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

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June 1, 1950
 £<4 /jro '

T h is p r o j e c t report, w ri tt en under the direction

of the candidate’s adv is e r a n d a p p r o v e d by him,
has been p r es e n te d to a n d a c ce pte d by the F acu lty
of the S c h o o l of E du c a ti o n in pa r ti al fu lfi llm en t of
the requirements f o r the de gre e o f M a s t e r of
Science in E ducation.

Date.... 1.9.50...........................

Adviser

Dean
 ii

PREFACE

The jun ior c o lle g e movement o f today has come a long way from merely

f u l f i l l i n g th e needs o f th e p r e -c o lle g e requirements o f former y e a r s.

More stu d en ts are spending time in a two year in s t it u t io n rath er than

going to work a fte r th e completion of a high sch o o l education. The

jun ior c o lle g e i s now o ffe r in g courses which are designed to prepare in

two y e a r s, se m i-s p e c ia lia ts who can meet th e demands o f employers.

Although emphasis s t i l l tends t o favor the needs o f th e four year

stu d en t, more e ff o r t i s being made today to recogn ize the g i f t s and

needs of th e in d iv id u a l who w i l l be confronted w ith a p ressin g problem

in two years *^
This course of study in plan t m icrotechnique, which i s presented

h ere, i s designed to meet the needs o f th e term inal stu d e n t. T h is,

however, does not mean th at the four year student has been n e g lec ted .

The su b ject m atter, although not new, has purposely been broken down

in to f i v e p arts so a s to more c le a r ly a n tic ip a te th e plan fo r each

succeeding step*

1 Vide:

Crawford, C. C ., Siem ens, C orn eliu s, I n g a lls , Rosco, and C. Gordon,

T. E .: Junior C ollege S y lla b u s* Los Angeles 43, C a lifo rn ia , 1949*

Stoddard, Alexander: Education fo r a l l American Youth. N ational

Educational A sso c ia tio n , Washington, D. C.
 ill

In order t o avoid d e ta ile d accounta o f th e variou s formulae used, a

complete l i s t o f reagen ts w i l l be found in th e appendix* Several fig u r e s

have been added throughout th e sy lla b u s t o g iv e emphasis and c la r it y to

e s s e n t ia l tech n iq u es.

As w i l l be n oted , an e f f o r t has been made t o concentrate upon

classroom a c t i v i t y in which th e doing i s emphasized. This approach i s

i n accord w ith th e modern philosophy of curriculum con stru ction i n which

th e s tr e s s has been placed on th e doing rath er than the knowing about*

" A ctivity l i e s a t th e ro o t o f a l l education* I t i s the

fundamental plank in th e fu n c tio n a l platform* Tour student
has t o do som ething, e ls e he c a n 't know anything* He lea rn s
through h is own s e l f - a c t i v it y * A ll learn in g i s a c tiv e rather
than passive* Doing must be th e foundation o f your fun ction­
a l method i f you hope to achieve r e s u lt s * 1' 2

This approach to education a ls o p la ces emphasis on "acts"and not

"facts"#

"Facts have meaning only because o f th e rela tio n sh ip s*

Facts a s ends in them selves, a s a l l to o commonly taugh t,
have no meaning* They do c o n s titu te learn in g responses
which are u s e fu l in th e world* Facts are le g itim a te ob­
j e c t s o f study i n school when th ey are them selves immed­
i a t e l y u s e f u l, o r , what i s more common, contrib ute m a ter ia lly
t o important major resp on ses*"3

2 Vide:

Crawford, C. G*: F unctional Education. C. C. Crawford,

Los Angeles 4 3 , C a lifo r n ia , 1949* Chapter V III - 3*

3 Vide:

Burton, W. H.: The Guidance o f Learning A c t iv i t i e s .

D. Appleton Century Company, New York, 1944*
 iv

This philosophy o f fu n ctio n a l education had i t s r e a l s t a r t with the

exp ression s o f a 20th century educator,, John Dewy, who even i n h is e a r ly

w r itin g s advocated doing a s a c r ite r io n for th e fu lfillm e n t of

ed u cation al growth*

”The advantage which th e a c t i v i t y o f man has in

appropriating and fin d in g meanings makes h is education
something e ls e than th e manufacture o f a t o o l or the
tra in in g o f an animal* The l a t t e r in crease e ffic ie n c y ;
they do not develop s ig n ific a n c e . The f in a l educational
importance of such occupations in play and work i s that
th ey a ffo rd th e most d ir e c t in str u m e n ta litie s fo r such
exten sion o f m eaning.” ^

I t i s intended th a t t h is sy lla b u s be placed in the hands of

stu dents a s a guide to a c tio n . I t i s hoped th a t t h i s approach w i l l be

a help to both teacher and student*

4 Vide:

Dewy, John: Democracy and Education* MacMillan Company,

New York, 1 9 1 ^ Page 243.
 V

TABLE OF CONTENTS

CHAPTER PAGE

PART I . PRELIMINARY STEPS

HOW TO PREPARE YOUR TISSUE FOR PROCESSING

1. ASSEMBLING TISSUE: How to c o ll e c t and subdivide t is s u e .......... . . . . 2

2. PRESERVING TISSUE: How to vin ta g e your t i s s u e , . . . ........... . ........... .*10

PART I I . ' NON-SECTIONING METHODS

HOW TO PREPARE SLIDES OF TISSUE THAT DO NOT
REQUIRE SECTIONING

3. SMEAR SECTIONS: Howto make smear s lid e s ....................... *.20

4* DISSOCIATION SECTIONS: How t o make te a sin g and macerated

s lid e s .................................................................. *25

PART I I I . SECTIONING METHODS

HOW TO PREPARE SLIDES BY SECTIONING

5. UNMBEDDED TISSUE: How t o produce s lid e s o f u n in filtr a te d

t is s u e .......... ..* 3 0

6. EMBEDDED TISSUE: Howto make s lid e s o f i n f i l t r a t e d t i s s u e . . . . . . . 3 5

PART IV. ATTACHMENT AND MOUNTING

HOW TO ATTACH AND MOUNT TISSUES ON THE SLIDES

7* ADHESIVES: How t o a f f i x t is s u e to th e s lid e . . .....................................4 9

8* MOUNTING TISSUE: Howto make s lid e s permanent .................... . . . . . 5 4

 vi

CHAPTER PAGE

PART V. STAINING
HOW TO SELECT THE PROPER STAINS

9. UNEMBEDDED TISSUE: How to s t a in u n in filtr a te d t i s s u e ...................... ,63

10* EMBEDDED TISSUE: How t o s ta in i n f i lt r a t e d t is s u e ............... ...........68

