in your tech n iq u e. Search p a r tic u la r ly f o r new s t a i n s .

EVALUATION: Samples o f check-ups which may be employed in ev a lu a tin g your progress in sta in in g embedded t i s s u e s . 1.

TRUE-FALSE:P lace X in the c o r r e c t space fo r tr u e or f a l s e . T

F

a.

( ) ( )

S ectio n s embedded in soap or by fr e e z in g should be sta in e d l i k e p a r a ffin s e c t io n s .

b.

( ) ( )

Haematoxylin s ta in s should be used fo r c e l lo i d in s e c t io n s .

c.

( ) ( )

X y lo l should be used t o d is s o lv e p a r a ffin .

d.

( ) ( )

Running up cr down a s e r ie s o f a lc o h o ls should precede s ta in in g .

e.

( ) ( )

You should not run your se c tio n s in an up a lc o h o l s e r ie s b efore c le a r in g in x y lo l or clove o i l .

f•

( ) ( )

Acetone should be used as a so lv e n t f o r c e llu lo s e a c e ta te .

g.

( ) ( )

Sm all brushes should be used t o .t r a n s f e r s e c tio n s ■whenever n ecessa ry .

RATING SCALE: Rate the fo llo w in g procedures in order of t h e ir sequence by p la c in g number 1 , 2 , 3 , It., £ , 6 , and 7* Use number 1 as the f i r s t s t e p . Caution: A ll ste p s are not l i s t e d . a.

In s ta in in g p a r a ffin se c tio n s the fo llo w in g order should be observed: ( )

Remove th e p a r a ffin by a bath i n x y l o l.

( )

P lace s e c tio n in th e d e sir e d s t a i n .

( )

Wash t is s u e s in two changes of ab solu te a lc o h o l.

( )

Run up or down the s e r ie s o f a lc o h o ls , depending on s ta in used.

( )

Clear in x y lo l or c lo v e o i l .

( )

Mount s e c tio n in balsam, and s to r e .

7k

CHAPTER 1 1 .

SPECIAL TISSUES

HOW TO STAIN SPECIFIC TISSUES A.

B.

MOTIVATION: B e n e fits th a t w i l l r esu lt-fro m lea r n in g more about s p e c if ic s t a i n s . 1.

VERSATILITY: Wide knowledge and experience makes f o r a freedom known on ly to the c o n sc ien tio u s te c h n ic ia n .

2.

ADVENTURE: Everyone l i k e s a change and lea r n in g a new and unusual technique i s a ch allen ge to am bitious stu d e n ts.

3*

FINANCIAL STATUS: M aterial returns mean more s e c u r ity and a share in the b e t te r th in g s in l i f e .

PRESENTATION: s t a in s .

Su ggestion s on the u ses and r e s u lts o f s p e c if ic DIRECTIONS

1.

BULK STAINS: a.

2.

5.

Use sa fr a n in . o f sa fr a n in .)

(See Chapter 12 fo r in s tr u c tio n s in the use How to

s ta in l i g n i f i e d c e l l w a lls .

Use sa fr a n in , c r y s ta l v i o l e t , or io d in e green. (See Chapter 12 fo r in s tr u c tio n s i n th e use o f e a c h .)

CUTINIZED CELL WALLS: a.

s t a in c e llu lo s e c e l l w a lls .

How to s t a in e h it in .

LIGNIFIED CELL WALLS: a.

How to

Use a cid fu c h sin , a n ilin b lu e, Bismarck brown, Congo red , D e la fie ld ’s haem atoxylin, f a s t green, or l i g h t green. (Follow in s tr u c tio n s f o r in d iv id u a l s ta in s in Chapter 1 2 .)

CHITIN: a.

b.

Use Bismarck brown Y, carmine, or Harris* haem atoxylin. (Follow in s tr u c tio n s f o r use in Chapter 1 2 .)

CELLULOSE CELL WALLS: a.

3.

How to s t a in t is s u e s in bulk*

How to

s ta in c u tin iz e d c e l l w a lls .

Use sa fr a n in , c r y s ta l v i o l e t , a cid fu c h sin , or e ry th r o sin . (See Chapter 12 fo r in s tr u c tio n on th e use of e a c h .)

7$

.

6

MIDDLE LAMELLA, s a*

7.

8.

.

.

.

How to s t a in achromatic f ig u r e s .

How to s ta in filam entous

How to s ta in cytoplasm .

Use e o s in , e ry th r o sin , f a s t green FCF, or orange G. in s tr u c tio n s in Chapter 1 2 .)

(See

How to s t a in p e c tin s .

Use iod in e s t a in .

(G ives y e llo w c o lo r .)

(See Chapter 1 2 .)

How to s ta in p r o te in s.

Use sa fr a n in .

PLASTIDS: a.

(See Chapter 12 fo r

Use iro n haem atoxylin, sa fr a n in , or f a s t green FCF. (Use in s tr u c tio n s in Chapter 1 2 .)

PROTEINS: a.

1U.

(See in s tr u c tio n in Chapter 1 2 .)

Use c r y s ta l v i o l e t or f a s t green FGF. in s t r u c t io n s .)

PECTINS: a.

13.

Use iro n haem atoxylin.

CYTOPLASM: a.

12

How to s ta in m itochrondria.

FILAMENTOUS FUNGI IN HOST TISSUES: fu n g i in h o st tissu e s * a.

11

Use iron haem atoxylin, sa fr a n in , c r y s ta l v i o l e t or carmine fo r acetocarmine smears. (See in s tr u c tio n in Chapter 12 fo r th e proper u se*)

ACHROMATIC FIGURE: a.

10

How to s ta in chromosomes.

MITOCHRQNDRIA: a.

9.

Use ir o n haem atoxylin or ruthenium red fo r fr e sh m a te r ia l. (See in s tr u c tio n in Chapter 1 2 .)

CHROMOSOMES: a.

How to s ta in the middle lam ella*

(See in s tr u c tio n s in Chapter 1 2 .)

How to s ta in p l a s t i d s .

Use c r y s t a l or methyl v i o l e t , or in s tr u c tio n s in Chapter 1 2 .)

iro n haem atoxylin. (See

76

SOURCES 1.

2.

BOOKS: A handy s e t o f referen ces which w i l l a id you i n your s ta in in g o f s p e c if ic t i s s u e s . a.

Chamberlain, pages (379-385.)

b.

Johansen, pages (6 5 -9 5 .)

c.