11* SPECIAL TISSUE: Howto s t a in s p e c if ic t is s u e .................. • • • • 7 4

12. INDIVIDUAL STAINS: How to use s p e c if ic s ta in s ........................................7 8

BIBLIOGRAPHY: Books and magazines . . . . . . ......................... . . . . . . . . . . . . . . . . . . 9 0

APPENDIX: Formulae fa r reagents ................. .............9 5

 PAGE
INDEX OF FIGURES

FIGURE 1 . Subdividing and embedding t is s u e . . . . . . . . . . . . ........... 6

FIGURE 2 . S u bd ivision of m assive c y lin d r ic a l organs • • • • • • • • • » • • • • • . « 7

FIGURE 3 . O rien tation o f k n ife and t i s s u e . P a ra ffin ribbons . . . . . . . . 4 2

FIGURE 4* Steps in sharpening a microtome k n ife ............. 44

FIGURE 5 . Spacing and arrangement o f se c tio n s ...........• ♦ . . • • • ♦ 5 2

FIGURE 6 . Suggested stainingw dish arrangement • • • • • • • • . . . . . . • • . . . • • ♦ ♦ 7 1

 1

PART I , PRELIMINARY STEPS

HOT TO PREPARE YOUR TISSUE FOR PROCESSING

The r e ta in in g o f str u c tu r a l d e t a ils o f c e l l s and t is s u e s i s

in flu en ced in grea t measure by th e co n d itio n o f the p lan t a t th e tim e

o f c o lle c t io n and treatm ent subsequent to c o lle c tio n * To know good

c o lle c t in g and preserving techniques means a g rea ter su ccess in a l l the

ste p s th a t follow * In the two chapters which fo llo w i s m a teria l th a t

w i l l be o f fundamental importance to you in developing your b o ta n ic a l

microtechnique*
 2

CHAPTER 1 . ASSEMBLING TISSUE

HOW TO COLLECT AND SUBDIVIDE TISSUE

A. MOTIVATION: Rewards t h a t w i l l be yours i f you lea rn how to

c o ll e c t and subdivide your t is s u e s p rop erly.

1* GREATER. SUCCESS LATERs The making o f g ood s lid e s m i l be

r e la te d t o your s u c c e s sfu l e f f o r t s a t t h i s time*

2* LESS DISTORTIONS Too la rg e and too sm all a sample w i l l g iv e

g iv e an unnatural s l i d e o f too l i t t l e or no value*

3* PERSONAL SATISFACTION: There i s p rid e in knowing a job i s

w e ll done*

B« PRESENTATION: Suggestions th a t w i l l f a c i l i t a t e in a b e tte r job

o f s e le c tin g and subdividing your tis s u e *

DIRECTIONS

1, SELECTION: How t o s e le c t your specimens*

a* Decide what you are going to work on*

b. P ick out only normal tis s u e * (Unless you are in te r e ste d

in abnormal con d ition s*)

c* Make c a r e fu l choice o f r ep resen ta tiv e m a teria ls in

p e r fe c t condition#

d* Spend time fin d in g th e most h ealth y tis s u e s *

2* LEAVES? How to c o ll e c t leaves*

^ a* Remove l e a f or l e a f l e t s by c u ttin g w ith a sharp k n ife a t

. th e p e t io le w ithout squeezing or p u llin g*

b* Transport th e t i s s u e fo r a short d ista n ce between sh eets

o f wet paper tow eling and keep them in a clo sed container*

c* Use a t i n can or ja r fo r the container*

d* Freshen up t is s u e s upon a r r iv a l a t th e lab oratory i n a

m oisture chamber b efore p rocessin g them#
STEMS: How to c o lle c t stems*

a* Remove from th e p la n t w ith a sharp k n ife w ithout in ju r y .

b. Keep t is s u e s fr e sh by standing them in a container o f

water or in th e r efrig e ra to r *

c* I f such storage i s not handy, cut th e steins in to th e

lo n g e st p ie c e s th a t w i l l f i t in to your con tain er without
being crushed or folded* Wrap th e p ie c e s promptly i n a
wet paper and sto r e in a c o o l spot*

d. Store dormant woody tw ig s, la rg e limbs and d isk s cut

from lo g s in th e r e fr ig e r a to r u n t il processing*

ROOTS: How to c o l l e c t r o o ts •

a* Do n ot c o ll e c t by p u llin g up th e p la n t.

b. Dig up th e p la n t and soak th e mass o f s o i l andro o ts in

water u n t i l thoroughly softened*

c. la s h the s o i l away c a r e fu lly and cut o f f the d esired r o o ts .

d* Brush g e n tly w ith a f in e brush to remove as much s o i l as

p o ssib le *

e* Wrap t h e p ie c e s and sto r e (a s in the case o f stem s) u n t il

processing*

FLORAL ORGANS: Hew t o c o lle c t f l o r a l organs*

a* Remove the e n tir e flow er or flow er c lu s t e r .

b* Wrap in wet paper*

c* Keep la rg e buds in a Mason ja r o f water u n t il d isse c tio n *

d. Do th e same fo r fr u its *
 u

6* LIVERWORTS AND MOSSES: How to c o l l e c t liv e r w o r ts and

mosses*

a. Remove m aterial w ith a mat o f th e su b strata covering

th e p lan t*

b* S tore th e p la n ts in a m oisture chamber u n t i l they are

turgid*

c* Saturate th e su b stra ta u n t i l a l l or most o f th e s o i l has

been removed*

d* D is s e c t th e p a rts d esired under a microscope and tran s­

f e r th e t is s u e s fo r processing*

7* AIGAE: How t o c o l l e c t a lg a e .

a* C o lle c t in th e water i n which th e p la n ts are growing*

b. Keep th e se p la n ts in t h e ir natu ral substratum (water)

( s o i l ) u n t i l th e moment o f k i l l i n g .

c* Avoid su b jectin g th e p la n ts to e x ce ssiv e h eat or to

d e s s ic a tio n during storage or tran sp ortation*

d* Keep in a c o o l p la ce i n subdued lig h t*

e. T ransfer fo r immediate processing*

8* FUNGI: How t o c o lle c t fle s h y fungi*

a. Transport and sto r e in lo o s e ly wrappedwaxpaper.

b. Wrap sm all fu n g i in m oist paper enclosed in waxed paper*

c. P rocess as soon as p o ssib le *

9* DISEASED: How t o c o ll e c t p a th o lo g ic a l m aterial*

a* Take care th a t the co n d itio n o f th e h o st t is s u e s are

n ot a lte r e d b y handling*

b. Prevent w iltin g o f m aterial by p la cin g in a m oisture

chamber*
 5

c. Avoid the development o f "bacteria, molds or other

secondary organisma.

d. Always c o lle c t along w ith the d isea se d m a teria l, p e r fe c tly

normal t is s u e fo r comparisons l a t e r .

10, SUBDIVISION: How to subdivide the m aterial p rior to

p ro cessin g . (See fig u r e s 1 and 2 .)

a. Remove your c o lle c te d p ie c e s and p lace them before you,

b. S e le c t the d esired fe a tu r e s and o r ie n t them so as to

e s t a b lis h planes in which se c tio n s are going to be cu t.

c. Out in to su ita b le working p ie ee s w ith minimum bruising*

compression or d e sic c a tio n .

d. In se rt the p ie ce s promptly in to the k i l l i n g flu id .

e. Record the necessary data concerning s p e c ie s , lo c a tio n ,

d a te , p a rts s e le c te d and k i l l i n g f l u i d used.
SOURCES

1. BOOK SHELF: Some referen ces which w i l l provide you w ith

a d d itio n a l inform ation, (Books th at are used fo r frequent
reference are c ite d as below . See the B ibliography which
b egins a t page 90 fo r the complete c it a t i o n .)

a. Chamberlain, Chapter (XVI-XXVII.)

b* HcClung, pages (157-158.)

c. S ass, pages (5 -2 2 .)

2, SPECIAL REFERENCES: Some sp e c ia l referen ces th at may help

you w ith some sp e c ia l d e t a i ls .

a. Smith, S . M.: "The P reservation of Fresh Water Algae”.

if

b. West, G. S ., and F r its c h , F. E .: B r it is h Fresh Water

A lgae. Cambridge U n iv ersity P ress, 1927.

c, Yamanouchi, Shigeo: Moronhology and Cytology of A lgae.

U n iv ersity of Chicago P ress, 1932.
 LONG NARROW LEAF

DIVISION OF A
& BROAD LEAF

LONG NARROW LEAF

D.

ANTHER OVARY OVARY

EMBEDDING OF LONG LEAF SECTION

FIGURE 1 .

SUBDIVISION AND EMBEDDING TISSUE

Adapted from S a s s. (See bibliography for complete referen ce)

 7

V ,

A.

TISSUE IN AXIS

B.

TRIMMED BLOCK

SECTIONS FROM A
LARGE LOG

D. .

TRIMMED BLOCK

FIGURE 2 .

' SUBDIVISION OF MASSIVE CYLINDRICAL ORGANS

* Adapted from S a ss. (see bibliography fo r complete referen ce)

 8

d* Gwynne, Vaughan, H.C.L., and Barnes, B.: The Structure

and Development of th e Fungi* Cambridge U n iversity P r e ss,
1927.
e. Campbell, D. H.: Mosses and F erns. MacMillan, New York, 1918.

f. Bower, F . V . : The Ferns, Cambridge U n iv ersity P ress, 1926.

g. Smith, G ilb e r t, M.: The Freshwater Algae o f the United

S t a t e s . McCraw H i l l , New York, 1933.

C« ACTIVITIES: Pro.jects which w i l l a id you i n g e ttin g experience

in c o lle c tin g and subdividing t i s s u e .

1. COLLECTING TISSUE: Gather tis s u e s of th e various typ es and

subdivide them in preparation fo r p ro cessin g .

2. SPECULATING: A fter having used the sugge ste d tech n iq u es,

attempt to p e r fe ct them and make u se o f some o r ig in a l methods
t o g e t b e tte r r e s u lt s . Check th ese r e s u lt s m th th ose already
completed by the methods suggested.

3. BEADING: Refer to th e suggested read in gs so as to make use

o f other methods of c o lle c t io n and su b d iv isio n . Check your
r e s u lt s vdth th e e x h ib ite d s lid e s presented by the in s tr u c to r .

D. EVALUATION: Samples o f ways i n which your c o lle c t io n and sub­

d iv is io n techniques may be checked.

1. TRUE-FALSE: P lace an X in th e correct space for true or f a l s e .

T F

a. ( )( ) The s e le c t io n o f normal r ep resen ta tiv e t is s u e

should always be foremost in your mind.

h. ( )( ) Folding and crushing should be avoided in

c o lle c t in g t i s s u e .

c. ( )( ) Large se c tio n s o f t is s u e should not be divided

in to sm aller p ie c e s .

d. ( )( ) Delay i n tra n sferrin g t is s u e from the source to

m oist container should be avoided a t a l l tim es.

e* ( )( ) You should e s t a b lis h the plane o f se c tio n in g a t

the time o f su b d iv isio n .
f. ( ) ( ) You should tra n sfe r the t is s u e s as soon as
p o ssib le to a f ix a t iv e f l u i d .

RATING SCALE: Plg.ce X i n each space fo r which your c o lle c t ­

io n and su b d iv isio n plans can q u a lify .

s. ( ) You should s e le c t healthy and normal t is s u e ,

b. () Algae should he transported in the water in which
th e plan t grew.

c. {, ) You should determine ahead of time the t is s u e you

are going to gather.

d. () You should record a l l neeessary data.

e. V) You should e lim in a te a l l odd or str a y t i s s u e s .

f. () B a c te r ia l development should he avoided in patho­

lo g ic a l m a ter ia l.
 10

CHAPTER 2 . PRESERVING TISSUE

HOW TO VINTAGE YOUR TISSUE

A. MOTIVATION: Success which you m i l ach ieve i f you master the

p r in c ip a ls o f proper p ro cessin g .

1* SUPERIOR SLIDES: Your fin is h e d product i s now in the most

c r i t i c a l and important phase,

2. NORMAL TISSUE: The aim o f a l l s lid e making i s to have a

product which resem bles liv in g m a te r ia l.

3. ECONOMY OP TIME: A ll ste p s from now on depend on the care

and e f f ic ie n c y th a t was taken during p r o c essin g .

B. PRESENTATION: Ideas th a t w i l l a s s i s t you to do th e b e st p o ssib le

job o f p ro c essin g .
DIRECTIONS

1. FIXATIVE: How to s e l e c t th e b e st fix in g and preserving

flu id s .

a. Decide upon what stru ctu res you are going t o stu d y.

b. Plan on the s t a in t o be used and i t s rea c tio n t o th e

f i x a t iv e .

c. Use the em pirical method and by means o f elim in a tio n make

a s e le c t io n .

d. S e le c t th e proper reagent according to the job to be done.