MeClung, pages (573-614.)

d.

S a ss, pages (6 0 -7 8 .)

SPECIAL REFERENCES: a.

C.

A d d ition al a id s for you.

See Chapter 9 s e l e c t io n s .

ACTIVITIES: Some lab oratory procedures which w i l l g iv e you experience i n sta in in g s p e c if ic t i s s u e s . 1.

TRYING: S e le c t a few o f your samples o f which you wish to s ta in some s p e c if ic t i s s u e s . P r a c tic e u n t il you f e e l sure of your tech n iq u e. Exchange id e a s and experim ental techniques w ith other stu d e n ts . Make use o f t h i s knowledge in your sta in in g methods.

2.

READING: Refer to th e magazine, S ta in Technology, and look fo r new id e a s in s ta in in g . Use th e se su g g estio n s in p e r fe c t­ in g your tech n iq u e.

3.

OBSERVING: Look over the many d iffe r e n t s l i d e s presented by th e in str u c to r fo r your observation and stu d y . Make c r i t i c a l comments which w i l l be u s e fu l t o you. Ask th e in str u c to r to help c r i t i c i z e your s lid e s of s p e c if ic t i s s u e s .

77

D.

EVALUATION; Some e v a lu a tio n instrum ents for p o s sib le check-up on your technique on s ta in in g s p e c ia l t i s s u e s . 1.

TRUE-FALSEi T

2.

3.

P lace X in th e co rrect space fo r tr u e or f a l s e .

F

a.

()( )

Safranin should be used fo r s ta in in g c h it in .

b.

() ( )

P ec tin should be sta in ed w ith io d in e .

c.

() ( )

Iron haem atoxylin should be used in sta in in g s p e c if ic t i s s u e s .

d.

()( )

You should use Bismark brown fo r bulk s ta in in g .

e.

() ( )

C rystal v i o l e t should be used to s ta in p la s t id s .

BEST ANSWER: p aren th eses.

P lace th e number of the b e st answer in the

a.

( ) Haematoxylin s ta in should be used fo r : ( l ) C h itin . (2) Bulk t i s s u e s . (3) C e ll w a lls . (4) Chromosomes.

b.

( ) Fast green s t a in should be used fo r : ( l ) P la s t id s . (2) Chromosomes. (3) Cytoplasm. (4 ) Middle la m e lla .

c.

( ) Bismark brown should be used fo r : ( l ) C h itin . (2) P e c tin . (3) Achromatic f ig u r e s . ( 4) C e llu lo se c e l l w a lls .

COMPLETION:

Supply th e m issin g word i n th e statem ents below . ________________ fo r s ta in in g m ito-

a.

You should use chrondcia.

b.

You should use f or s ta in in g p e c tin , in order t o get th e b e st c o lo r com bination.

7a

CHAPTER 1 2 .

INDIVIDUAL STAINS

HOW TO USE SPECIFIC STAINS A.

B.

MOTIVATION: Advantages th a t w i l l be yours i f you can use s p e c if ic s t a in s properly. 1.

CLEARER SLIDES: Well sta in e d s l i d e s enable one t o see stru ctu res which would otherw ise be in v i s i b l e or n early s o .

2.

GREATER POSSIHLILITIES: Toknow the p o s s i b i l i t i e s i s t o understand t h e ir a c tio n on s p e c if ic t i s s u e s .

3.

FUTURE REFERENCES: Cnee a technique has been p e r fe cte d i t i s a sim ple procedure t o rep eat i t w ith l i t t l e d i f f i c u l t y .

o f s t a in s

PRESENTATION; H elpful su g g estio n s which w i l l make your use o f s p e c if ic s t a in s a l o t e a s ie r . DIRECTIONS 1.

2.

SELECTION:How t o s e l e c t

th e proper s t a i n s .

a.

Decide what stru ctu res you d e sir e to s t a i n .

b.

Decide what purpose i s going t o be made o f th e s l i d e .

c.

P ick one or two s ta in s and p e r fe c t your technique on th e se b efore tr y in g a g rea t number.

d.

Use th e em p irical method, elim in a tin g th e poor s t a in s and u sin g th e good o n es.

HEMATOXYLIN: a.

How t o use hem atoxylin s t a in s .

Heidenhain’s iron -alu m . (Limited t o th e middle la m ella o f xylem t i s s u e .) (L ig n ifie d t i s s u e and n u c le i s ta in b la c k .) (1)

Proceed from th e method o f se c tio n in g and use th e in s tr u c tio n s presented th e r e .

(2)

Be sure to bring a l l s e c tio n s down t o w ater, by means o f descending a lc o h o ls , b efore going on.

\

79

(3)

Whsh thoroughly w ith w ater, and f i n a l l y r in s e in d i s t i l l e d w ater.

(4 )

Use a 2%, J%, or h% s o lu tio n o f th e s t a in depending on th e d e lic a c y o f th e t i s s u e . (Weak fo r d e lic a t e , and strong fo r robust t i s s u e .)

( 5)

Place in th e s t a in fo r not more than 2 hours.

(6)

Wash thoroughly i n running water fo r 5 m inu tes.

(?)

D estain i n a 2% f e r r ic ammonium sulphate or f e r r ic c h lo rid e s o lu tio n .

(8)

D estain in g tim e depends on th e th ick n ess o f th e t i s s u e . (Ohserve t is s u e s during th e d esta in in g under th e m icroscope u n t il d esir ed e f f e c t i s o b tain ed .)

(9)

la sh in running water fo r a t l e a s t 30 m inu tes.

(10) Dehydrate in 50%, 70%, and 95% a lc o h o ls allow in g a t l e a s t 5 minutes in each . ( U ) T ransfer t o a b so lu te a lco h o l fo r 5 m in u tes. (12) Transfer to x y lo l fo r two changes o f 5 m inutes ea ch . (13) Proceed t o mounting mediums. b.

(See Chapter 8 .)

D e la fie ld * s (L ig n ifie d t is s u e s and n u c le i s t a in b la c k .) (1)

Follow in s tr u c tio n s given above under 2 . , a . - ( l ) and ( 2 ) .

(2)

Transfer to s t a in fo r about 10 m inu tes.

(3)

Treat th e t i s s u e w ith a a c id u la ted w ater. (Add about two drops o f hydrochloric a c id to each 100 c c . o f w a ter.) (a)

Treat here fo r a few m inutes u n t il se c tio n s turn a p a le p in k ish -p u rp le.