(1) Absolute e th y l a lc o h o l. (To be used a s a fa ir

so lu tio n i f immediate r e s u lt s are d esired on a l l
t i s s u e .)

(2) Form alin.

(a) Use on ly chem ically pure s o lu tio n s .

(b) Use a s a f a ir f i x a t iv e bub w ith unpredictable

r e s u lt s because o f shrinking and o v e r fix a tio n .
 11

(c ) Use on t is s u e in which th e f a t s , p r o te in s, and

o i l s are t o be preserved.

(3) G la cia l a c e t ic a c id , (use a s a weak f i x a t iv e in

which f a t- s o lu b le a c id are used to counteract th e
sw e llin g a c tio n .

(4) B onin's S o lu tio n , A lle n 's M od ification .

(a ) Use when th e b r illia n c y o f H eidenhain's

hem atoxylin i s d e sir e d .

(b) S e le c t fo r chromosome counts.

(5) Carnoy's F lu id .

(a) Beware o f extreme shrinkage.

(b) U sefu l in p en etratin g hairy and c u tin iz e d

s u r fa c e s .

(c) E sp e c ia lly u s e fu l a s a pretreatm ent before

chrom-osmo-acetic m ixtures.

(6) Chrom-acetic S o lu tio n s. (Weak, medium, and str o n g .)

(a) E x cellen t power o f p en etra tio n .

(b) P reserves th e chromatic in th e coarser a sp e c ts.

(c) F a ils t o p r e c ip ita te some o f the element o f

cytoplasm .

(d) P reserves th e prophases with the metaphases o ften

dumped and th e chromosomes contract lo n g itu d i­
n a lly .

(?) Farmer's F lu id . (Remarks under Garnoy's f lu id apply

here*)

(6) Chrom-osmo-acetic F lu id s.

(a) G ives by fa r the more accurate rep resen ta tio n o f

most o f the elem ents in p lan t c e l l s .
 12

(b) Caution i s su ggested here as th e s o lu tio n i s un­

sta b le and each chemical should be kept separate
u n t il the tim e o f u se.

(9) Gib3on*s F lu id .

(a) Recommended fo r f le s h y fb n g i.

(b) F lu id i s not s t a b le .

(10) Navashin F lu id -B e llin g fs M odified. (E xcellen t fo r

smears o f a n th ers, buds, and root t i p s . )

(11) Schaudinn's F lu id . (Widely used for U n ic e llu la r

a lg a e .)

(12) Strom sten's F lu id . (General a l l around f i x a t i v e .)

2. KILLING AND FIXING: How to use k i l l i n g and fix in g a g en ts.

(Immerse t is s u e in four tim es i t s volume o f f l u i d .)

a. How to use Absolute e th y l a lc o h o l.

(1) Do not f i x fo r over an hour a f t e r immersion.

(2) Pour o f f f l u i d a f t e r f ix a t io n and im mediately add

fr e s h ab solu te e th y l a lc o h o l, changing each on e-h alf
hour for two hours.

(3) Proceed to th e embedding.

b. How to use form alin.

(1) Immerse for an in d e f in ite tim e.

(2) F ix marine algae in a 6 t o 10% s o lu tio n .

(3) Proceed t o dehydration.

c. How to use g la c ia l a c e t ic a c id .

(1) Place t is s u e in the f lu id fo r not more than 15

m inu tes.

(2) Wash w ith 30 to 50% e th y l a lco h o l u n t i l f ix a t iv e i s

washed com pletely away.
 13

(3) Transfer t i s s u e t o a 10% a lc o h o l fo r sto r a g e ,

(4) Proceed t o dehydrate*

d. How to use B o u lin 's s o lu tio n , Allen* s m o d ific a tio n ,

(1) P lace t is s u e in th e f lu id and a llo w fo u r hours for

f i x in g .

(2) Remove t is s u e and make se v e r a l changes o f 10% e th y l

a lc o h o l over two days u n t i l no mere y e llo w c o lo r
rem ains,

(3) Allow th e t is s u e t o remain in 10% e th y l a lco h o l u n t il

ready fo r dehydration.

e. How t o use Carney's f l u i d ,

(1) F ix t is s u e s for two t o th ree hours o n ly ,

(2) F ix ro o t t i p s fo r o n ly 1$ m in u tes,

(3) Wash in two changes o f 95% e th y l a lc o h o l and proceed

as q u ic k ly as p o s sib le t o the p a r a ffin embedding,

f. How t o use chrom -acetic s o lu tio n s , (weak, medium, and

str o n g .)

(1) Weak: f i x filam entous a lg a e , fu n g i, fe r n s , and

mosses fo r 20 m inutes,

(2) Medium: f i x ro o t t i p s and sm all ova ries for 12 hours,

(3) Strong: f i x woody m a teria l and tough le a v e s for 24

hours,

(4) Wash a l l t is s u e s in running water and proceed to

dehydrate,

g. How t o use chrom-osmo-acetic f l u i d s , (weak and s tr o n g .)

(1) Weak: use for d e lic a t e t is s u e s fo r 4 to 5 hours,

(2) Strong: use fo r more r e s is ta n t t is s u e s fo r 12 to 24

hours.
 u

(3) la sh a l l t i s s u e s in running water and proceed t o

dehydration.

h. How t o use Gibsonf s f l u i d .

(1) Allow f l u i d t o r e a c t fo r 18 to 20 hours.

(2) Wash out thoroughly w ith 50£ e th y l a lc o h o l to remove

mercuric d e p o sits from the se c tio n s*

(3) Proceed t o dehydration.

i. How to use B e llin g *s m odified Navash in f l u i d .

(1) F ix smear fo r 12 hours com pletely immersed.

(2) Transfer smears to a stend er o f 0 . 5% aqueous chromic

a c id fo r not longer than 10 minutes t o remove form alin

(3) Proceed to s ta in in g .

J. How to use Schaudinn, s f l u i d .

(1) F ix fo r se v er a l hours.

(2) Wash out w ith medium stren gth a lco h o l t o which has
been added io d in e s o lu tio n t o remove mercury d e p o s its .

(3) Proceed t o dehydration.

k. How to use Stromsten*s f l u i d .

(1) Immerse t is s u e fo r an in d e f in it e p e r io d .

(2) Wash t is s u e out thoroughly w ith 9Q£ e th y l a lco h o l

Ju st before embedding.