(b)

Transfer q u ic k ly t o water and wash i n running tap water u n t i l th e s e c tio n s acquire a r ic h purple c o lo r .

80

c.

(4)

Pass through 50$, 70$, and 95$ a lc o h o ls . minutes in e a ch .)

(5)

I f d e sir e d , mount d ir e c t ly from 95$i n diaphane. ( I f t h is i s not d e sir e d , proceed as in above 2 . , a . ( 1 1 ), (1 2 ), and (1 3 ).

Mayer*s haem-alum.

(A few

(R esu lts a s above.)

(1)

Follow su g g estio n s under 2 . , a . - ( l ) and ( 2 ) .

(2)

S ta in fo r not more than 10 m inutes. i s u s u a lly p le n ty .)

(4 or 5 m inutes

(3) Vfesh thoroughly i n w ater. ( 4 ) Transfer t o 10$ g ly c e r in and fo llo w th e Venetian tu rp en tin e method o f mounting in Chapter 8 . d.

E h rlic h ’s hem atoxylin.

(R esu lts a s above.)

(1) Follow su g g e stio n s under 2 . , a . - ( l ) and ( 2 ) .

2.

(2)

S ta in 5 t o 30 m inutes.

(3)

»Vash out the e x cess s ta in w ith 50$ a lc o h o l.

(4)

Clear and mount.

CARMINES: a.

(See Chapter 8 .)

How to use carmine s t a in s .

Greenacher*s borax acarm ine. (1)

S ta in m a teria l i n bulk or in s e c tio n s i n 50$ a lc o h o l 1 -3 d ays. (Short tim e for s e c t io n s , 2-3 hou rs.)

(2)

Treat with an a c id a lco h o l (5 0 cc . o f 70$ a lco h o l p lu s 2 drops o f hydrochloric a c id .)

(3)

Treat u n t il th e c o lo r becomes c le a r r ed . tak e a few hours or 2 t o 3 d ays.)

(4)

Dehydrate in 50$, 70$, 95$ a lc o h o ls for 6 to 24 hours.

( 5)

Proceed t o c le a r in g in x y l o l.

(6)

F ollow in s tr u c tio n s fo r mounting.

(This may

(See Chapter 8 .)

ai

b.

c.

3.

Alum carm ine. (1)

S ta in fo r 12 t o 24 hours.

(2)

Ihsh i n w ater.

(3)

Continue as in above. and (6) . )

Mayer1s carmalum. (1)

S ta in as w ith alum carmine.

(2)

Use fo r bulk m a teria l and for a c o u n te r -sta in on th e s l i d e w ith Bismark brown.

ANILINS: a.

( 2 . , a . - ( 2 ) , (3)> (4)> ($)»

How t o use a n ilin s t a i n s .

Safranin (L ig n ifie d t i s s u e , c u t i c l e and cork s t a in r e d .) (1)

G en erally used w ith other s t a i n s .

(2)

Use sa fr a n in alon e as fo llo w s: (a)

Pass s e c tio n s through 50%,

and 70% a lc o h o l.

(b) Transfer t o th e s t a in fo r a minimum o f 2 hours and a maximum o f 24 h ou rs. (c)

Pass through 50%, 70%, 85%, 95% and 100% a lc o h o ls .

(d) Use c lo v e o i l or x y lo l fo r c le a r in g . (e) b.

Mount.

(See Chapter 8 .)

Acid fu c h sin (C e llu lo se and collenchyma s ta in s p in k .) (1)

Proceed from a t l e a s t a 70% a lc o h o l s o lu tio n .

(2)

S ta in 1 t o 2 hours.

(3 )

D iffe r e n tia te in a satu rated s o lu tio n of p ic r ic a c id in 70% a lc o h o l fo r about 30 secon d.

(4)

Rinse in 70% a lc o h o l.

(5)

Proceed through the regu lar s e r ie s o f up a lc o h o ls and mount. (See Chapter 8 .)

82

c.

Congo red (G enerally used w ith m alach ite green or a n ilin b lu e .)

d.

Eosin (C e llu lo se and s o f t t i s s u e s s t a in p in k .) (1)

For m aterial mounted i n whole i n g ly c e r in , g ly c e r in j e l l y or V enetian tu r p e n tin e .

(2)

S ta in overnight or b e tte r fo r 24 hours.

(3)

Treat v&th a 2# aqueous s o lu tio n o f a c e t ic a c id 5 t o 10 m in u tes.

(4)

Transfer to 10$ g ly c e r in w ithout washing in w ater.

(5)

Mount i n g ly c e r in j e l l y .

fo r

(See Chapter 8 .)

e.

E rythrosin (Cytoplasm s t a in s p in k .)

f.

G entian v i o l e t (L ig n ifie d t i s s u e s , c u t i c l e and cork s t a in s v io le t .)

g.

(1)

Transfer s l i d e s which have been brought up th e s e r ie s o f a lc o h o ls t o th e down s e r ie s o f a lc o h o ls .

(2)

S ta in u n t i l th e d esired c o lo r i s o b tain ed .

(3)

Dip s l i d e s in water t o remove th e ex cess s t a i n .

(4)

Transfer d ir e c t ly from water t o a 95$ a lc o h o l fo r 2 t o 3 secon d s.

(5)

Transfer to a b so lu te a lc o h o l fo r 5 t o 6 m in u tes.

(6)

Clear in c lo v e o i l or x y l o l .

Mount.

(See Chapter 8 .)

Light green (C e llu lo se and collenchyma s t a in g reen .) (1)

h.

(Use as double s t a i n .)

Use w ith sa fr a n in , a s a double s t a i n .

A n ilin blue (C e llu lo se w a lls and collenchyma s t a in b r i l l i a n t b lu e .) (1)

Use w ith sa fr a n in .

83

4.

DOUBLE STAINS: a*

How t o use double s t a i n s ,

Safranin and D e la fie ld , s hem atoxylin. (L ig n ifie d t i s s u e s , n u c le i, c u t i c l e and cork s t a in r ed . C e llu lo s e , collenchym , chromatophores s t a in p u rp le,) (1)

Run up th e s e r ie s o f a lc o h o ls t o 95$*

(2)

P lace s e c tio n i n 95$ a lc o h o l fo r 3 to 5 minutes*

(3)

Transfer t o sa fr a n in , 12 to 24 hours*

(4)

Transfer to 50$ a lc o h o l u n t i l c o lo r i s r ig h t , 2 to 10 m inu tes,

(5)

P lace in water fo r 5 minutes w ith frequent changes,

(6)

P lace in D e la fie ld 's s t a in fo r 3 to 30 m in u tes.

(7)

Wash i n water 5 to 10 m inutes in frequent changes,

(8)

Whsh i n water s l i g h t l y a cid u la ted fo r $ t o 10 m in u tes,

(9)

Whsh in running water fo r 20 t o 30 m in u tes,

(10) P lace i n 50$, 95 $ and 100$ a lc o h o ls . (11) P la ce in X ylol fo r 5 m inutes, (12) Proceed to balsam mounting, b.

(se e Chapter 8 .)

Safranin and a n a lin b lu e , (1)

F ollow ste p s(l)a n d (2 )a b o v e . F ie ld * s hem atoxylin.)

(Safranin and D ela-

(2)

S ta in in sa fr a n in fo r 24 hours,

(3 )

P lace in 50$ a lco h o l u n t i l s t a in becomes weak in c e llu lo s e w a lls , but not removed com p letely.

( 4)

P lace i n a n i l i n blue fo r 2 to 10 m inutes,

(5)

Send through a 95$ a lco h o l fo r 2 to 5 secon ds,

(6)

P lace in a 95$ s l i g h t l y a c id u la ted a lco h o l fo r 5 secon d s.

84

c.

d.

e.

(7)

P lace in 95$ a lc o h o l w ith a tr a c e o f sodium carbonate fo r 1 t o 2 minutes*

(8)

P lace i n ab so lu te a lc o h o l fo r 1 t o 5 minutes*

(9)

Proceed a s i n above. (11)(1 2 ) D e la fie ld 's hem atoxylin.)

(Safranin and

Safranin and l ig h t green* (1)

Proceed from a 95$ a lc o h o l s o lu tio n .

(2)

P lace in safran in for 2 t o 24 hours*

(3)

Use a 50$ a lc o h o l far d iffe r e n tia tio n *

(4)

Dehydrate i n 95$ and 100$ a lc o h o l.

(5)

P lace in l ig h t green, minutes*

(6)

Proceed t o c le a r and mount i n balsam .

(In clo v e o i l ) fo r 3 to 30 (See Chapter 8 .)

M alachite green and Congo red* (1)

Proceed from a water so lu tio n *

(2)

P lace s e c tio n in an aqueous s o lu tio n o f m alachite green fo r 6 hours*

(3)

Vl&sh thoroughly in water*

( 4)

P lace in a 1$ Congo red aqueous s o lu tio n for 15 minutes*

(5)

Wash in water and r in se in 80$ a lc o h o l and tr a n sfe r q u ick ly t o ab solu te alcoh ol*

(6)

Proceed t o c le a r in g and mount in balsam . Chapter 8*)

(See

Iodine green and a c id fuchsin* (1)

Proceed from a water s o lu tio n .

(2)

P lace i n an aqueous io d in e green s o lu tio n fo r 12 to 24 hours,

85

(3)

Wash in water u n t i l s ta in i s alm ost a l l washed away,

(4)

S ta in w ith an aqueous a c id fu c h sin so lu tio n fo r 2 to 10 minutes*

(5)

P lace t is s u e i n a 95$ a lc o h o l and immediately pour o f f and add ab so lu te fo r a few seconds*

(6)

Proceed t o c le a r in g and mounting i n balsam* Chapter 8*)

(See

f.

Methyl green and a c id fuchsin* (See d ir e c tio n s under io d in e green and acid- fu c h s in ,)

g*

Safranin and gen tian v io le t * (1)

Bring s l i d e s down to 70$ a lc o h o l* ( Or freehand s e c tio n up to 35$ a lc o h o l.)

(2)

S ta in in sa fra n in o v e rn ig h t,

(3)

Rinse in 50$ a lco h o l u n t il s t a in i s reduced to lig h t pink*

(4)

Rinse in water and s t a in 5 to 10 minutes in aqueous gen tian v i o l e t or c r y s ta l v i o l e t .

(5) Rinse in water; dehydrate i n 95$ and 100$ alcoh ol* (6) h*

D iffe r e n tia te and proceed t o mount i n balsam* Chapter 8 .)

(See

Safranin and f a s t green* (1)

See number 1 above*

(See sa fr a n in and gen tian v io le t * )

(2)

S ta in in aqueous sa fr a n in fo r 1 to 12 hours.

(3)

R inse i n water u n t il water becomes c o lo r le s s ,

(4)

Send through baths of 50$ and 95$ acetone*

(5)

S ta in i n a 95$ a lc o h o l f a s t green s o lu tio n for 10 seconds*

(6)

P lace i n a s e r ie s o f two baths o f anhydrous acetone* (For a few seconds i n each o n e,)

86

(7) i,

j,

Safranin and f a s t green FCF, (1)

Bring down t o a t le a s t a 35% a lc o h o l s o lu tio n ,

(2)

Mordant th e s e c tio n s 1 to 3 hours i n a 1% iron** ammonia alum,

(3)

Ih sh i n d i s t i l l e d water fo r 5 m in u tes,

(4)

S ta in i n 1/10% hem atoxylin u n t il dark enough,

(5)

Wash 15 minutes in tap w ater,

(6)

Dehydrate t o 70% a lc o h o l,

(7)

C ou n ter-stain w ith a lc o h o lic e o s in ,

(8)

Complete dehydration, c le a r and mount,

Follow in s tr u c tio n under s a fr a n in -lig h t green ,

Auramine and a n ilin b lu e , (1)

m.

F ollow in s tr u c tio n under s a fr a n in -lig h t green ,

Safranin and Orange G. (1)

1,

(See,Chapter 8 ,)

Safran in and a n ilin b lu e , (l)

k,

P lace i n c a r b o l-x y lo l and proceed t o c le a r in g and mounting in balsam* (See Chapter 8 .)

F ollow in s tr u c tio n under s a fr a n in -lig h t green,

Iod ine green and a c id fu c h s in , (1)

Bring up or down th e s e r ie s o f a lc o h o ls so th a t t i s s u e i s c o rr ec t t o a 70% a lc o h o l s o lu tio n ,

(2)

Soak t i s s u e i n io d in e green fo r se v e r a l hours,

(3)

la s h in a 50% a lc o h o l s o lu tio n ,

(4)

C ou n ter-stain w ith a c id fu c h sin (b e st d ilu te d w ith 1 to 5 p a r ts o f 70% a lc o h o l) fo r 2 t o 3 minutes*

(5)

R inse and d if f e r e n t ia t e i n a s e r ie s o f ascending a lc o h o ls .