3. DECALCIFYING: How t o d e c a lc ify t i s s u e .

a. P lace th e t is s u e i n a d ilu te s o lu tio n o f hydrochloric a c id .

b. Change the f lu id f r e e ly u n t il a l l lim e has been d is s o lv e d .

c. Yfesh the, t i s s u e in running w ater.

d. P lace in storage f l u i d . (7Q£ e th y l a lc o h o l.)

 15

*4-. BESILICIFYCATI01T: How to d e s i l i c i f y p lan t tissu e s *

a. P lace the specimens to be tr e a te d in a wax v e s s e l and

flo o d with, h yd ro flu o ric a c id .

t>. Let the t is s u e stand fo r se v er a l days in f l u i d .

c. Wash the t is s u e in running water and store u n t i l ready

fo r dehydration.

5* SPECIFIC DEPOSITS: How to preserve s p e c if ic c e l l d ep osits#

a . ■In u lin .

(1) Sub-divide fr e s h ly cut m a teria l in to b lock s o f about

1 cm.
(2) Treat in se v er a l changes o f ab solu te a lco h o l to re­
move water q u ick ly . (Be sure to cut t is s u e s w ith
k n ife flood ed w ith ab solu te a lc o h o l. )

b. Latex.

(1) C o lle c t t is s u e s quickly to prevent lo s s o f f l u i d

b efore f ix a t io n .

(2) Store in 70$ a lc o h o l.

c. R esin.

(1) Immerse t is s u e in a satu rated aqueous copper a ceta te

so lu tio n fo r one to sev era l weeks*

(2) Wash e x cess eopper away w ith water and preserve in

a 50$ a lc o h o l.

d. Starch.

(1) Preserve in a lco h o l rath er than in form alin.

( 2) I f starch in good co n d itio n i s d esired preserve in

a c id s th a t hyd rolyze.
 16

7* DEHYDRATION: How to dehydrate plant M aterial.

a. Do not a llo w t i s s u e s to dry between the tr a n s fe r s to th e .

dehydrating f lu id s .

b. Use only sm all p ie c e s (2 to 6 am. cubes) of t is s u e when

ever p o s s ib le .

c. Fix and harden in the f lu id which i s 10 to 50 tim es th at

o f th e f l u i d .

d. Wash t is s u e in g e n tly running water before s ta r tin g de­

hydration. (Do not scrape o f f fo r e ig n m a tter.)

e. Keep the reagen ts and preparations from th e d ir e c t sun­

lig h t.

f. Label a l l v e s s e ls o f t is s u e c a r e fu lly . (S ta te co n ten ts,

f lu id s used, and the d a te .)

g. Keep a c a r e fu l record of the reagents used on each

separate t is s u e and the time changed.

h. P lace form ol-alcoh ol fix e d m a teria ls in 70% a lco h o l a fte r

properly washing.

i. Place filam entous organisms in a 5% g ly c e r in so lu tio n

and a llo w t o evaporate u n t il a th ic k syrup forms.

(1) When ready t o embed, wash in 9556 e th y l-a lc o h o l.

j. P lace m ateria ls fix e d i n chrom -acetic and chrom-osmo-

a c e t i c , a fte r a complete washing, in a s e r ie s o f th ree
to four e th y l-a lc o h o ls (15%, 25%, and 50% and 70%) for
th ree to s i x hours in each.

k. P lace a l l other t is s u e s to be embedded in a s e r ie s of

a lc o h o lic baths (e th y l - 15%, 25%, 50%, and 70%) for
a period of th ree hours s t a r tin g with the n ext high est
a lc o h o l th a t the f ix a t iv e had contained.

1. An a lte r n a tiv e method i s to dehydrate in so lv e n ts o f

p a r a ffin . (Three-butyl a lc o h o ls . The Dioxan Method.
Chapter 8 .)

m. Proceed to embedding or t o storage f l u i d s .

 17

8. STORAGE: How to sto r e t i s s u e .

a. Place t is s u e s in a 10% e th y l a lco h o l for a l l general

sto r a g e .

b. P la ce prolonged sto red t is s u e in th e above p lu s 5 to 20%

g ly c e r in s o lu tio n .

SOURCES

1. BOOKS: Some r e la te d readings which w i l l be of a ssista n c e

t o you.

a. Chamberlain, pages (18-41); (2 7 -4 8 .)

b. MeClung, pages (158-163); (215-220.)

c. Johansen, pages (2 7 -4 8 .)

d. S a ss, pages (1 5 -2 2 .)

2. SPECIAL REFERENCES: A d ditional a id s i n your m astery.

a. Haup,4A. W.: "A G ela tin F ix a tiv e fo r P a ra ffin S e c tio n s."

S ta in Technology. 5: 97-98. 1930.

b. H i l l , J . H.: "A Method fo r Dehydration o f H isto lo g ic a l

M aterial." B otanical G a zette. 51: 255-256. 1916.

C. EVALUATION: Samples o f check-ups which may be a help in your

mastery o f th e p rocessin g o f t i s s u e .

1. RATING SCALE: P lace an I i n each space fo r which your

p ro cessin g technique q u a lify s .

Yes No

a. ( ) ( ) You should use th e reagent th a t does the Job b e st

b. ( ) ( ) You should reserv e starch es in a lco h o l in ste a d

o f form alin.

c. ( ) ( ) You should save r e s in s by immersing the t is s u e

in a satu rated copper a c e ta te s o lu tio n .
 18

d. ( ) You should keep a record fo r th e progress and

th e time for each s te p .

e. ( ) You should use p ie c e s 2 to 6 mm. cubes.

f. ( ) You should dehydrate in progressive steps of 15,

25, 50, and 70% alcohols.

g. () Chrom-osmo-acetic fluid should be used for the

most truthful representations.

h. ( ) The experim ental technique should be used .

2. TRUE-FALSE: P lace an X i n the co rrect space fo r tru e cr f a l s e .

T F

a. () You should not consider the stain to be used when

selecting a fixative.

b. () You should make normal tissue your major aim.

c. () You should use form alin a s a f ix a t iv e because

th ere i s never a chance o f shrinking or over­
f ix a t io n .

d. () You should use Navashin's f lu id for smear se c tio n s,

•. ( ) You should use one o f th e b e st gen eral storage

flu id s .

f. ( ) You should add a 5 to 20% solution of glycerin

to the alcohol for prolonged storage.

g. ( ) You should use hydrofluoric acid far desilici-

fication.
 19

PART I I . NON-SECTIONING METHODS

HOW TO PREPARE SLIDES OF TISSUE THAT DO NCT REQUIRE SECTIONING

S k i l l in making s e c tio n s by te a s in g with n eed les and in making

d e lic a t e d is s e c tio n s under the m icroscope i s n ecessary in any worth­

w h ile in v e s tig a tio n which d ea ls w ith th e str u c tu r e and development o f

p la n ts . Such developed techniques not o n ly giv e a broader view o f

stru ctu res in a l l dim ensions, but a ls o help to in te r p r e t sta in ed micro­

tome preparations a s w e ll a s t o g iv e th e in v e s tig a to r an id ea o f whether

th e m a teria l i s worth a l l th e lab or o f making permanent mounts.

 20

CHAPTER 3 . SMEAR SECTIONS

HOW TO MAKE SMEAR SLIDES

A. MOTIVATION: Galas th a t w i l l be yours i f you lea rn t o prepare

smear s l i d e s .

1. RICH EXPERIENCE: New horizons a re always beckoning the

am bitious student *

2. ACADEMIC ACHIEVEMENT: P er fe c tio n of an unusual technique

means a higher s c h o la s tic stan d in g.

3. FINANCIAL RETURNS: The f i e l d o f advancement i s always open

t o th o se who can use a wide v a r ie ty o f tech n iq u es.

B. PRESENTATION: P r a c tic a l s uggestio n s to help you in th e d e t a ils o f

making smear s l i d e s .

1. PREPARATION: How to prepare your s l i d e s fo r smear s e c t io n .

a. Use s lid e s which are chem ically d e a n .

b. New s l i d e s should be given a long immersion in sulphuric

acid-potassium bichromate m ixture.

(1) Rinse in running water and p lace for a sh ort time in

a strong a lc o h o l t o which a l i t t l e ammonia has been
added.

(2) R inse again and dry with an a b s o lu te ly clea n c lo th

fr e e from sta rch and l i n t .

2. JOHANSEN'S METHOD: How to make smears by the Johansen Method.

a. Use smear methods fo r th e study of chromosome cou n ts.

b. S e le c t fr e sh anthers or onion root t i p s .

c. Use Navashin*s f ix a t iv e on the t i s s u e . (See Chapter 2 .)

 21

d. Use P e tr i d ish es fo r carrying out the f ix a t io n .

(l) Use some sle n d er g la s s rod s, cut to th e proper len g th

a t op p osite ends o f th e lower h a lf of the d ish to
keep th e s l i d e o f f the bottom.

e. P lace the t is s u e to be used (about 2 mm. long) on the

s l i d e and with a clean s c a lp e l, q u ick ly and ev en ly crush
and spread th e m a teria l cnrer th e cen ter o f th e s l i d e .

f. Immediately in v e r t th e s l i d e and p lace in th e P e tr i d ish

so th a t th e e n tir e smear surface comes i n instantaneous
contact w ith the k i l l i n g f l u i d .

g. Leave in the f i x a t iv e fo r 4 hours.

h. A fter fix a t io n i s com pleted, turn th e s l i d e r ig h t s id e up

and w ith forcep s remove th e t is s u e fragments and other
th ic k p ie c e s o f debris th a t might unduly e le v a te the
c o v e r slip when mounted.

i. P lace i n a low sta in in g d ish and wash for about 15 minutes

i n g e n tly running w ater.

j. Examine th e t i s s u e under the microscope fo r a l l the

d esir ed m a te r ia l.

k. Proceed t o s ta in in g . (See Chapter 9 .)

3. TUAN'S MODIFIED HEMATOXYLIN METHOD: How t o make sugars by

th e Tuan m odified hem atoxylin method.

a. S e le c t fr e sh anthers or fr e sh onion root t i p s .

b. Use fo r th e study o f th e metaphase chromosomes.

c. Make smears as i n above. (See Johnson Method above.)

d. F ix and k i l l in Navashin's f l u i d or i n a chrom-osmo-acetic

mixture fo r about 20 m in u tes. (See Chapter 2 .)

e. Wash i n running water fo r 20 m inutes.

f• Bleach i f n ecessary w ith d ilu te d hydrogen peroxide and then

wash a g a in .

g. Mordant in 2$ ferric ammonium sulphate for 20 minutes.

 !
22

h. T
$ash in running viat e r for 6 to 10 minutes and r in s e in
d i s t i l l e d w ater.

i. Proceed to s t a in in g . (See Chapter 9 .)

4. CAPINPIN'S BRAZILIN METHOD: How to make saears by the

Capinpin's B r a z ilin method.

a. S e le c t fre sh onion ro o ts or a n th er s.

b. Use fo r the diaphase chromosomes.

c. Make th e smears as in the Johansen method.

d. Use th e Navashin f lu id fo r f ix a t io n . (See Chapter 2 .)

e. Wash in water fo r about 15 minutes u n t i l the fix a t iv e

has been removed?

f. Pass through 1 5 , 3 0 , 50, and 7056 a lc o h o ls allow in g 10 to 15

m inutes in each . Allow to remain overnight i n th e 70%
a lc o h o l,

g. Mordant overnight i n a fr e s h ly prepared 1% s o lu tio n o f

f e r r ic ammonium su lph ate in 7056 a lc o h o l.

h. Ifesh about 45 minutes i n se v e r a l changes o f 70£ a lc o h o l,

5. MISCELLANEOUS METHODS: How to make smears by va rio u s methods.

(See Johansen fo r in form ation .)

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

b. McCallum’s Iron-propionocarmin method,

c. M cClintock's Permanent acetocarm in method.

d. Z ir k le ts method,

e. Whrmke1s method •

f. H illa r y 's method.

 23

SOURCES

1. BOOK SHELF: Works in which you v d ll fin d a d d itio n a l

a s s is t a n c e .

a. Johansen, pages (155-169.)

b. MeClung, pages (165-173.)

c. S a s s, pages (110-111.)

2, SPECIAL REFERENCES: Some a d d itio n a l a id s which may help

you.

a. McClintock, Barbara: "A Method fo r Making Acetocarmin

Smears." S ta in Technology. 4: 53-56. 1929.

b. B e llin g , John: "On Counting Chromosomes in P o llen

Mother C e lls 11. American N a tio n a l. 55: 576-564. 1921.

c. Mann, A lbert: "The Preparation o f Unbroken P o llen

Mother C ells and Other C e lls fo r S tu d ies in M ito sis" .
S c ien ce . 36: 151-153. 1912.

C. ACTIVITIES: P ro je cts th a t w i l l help you c u ltiv a te a c le a r and

co rrect technique fo r smear s lid e making.