87

n.

5.

(6)

F in ish dehydration i n a b so lu te a lc o h o l.

(7)

Beware o f to o much green .

(8)

Clear and

8 .)

Bismark brown and lig h t green* (1)

Bring t is s u e up or down th e a lc o h o l s e r ie s to a 70$ a lc o h o l s o lu tio n .

(2)

S ta in very s l i g h t l y w ith Bismark brown.

(3)

D estain w ith a 50% a lc o h o l s o lu tio n .

(4)

C ou n ter-stain in l i g h t green fo r 1 t o 5 m inu tes.

(5)

D iffe r e n tia te w ith 70 % a lc o h o l u n t il th e d esir ed c o lo r i s obtain ed .

(6)

Dehydrate i n th e s e r ie s o f a lc o h o ls to 100$ a b s o lu te .

(7)

Clear and

TRIPLE STAINS: a.

mount i n balsam . (See Chapter

mount in balsam . (See Chapter

8 .)

How to use t r i p le s t a i n s .

S a fra n in , c r y s ta l v i o l e t , and orange G. (1)

S ta r t s l i d e s in a 70$ a lc o h o l. (P lace freehand s e c tio n s in a 35$ a lco h o l f i r s t . )

(2)

S ta in i n sa fr a n in fo r 4 t o 24 hours.

(3)

Change water washes th ree tim e s.

(4)

P lace i n c r y s t a l v i o l e t fo r 10 minutes to 1 hour.

(5)

Make th r ee water eash changes.

(6)

Dip in to a 50$ a lc o h o l and then w ith a p ip e tte wash w ith a 95% a lc o h o l.

(7)

Hake 2 to 3 changes i n 100$ a lco h o l and flo o d s e c tio n s w ith orange G.

(8)

Drain o f f th e orange G and r in s e w ith a p ip e tte w ith clo v e o i l .

(9)

Rinse m th c lo v e o i l or x y lo l and proceed to mounting. (See Chapter 8 .)

88

SOURCES 1*

BOOKS: A d ditional m a teria l which would be o f a s s is ta n c e i n your work. a.

2. C.

D.

See Chapter 9 , 1 0 , and 11 fo r d e t a i l s .

SPECIAL REFERENCES:

(See Chapter 9 .)

ACTIVITIES: Some la b oratory experience which g iv e s you v e r s a t i l i t y in u sin g s p e c if ic s t a i n s . 1.

EXPERIMENTING: Try as many o f th e su ggested s t a in s as p o s s ib le and compare your r e s u lt s w ith th o se which your in s tr u c to r has l a id o u t. Try th e em p irical method on your s lid e s and fin d out th e l im it s and exten t o f th e s t a i n s . Get your in s tr u c to r t o c r i t i c i z e your r e s u l t s and su g g est m o d ifica tio n s or a d d itio n s in your tech n iq u e.

2.

PERFECTING: P er fec t your technique on two or th ree d iff e r e n t s t a in s and by t h i s experience a id your c la s s mates in a r riv in g a t your f in e r e s u l t s .

3.

READING: Your b e st newer p o s s i b i l i t i e s are a v a ila b le i n th e m agazine, S ta in Technology. R efer t o t h i s magazine and search fo r techniques which you may be over look in g in your methods. D iscu ss th e se p o s s i b i l i t i e s w ith your fe llo w stu d en ts and in s tr u c to r . I f th e se methods have value to you, incorporate them i n your tech n iq u e.

EVALUATION: Some check-ups th a t w i l l a s s i s t you i n making your technique a p o lish ed procedure. 1.

RATING SCALE: S e le c t some o f your b e st s l i d e s and compare th e s ta in in g s w ith s l i d e s prepared by a B io lo g ic a l Supply House. Rate y o u r s e lf ( 1 ,2 ,3 ,4 , and 5 .) Use number 5 a s th e b e st r a tin g . Have your in s tr u c to r compare your r e s u lt s and g e t h is comments.

2.

RATING SCALE: S e le c t some o f your p r iz e s e c tio n s and r a te them by means of numbers, ( l , 2 , 3 , 4 , and 5») Use number 5 fo r th e h ig h est r a t in g . Having ra ted y o u r s e lf ta k e them t o your in s tr u c to r and se e how you a g r e e . Repeat th e procedure u n t i l you both a g r e e .

3.

RATING SCALE: Compare some of your p r iz e s e c tio n s w ith th o s e o f your neighbor by, l e t t i n g him r a te yours and v ic e -v e r s a . Rate them a s above. Let your in str u c to r check your r a tin g s .

89

BIBLIOGRAPHY

90

BIBLIOGRAPHY

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BOOKS

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1938*

2.

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3*

Bower, F.

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4.

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5.

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6.

Campbell, D. H.:

7.

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McGraw-Hill.

Cambridge U n iv ersity P r e s s .

Mosses and Ferns.

MacMillan.

Hew York, 1926.

John W iley The (Referred

1918. 1933*

8.

Conn, H. J .: H istory o f S ta in in g * Commission on Standardization o f B io lo g ic a l S ta in s . Geneva, New York. 1933*

9.

Cowry, E. V .: M icroscopic Technique i n B iology and M edicine. The W illiams and W ilkins Company, 1943*

10.

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Morphology o f Vascular P la n ts.

McGraw-Hill.

11.

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An In trod u ction t o P lant Anatomy.

12.

Gage, S . H.: The M icroscope. 16th Ed. Ith a ca , Hew York. 1935.

13*

Guyer, M ichael, F .: Animal M icrology, 4th Ed. U n iv ersity o f Chicago P r e ss. Chicago, I l l i n o i s . 1936. (R eferred t o as G uyer.)

14.

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15.

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91

16.

Loomis, W. E. and C. k i S h u ll.: McGraw-Hill. New York, 1937.

17.

McClung, G. E»: Handbook o f M icroscopical Technique. 2nd Ed. Paul B. Hoeber, I n c ., Harper. Mew York, 1937* (Referred t o as McClung.)

I:I S .

Methods i n P lant P h ysiology.