1. SELECTING: Pick one or more o f the methods o f making smears

and fo llo w through th e technique u n t il s a t is fa c to r y r e s u lt s
have been obtain ed . S e le c t the method i n which you e x c e l.

2. ASSISTING: P erfect your technique by rep eatin g the method,

and ask your in s tr u c to r t o go over your r e s u lt s w ith you.

3. READING: Refer t o the magazine, S ta in Technology, and

search fo r new or valu ab le su ggestion s which you could in ­
corporate in your tech n iq u e.

D. EVALUATION: Samples o f some typ es o f check-ups which may be

a p p lied to your handling o f smear s l i d e s .

1. COMPLETION: Supply th e m issin g word in the sta tem en ts,

a. You should u s e s l i d e s th a t are c le a n .

 24

b. You should use .f ix a t iv e fo r smears by the

Johansen method.

You should use Capinpin's B r a z ilin method for

chromosomes.

d. You should use the Tuan's Hematoxylin method for

chromosomes.

e. New s lid e s should be properly washed in a c id .

f* P e tr i d ieh es should be used fo r fixation.

2. RATING SCALE: Place X i n each space whereby your smear

s lid e technique can b e n e fit you.

Yes No

a. ( ) ( ) You should gain a r ic h experien ce from th is method.

b. ( ) ( ) Navashin's f ix a t iv e should be used w id ely.

c. ( ) ( ) The smear technique should be used for

chromosome s t u d ie s .

d. ( ) ( ) You should gain fin a n c ia l returns for t h i s

exp erien ce.

e. ( ) ( ) You should consider the co n d itio n o f your

s lid e s as h ig h ly im portant.

3* BEST ANSWER: P lace th e number o f th e b e st answer in th e

p aren th eses.

a. ( ) For th e b e st smear s lid e s in th e study o f the

diaphase in c e l l d iv is io n you should use the
( l ) Capinpin's method. (2) Tuan method.
(3) Johansen method. (4) Whrmfee method.

b. ( ) In making smear s l i d e s you should use:

(1) Old r o o ts . (2) Stems. (3) Fresh an th ers.
(4) Developed f r u i t s .
 25

CHAPTER 4 . DISSOCIATION SECTIONS

HOW TO MAKE TEASING AND MACERATED SLIDES

A, MOTIVATION: B e n e fits th a t accrue from s k i l l f u l l y made s lid e s o f

m a teria l th a t has been tea sed or macerated.

1. WELL PAID CAREER: Employers are always on the lo o k -o u t for

e x c e p tio n a lly tra in ed and s k ille d men.

2* BETTE? EDUCATION: The wider your scope o f ex p erien ce, the

more complete has been your train in g*

3* FINANCIAL SECURITY: Independence and se c u r ity are d ir e c t ly

lin k e d w ith p e r fe cte d techniques and p r a c tic e s lea rn ed .

B. PRESENTATION: Some p o in ters th at w i l l help you i n th e proper

methods o f te a sin g and macerating t is s u e fo r s l i d e s ,

DIRECTIONS

1* TEASING: How t o make s lid e s by th e te a sin g method*

a* Use fo r t i s s u e f ib e r s and fo r stomata exam ination o f whole

mounts*

b. Use two n eedle se ts* (One p air w ith extrem ely f in e p o in ts

fo r f i n a l sep aration and a p air o f blu n t poin ted fo r rough
work*)

c* Be sure th a t th e n eed les are d e a n and sharp* (Sharpen

w ith a fin e carborundum hone*

d* E ither f i x t is s u e before or a f te r th e te a s in g ,h a s been

com pleted. (S e le c t one o f th e sim ple f ix a t iv e s i n Chapter 2 .)

e. On th e sta g e of a d is s e c tin g m icroscope, p la c e a b it o f

t is s u e in a drop o f medium from which i t was taken*
(G enerally taken fo r th e 70% a lco h o l storage f lu id mentioned
i n Chapter 1*)

f. With one o f th e sto u t n eed les in th e l e f t hand, hold th e

m a teria l down firm ly*

g. With the oth er needle p u ll th e m a teria l apart by sta r tin g

a t one o f th e edges and working toward th e cen ter a s the
m a rgin al.stru ctu res a re separated*
 26

h. Do not fo rce str u c tu r es a p a rt, as the d e lic a t e m aterials

w i l l be in ju re d ,

i. Comb out th e separated f ib e r s c a r e fu lly . (Keep t is s u e

wet with th e storage f l u i d .)

j. Lay a sid e the s to u t n eed les and p ick up the fin e n e e d le s.

k. Separate th e shreds o f c a r e fu lly divid ed m aterial u n t il

th e in d iv id u a l fib e r s d esired are is o la t e d .

1, Once you are s a t i s f i e d w ith your s e le c t io n tr a n sfe r th e

m aterial on a clean s lid e t o a compound microscope fo r
more complete exam ination.

m. Remove any la rg e p ie c e s of m a teria ls th a t remain undivided

and tr a n sfe r the d is s o c ia te d m aterial to th e proper re­
agent fo r mounting. (Chapter S .) (Canada balsam i s
suggested a f t e r dehydration.)

n. I f th e m aterial i s such th a t sta in in g may a id in i t s

study se e Chapter 9*

2. MACERATION: How t o make s l i d e s by the m aceration method.

a. Use fo r the study of epiderm is, h a ir s , or stom ata.

b. G enerally use in making whole mounts. (See Chapter 8 .)

c. E ither f i x t is s u e before or a f t e r the m aceration has been

com pleted. (S e le c t one o f the sim ple f ix a t iv e s in Chapter 2 .)

d. I f the m aterial i s dry, b o il i t in water u n t il thoroughly

satu rated before f i x i n g . (Use a pump with an a sp ira to r i f
n e c e ssa r y .)

e. I f the t is s u e has not already been divid ed (as su ggested

i n Chapter 1) d iv id e th e m aterial in to s liv e r s not th ick er
than a to o th p ic k .

f. Treat w ith one o f th e fo llo w in g m acerating p r o c e sse s.

(1) S c h u ltz e 's Method:

(a) Cover the m aterial w ith concentrated n i t r i c a c id .

(b) Add a few c r y s t a ls o f potassium c h lo r a te .

 27

(c ) Heat the mixture and t is s u e on a sand bath , in a

c lo s e d hood, u n t il th e m aterial i s bleached w h ite.

(d) Wash the t is s u e s thoroughly, and shake w ith g la s s

beads u n t il the m aterial d is in te g r a te s .