M ille r , D. F . and G. W. Blaydes: Methods and M aterials fo r Teaching B io lo g ic a l S c ie n c e . McGraw-Hill. New York, 1938.

19.

Photomicrography.

Shstman Kodak Company.

20.

S a s s, J . E.s Elements o f B otanical M icrotechnique. New York, 1940. (Referred t o a s S a s s .)

21.

Sm ith, G ilb ert M.: The Freshwater Algae o f th e U nited S t a t e s . MCGraw-Hill. New York, 1933.

22.

Smith, G ilb er t M.; 1938.

23.

T obias, C. J .: The Stu dent1s Manual o f M icroscopic Technique. American Photographic P u b lish in g Co. 1936. (R eferred t o a s B ia s .)

24.

West, G. S . and F . E. F r itsc h j B r itis h Fresh Yfater A lgae. Cambridge U n iv e r sity P r e ss. ,1932.

25.

Yamanouchi, Shigeo: Morphology and Cytology o f A lgae. U n iv e r sity o f Chicago P r e ss. 1932.

Cryptogamic Botany.

R och ester, New York. McGraw-Hill.

McGraw-Hill.

New York,

92

B.

MAGAZINES

1*

B ea tty, A. 7 . : "A Method o f Growing and fo r Making Permanent S lid e s o f P o lle n Tubes"* S ta in Technology. 12: 1 3 -1 4 . 1937.

2.

B ellin g * John: "On Counting Chromosomes i n P o lle n Mother C e lls ." American N a tio n a l. 55: 573-574. 1921.

3.

B en ed ict, H. M.: "An Imbedding Medium fo r B r it t l e or Woody T issu e s." B otan ical Gaz. . 52: 232. 1911.

4.

Conn, H. J . : "An In v e s tig a tio n o f American S ta in s ." B a c ter io lo g y . 7: 127-148. 1922.

5. .

Conn, H. J . : 1929.

6.

C r a fts, A. S .: Technology. 6:

7.

C row ell, Ivan H.: "Cutting M icroscopic S e c tio n s o f Wood Without Previous Treatment in H ydrofluoric A cid." S ta in Technology. 5: 149-150.

8 i. 9.

"Haematin and Acid Fusion."

Journal o f

S ta in Technology. 4:

"A Technique fo r Demonstrating Plasmodesma." 127-129. 1931.

97.

S ta in

Davenport, H. A. and R. L. Swank: "Bnbedding w ith Low V is c o s ity N it r o c e llu lo s e •" S ta in T ech nology.,9: 137-139• De Zeeuw, R .: "The Value o f Double I n f i l t r a t i o n i n B otanical M icrotechnique." Papers Michigan Academy o f S c ien ce . 1: 83-84. 1923.

10.

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U .

Dickson, B. T .: "The D if f e r e n t ia l S ta in in g o f P lant Pathogen and H ost." N. S . , 52: 6 3 -6 4 . 1920.

12.

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13.

Hance, R. T .: "A New P a ra ffin Embedding M ixture." 77: 3 5 3 . 1933.

14*

Haupt, A. W.: "A G ela tin F ix a tiv e fo r P a ra ffin S e c tio n s." Technology. 5: 97-98. 1930.

15.

H i l l , J . B .: "A Method fo r Dehydration of H is to lo g ic a l M ateria l." B otanical Gaz. . 51: 255-256. 1916.

S c ie n c e . S ta in

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16. 17*

H oskins, J . H .: "Transfer Method fo r Thin Rock S e c tio n s* 1" B o ta n ica l Gaz., 89s UlU-Ul5. 1930. J e ffr e y , E . C*s “Improved Method o f S o ften in g Hard T issu es.® . B otan ical G a z., 86s U56-U58. 1928.

18*

Kohl, E . J*, and C* M. Jamess “A Method f o r Ripening Hematoxylin S o lu tio n s Rapidly.** S c ie n c e , 7ht 2li7. 1931

19*

Lang, A. G. t **A S ta b le H igh -con trast Mordant fo r Hematoxylin S ta in in g .1* S ta in Technology, U s 11*9-153* 1936.

20.

Mann, A lb erts “The Preparation o f Unbroken P o lle n Mother C e lls and Other C e lls fo r S tu d ie s in M itosis.** S c ie n c e , 36s 151-153* 1 912.

21.

M cClintock, Barbaras "A Method fo r Making Aceto-carmine Smears Permanent.** S ta in Technology, Us 53-56* 1929.

22.

McWhorter, F . P ., and E. W eier: “P o ssib le Uses o f Dioxan in B o ta n ica l Micro technique • “ S ta in Technology, 11s 107-119* 1936.

23.

Plowman, A* B .s “C e llo id in Method With Hard Tissues.** G a z., 3 7 t U56-U61. 190U.

2U.

S tew art, A. B .s “The Mounting o f C e llo id in S e c tio n s in S e r ie s .* S cien ce, U2* 872. 191U*

25.

S te e r e , W. C .t **A Hew and Rapid Method fo r Making Permanent Aceto-carmine Smears.** S ta in Technology, 6 s 107-111. 1931

26.

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27.

W a lls, G. L. s "A Rapid C e llo id in Method fo r th e Rotary Microtome.® S ta in Technology, l i t 89—93* 1936.

28.

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29*

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30.

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B o ta n ica l

APPENDIX

95

APPENDIX FORMULAE FOR REAGENTS . ( As adapted from Chamberlain. ) A.

FIXING AGENTS. 1.

Absolute a lc o h o l.

2*

Bouin*s S o lu tio n , A llen M odification ! a. b. e. d.

3.

3 p a rts 3 p arts 1 part

A bsolute a lc o h o l • • • • . • • « • • • • • • • • . • • • • • G la c ia l a c e t ic a c id

3 p arts 1 part

Commercial form alin • • • • • • • • • • • . . . . . • • • Vfoter .......... ..

3 t o 10 c c . 100 c c .

Chromic a c id ........ ................................ G la c ia l a c e t ic a c id ................................. .. Water ........ ......................... ..

1 g. 1 cc. 100 c c .

Chromic a c id 10$............................................. .. A cetic a c id ...................................... Water

2 .5 c c . 10$ so lu tio n 5 c c . t o 100 c c .

Medium chromic a c id : a. b. c.

1

A bsolute a lco h o l Chloroform G la c ia l a c e t ic a c id

Weak chrom o-acetic s o lu tio n : a. b. c.

8.

p a rts p arts p a rts part

Strong chrom o-acetic so lu tio n : a. b. c.

7.

75 15 10 1

Formalin: a. b.

6.

*•••«••••

Farmer*s f lu id : a. b.

5.

P ic r ic a c id , aqueous s o lu tio n Form alin, (C. P .) G la c ia l a c e t ic a c id Urea . . • • • .......... ..

Carnoy*s flu id s a. b. c.

4*

(Used alon e w ithout any m ix tu res.)

Chromic a c id 10$... .......................... .. A cetic a cid .......... ................... Water ....................................

7*0 c c . 10$ s o lu tio n 10 c c . to 100 c c ,

Chamberlain, C harles, J .: Methods in P lant H isto lo g y . U n iv ersity of Chicago Press"! Chicago, I l l i n o i s , 1928.

The

96

9.

Chrom-osmo-acetic f lu id : a*

Flemming1s f l u i d (weak.) (1) 1% chromic a c id (in water) (2) 1% g l a c i a l a c e t ic a c id (in water) (3) l a t e r (4) 1% osmic a c id (in water)

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

25c c . 10 c c . 55 c c . 10 c c .

Keep th e m ixture ( l , 2 , 3) madeup, andadd(4) as the reagent i s needed fo r u s e , sin c e i t does not keep. b.

10.

Flemmingt s f lu id (Stronger): (1) 1% chromic a c id (2) 2J6 osmic a c id (3 ) G la c ia l a c e t ic a c id • • • •

45 c c . 12 c c . 3 cc.

Naswaschin's flu id : a. b. c. d. e.

Chromic a c id 10^ G la c ia l a c e t ic a c id D i s t i l l e d water Commercial form alin D i s t i l l e d water

1 .5 ................ 1 0 .0 .......................................... 90.0 4 0 .0 6 0 .0

cc. cc. cc. cc. cc.

Mix equal p a rts o f (a , b , c) and add ( d , e ) j u s t before u s in g . 11.

Gibson1s f l u i d : a. b. c. d. e.

12.

42 60 IS 2

cc. cc. cc. cc.

U cc.

Schaudinn's f lu id : a. b. c. d.

13.

95% a lc o h o l .............................. la t e r G la c ia l a c e t ic a c id Concentrated n i t r i c a c id Corrosive sublim ate (Saturated s o lu tio n in water ) . . . . . . . . . . . . .

D i s t i l le d water Sodium c h lo r id e Mercuric c h lo rid e A bsolute a lc o h o l

200 1 .2 ......1 0 .0 100.0

cc. gm. gm. cc.

Strom sten, s f lu id : a. b. c.

Chromic a c id 10% A cetic a c id 10$ lite r

16 cc* 100 c c . 54 c c .

When ready fo r u s e , add 2 p a r ts o f the above t o 1 part o f form alin and im m ediately immerse th e t i s s u e s .

97

STAINS. 1.

D e la fie ld * s haem atoxylin. To 100 c . c . o f satu rated so lu tio n o f ammonia alum add, drop by drop, a s o lu tio n o f 1 g . of haem atoxylin d isso lv e d i n 6 c . c . o f ab solu te a lc o h o l. Expose to a ir and lig h t far one week. F i l t e r . Add 25 c . c . of g ly ce rin and 25 c . c . o f m ethyl a lc o h o l. Allow to stand u n t il th e color i s s u f f ic i e n t ly dark. F ilt e r and keep i n a t i g h t l y stoppered b o t t l e . Let the s o lu tio n stand fo r a t l e a s t two months before u sin g .

2.

E rlich *s Haematoxylin. a. b. c. d. e.

D i s t i l le d w a t e r .50 c . c . Absolute a l c o h o l ................................................ 50 c . c . G ly c e r i n ... 50 c . c . G la c ia l a c e t ic a c i d . . ....................................... 5 c . c . Haematoxylin.............................................................1 g . Alum in e x c e s s .

Keep i t in the dark u n t il the c o lo r becomes a deep red . w e ll stop pered, i t w i l l keep i n d e f in it e ly . 3*

If

Mayer' s haem-alum. Haematoxylin, 1 g . d isso lv e d w ith heat in 50 c . c . o f 95$ a lc o h o l and added to a so lu tio n of 50 g . of alum in a l i t e r o f o f d i s t i l l e d w ater. Allow th e mixture t o co o l and s e t t l e ; f i l t e r ; add a c r y s ta l of thymol to preserve from m old.(Lee) I t i s ready for use as soon a s made up. U nless attack ed by mold, i t keeps i n d e f in it e ly .

4.

H aidenhain's iron -haem atoxylin. S o lu tio n Ai l g to 4$ aqueous so lu tio n of ammonia su lph ate o f ir o n . Use the f e r r i c ( v i o l e t ) c r y s t a l, not th e ferrou s(green ) c r y s t a ls . S o lu tio n Bs of s o lu tio n o f haem atoxylin in d i s t i l l e d w ater. The c r y s ta ls of haem atoxylin w i l l d is s o lv e in the d i s t i l l e d water in about 10 days; the s t a in reaches i t s g r e a te s t e f f ic ie n c y in about 6 weeks. About 3 months from the time i t i s made up, i t b eg in s to d e te r io r a te . A s t a in made by d is ­ so lv in g the c r y sta l in stron g a lco h o l and then d ilu tin g with water so a s to g et a p r a c tic a lly aqueous s o lu tio n i s not so good.

5

Greenacher*s borax carmine. a. b. c.

C arm ine............. Borax.................... D i s t i l le d water

3 g. 4 g. 100 c .c *

D isso lv e the borax i n water and add th e carmine, which i s q u ick ly d is so lv e d w ith th e a id o f g e n tle h e a t. Add 100 c . c . o f 70% a lc o h o l and f i l t e r ( S t ir lin g )* 6.

Alum carmine. a*

7.

A k% aqueous s o lu tio n o f ammonia alum i s b o ile d 20 m inutes w ith 1%o f powdered carmine. F il t e r a f te r i t c o o ls (L ee)*

Alum c o c h in e a l. a* b* c*

Powdered co ch in ea l ............................................. 50 g . A lu m 5 g* D i s t i l l e d water 500 c . c .

D isso lv e the alum in w ater, add th e c o c h in e a l, and b o ilj evaporate down to tw o -th ird s o f th e o r ig in a l volume and f i l t e r * Add a few drops o f c a r b o lic a c id to prevent mold ( S t i r l i n g ) . 8*

Mayer's carmalum. a. b. c.

Carminic a c i d .......................................................... 1 g . A lu m 10 g* .....2 0 0 c.c. D i s t i l le d water

D isso lv e with heatj decant or f i l t e r , and add a c r y s ta l c£ thymol to avoid mold. A good s ta in fo r Volvox* 9.

A ceto-carm ine• a*

10.

Heat a aqueous s o lu tio n o f g la c ia l a c e t ic a c id to the b o ilin g p o in t, w ith an ex cess of powdered carmine. Cool and f i l t e r .

E osin . a. b.

E o s i n ...................................................... Water or 70^ a l c o h o l ......................

1 g. . . . . 1 0 0 c*c«

99

11.

A n ilin blue* a* b.

12.

Methyl g reen . a* b. c*

13.

Light green 1 g» Glove o i l ................................................................... 75 c . c . Absolute a l c o h o l 25 c . c .

Acid fU chsin T fe te r

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

1 g. 100 c . c .

S a f r a n in 1 g. A lcoh ol, 5056..............................................................100 c . c .

Safranin (B) • a. b.

19.

Light green ............................................................... 1 g . Glove o i l .100 c . c .

Safranin (A). a. b.

IS .

1 g. 100 c . c .

Acid fu c h sin . a. b.

17.

Light green

90% a lc o h o l

Light green (C). a. b. c.

16.

1 g. ......................................... 1 c . c . ..1 0 0 c* c .

Light green (B). a. b.

15.

Methyl green G la c ia l a c e t ic a c id Viater

Light green (A). a. b*

14 .

A n ilin blue .......................................................... 1 g . &5% i r 90% a lc o h o l 100 c.c*

Safranin Water

1 g. 100 c . c .

Safranin (C)• a. b.

S a f r a n in 1 g. A n ilin (3 % a n i l i n o i l in w a t e r ) . . . . .............. 100 c . c .

100

20*

Gentian v i o l e t . a. b. c. d.

21.

Gentian v i o l e t .................................................... l g . 95% a lc o h o l 20 c . c . W a te r ............... 80 c . c . A n ilin o i l ............................................................... .. 3 c . c .

Orange G. a. b.

Orange G. Water

1 g. .100 c . c .

For most purposes a so lu tio n in clo v e o i l i s p r e fe ra b le. I t i s e a s ie r to g e t a so lu tio n by d is s o lv in g 0 .1 g . of orange in 100 c . c . o f ab so lu te a lc o h o l; then add 100 c . c . o f clo v e o i l . Let th e a b so lu te a lc o h o l evaporate u n t il there i s l e f t about 100 c . c . o f th e s o lu tio n . 22.

Bismark brown. a. b.

23*

Congo r e d . . . ............................................................. ^ g. D i s t i l l e d w ater . . . . . . . 1 0 0 c .c .

E r y th r o s in . a* b.

C.

2 g. 100 c . c .

Congo r e d . a. b.

24.

Bismark brown .................. 70% a lc o h o l

E ry th ro s in .................... 1 g. D i s t i l l e d w a ter o r 70% a l c o h o l ..................... 100 c . c .

25.

M alachite green. (Make a 1-3$ so lu tio n ) (Alcohol or aqueous).

26.

Iodine green.

(Make 1% s o lu tio n in 70% a lc o h o l) .

27.

C rystal v i o l e t .

28.

Fast green.

(Same a s gen tian v i o l e t ) .

(1-2$ s o lu tio n in 95$ a lc o h o l) .

MISCELLANEOUS. 1.

Mayerl s albumen f i x a t iv e . a. b. c.

White o f egg (a c tiv e p r in c ip le ) G lycerin (to keep i t from drying u p ) S a lic y la t e o f soda or c a rb o lic a c i d Shake w e ll and f i l t e r through lin e n .

50 c . c . 50 c . c . 1 g.

Land*s gum f i x a t i v e . a. b. c.

Guta arab ic .............. Potassium dichromate W ater . . . . . . . .............

1 g. 1 g. 98 c . c .

D isso lv e th e gum in water and add the dichromate o f potash; or d is s o lv e the gum in h a lf th e qu an tity o f water and th e dichromate of potash i n th e other h a lf , and mix ju s t before u s in g . LePage*s liq u id glue may be used in s te a d o f th e gum a r a b ic . Haupt*s g e la tin e f i x a t i v e . a. b. c. d.

G e la tin 1 g. .............................................. 2 g. Phenol G ly c e r in .................................................................... 15 c . c . Water 100 c . c .

D isso lv e th e g e la t in in d i s t i l l e d water a t 30°C .; add 2 g . phenol c r y s t a ls and th e g ly c e r in . S t ir w e ll and f i l t e r . In smoothing rib b on s, flo o d th e s l i d e w ith 29S form alin, which makes th e g e la t in in s o lu b le . C e llo id in . fc U L -.- .i

a.

.

-

To.makea 2$ s o lu tio n , add one ta b le t o f Sobering*s c e l l ­ o id in and enough e th e r -a lc o h o l (equal p arts a b so lu te a lco h o l and eth er) t o make the whole weigh 2,000 g .

C e llu lo s e a c e ta te . a. b.

C e llu lo se a c e ta te Pdre acetone ........................... lis-'ir ■r.r*' G lycerin j e lly *

a.

. . 1 2 g. 100 c .c .

One part o f f in e s t French g e la t in i s l e f t for 2 hours in 6 p arts o f d i s t i l l e d w ater. Add 7 p arts o f g ly c e r in and fo r every 100 g . o f mixture add 1 g . o f concentrated c a r b o lic , a cid • Warm th e whole m ixture for 15 m inu tes, s t ir r in g a l l th e tim e, u n t i l a l l th e fla k e s produced by th e c a rb o lic a c id have’ disappeared. F i l t e r w hile s t i l l warm, through a fine-m eshed c h e e se c lo th .

V enetian tu r p e n tin e . * . *

a.

To.make a 10$ s o lu tio n , add 90 c . c . of ab solu te a lco h o l to 10 c . c . o f th ic k V enetian tu r p e n tin e . S t ir i t w ith a g la s s rod .

102

8•

G leaning f l u i d . a. b. c.

Dichromate o f p o t a s h 20 g . Sulphuric a c i d ......................................................... 30 c . c . Water 250 c . c .

H8IVERSITY OF SOUTHERN CALIFORNIA UERAR?