(e ) Use the te a sin g method i f n ecessary.

(f) In crease or decrease the duration o f heating

u n t il a l l unbroken c e l l s can be is o la t e d .

(2 ) J e ffr e y 's Method:

(a ) Mix equal volumes o f 10 % chromic a c id and 10%

n i t r i c a c id .

(b) Treat th e t is s u e in th is mixture fo r 1 t o 2 days

a t 30 to liO°C.

(c ) Wash and shake the t is s u e w ith g la s s beads.

(d) Use the te a sin g method i f n ecessary.

(3) Harlow's Method:

(a ) Treat the subdivided and b o ile d m aterial in

chlorine water fo r 2 hours.

(b) Wash in water thoroughly.

(e ) B o il in a 3 % sodium su lp h ite fo r 15 m inutes.

(d) Wash and shake with g la s s beads.

(e ) Use the te a sin g method i f n e c essa ry .

(f) Repeat th e c h lo rin a tio n and su lp h ite bath i f

n ecessary.

g. Proceed from any of the maceration treatm ents, by washing

th e pulp thoroughly by d ecan tation . (Use a c en tr ifu g e
to aid w ashing.)

h* Unstained m aterial can be mom ted in w ater and g ly c e r in ,

as su ggested i n Chapter 8 . (Semi-permanent s l i d e s . )

i. Unstained or sta in e d m aterial can be s u c c e s s fu lly mounted

by the dioxan or hygrobutol methods as su ggested in
Chapter 8 . (Permanent s l i d e s . )

3* For sta in in g see Chapter 9 .

 28

SO
URC
ES

1, B
OOKSHELF: Suggested readings for your assistance.
a. Chamberlain, pages (lL4~l45.)
b. Johansen, pages (l0h-105.)
c. M
eClung, pages (l73-17^.)
d. Sass, pages (111-112.)
e. Tobias, pages (43-^.)
2. SPECIALREFERENCES: Belated readings.
a. Jeffrey, E. C.: "Improved Method of Softening Hard
tissues". Botanical Gazette. 86: 456-^57. 1928.

0, ACTIVITIES: Som e learning experiences to enhance your skill in

various dissociation techniques.
1. TRYING : Select some of your tissues previously collected
and proceed to follow the various dissociation techniques,
2. PERFECTING: Having tried the methods and having found out
the possibilities of the technique, select one or two
methods and perfect a smooth running technique. Ask your
instructor for criticism and suggestions.
3. REA DING: Refer to the magazine, Stain Technology, for addi­
tional possible aids and incorporate them in your procedure.

D. EVALUATION: Samples of check-ups which m ay be applied to your

mastery of the dissociation methods of slide making.
1. RATINGSCA LE: Place an Xin the space for which your know­
ledge of the dissociation method qualifies.
Yes No
a, ( ) ( ) You should use this technique for tissue fibers
and stomata examination,
b, ( ) ( ) Great care should be used to carefully separate
tissue so as not to tear delicate fibers,
c, ( ) ( ) Before mounting, a careful examination should be
made under the compound microscope.
 29

PART III. SECTIONINGMETHODS

H
OWTOPREPA RE SLIDES B
YSECTIONING

To make use of all the potentialities of your tissue, sectioning

methods offer the grestest possibilities. The next two chapters take
you through the very simplest cutting methods into very complex and
exceptionally specialized techniques which will prove useful in broaden­
ing your- skill. Prom the simplest to the more difficult, there is only
one short cut; practice, practice, and practice.
 30

CHAPTER 5 . UNEMBEDDED TISSUE

HOW TO PRODUCE SLIDES OF UNINFILTRATED TISSUE

A. MOTIVATION; Rewards th at w i l l be achieved i f you understand how

t o make u n in filtr a te d s l i d e s .

1* BETTER CHANCES FOR EMPLOYMENT: A wide knowledge makes for

more op p ortu n ity.

2. WIDER KNOWLEDGE OF SLIDE POSSIBILITIES: V e r sa lity o f methods

and techn iqu es are e s s e n t ia l fo r g rea ter su c c e s s.

3. PERSONAL SATISFACTION: There i s grea t prid e and personal

f e e lin g i n knowing a job i s w e ll done.

B« PRESENTATION: Suggestions th a t w i l l enable yon to make good

u n in filtr a te d s lid e s o f better quality.

1. GRINDING: How t o prepare s lid e s o f f o s s i l p la n ts by gr in d in g .

a. Cut th e m a teria l in th in sla b s by means o f a temper hacksaw.

b. Cut th e sla b s i n th e d esired p la n es.

c. Grip sm all ir r e g u la r p ie c e s o f hard m a terial by p la cin g

them f i r s t i n a s o f t mass o f wood pu tty u n t il hardened.

d. Grind and p o lis h one fa ce o f th e specimen a s t h in as

p o s sib le in th e d esired plane o f s e c tio n in g . P o lish th e
su rface by rev o lv in g the s e c tio n on brass d is c s which have
been kept wet and l ib e r a l l y powdered w ith f in e carborundum
d u st.

e. Clean th e p o lish ed su rface w ith w ater, rubbing g e n tly

w ith a c lo th fr e e from l i n t .

f. Cement th e p o lish e d su rface o f th e specimen to a p ie ce o f

c le a n p la te g l a s s . (R e sin -la n o lin cement or s h e lla c .)

g. When hardened, continue to grind t o a very th in s e c tio n .

h. Wash and dry w ith a lco h o l and s l i d e o f f th e grinding s l i p

which has been w e ll cleaned w ith x y l o l .

i. Mount w ith th ic k warm balsam.

 31

j • Cover th e se c tio n on th e s lid e w ith a cover g la s s and

weight down to squeeze out excess f lu id and a llo w t o harden.

2. PEELING: How t o prepare s lid e s o f f o s s i l plant in aterial by

p e e lin g .

a. Choose th e plane o f th e specimen and p o lis h t h i s su rface

as i n above.

b. Etch th e su rface w ith 5$ hydrochloric a c id in water fo r

30 to 60 m inu tes.

c. Flow over th e etched su rface fo r 20 seconds a liq u id o f

n it r o c e llu lo s e 20 grm, t e c h ., b u ty le a c e ta te 200 e c . ,
m ethyl ph thalate l c c . , tolu en e or x y lo l 10 to 20 c c .

d. Allow the f ilm to remain on th e su rface fo r 43 hours.

e. Loosen m arginally by a sharp blade and p e e l o f f th e specimen*

f• Wash in d ilu te hydrochloric a c id , w ater, and then dry

between b lo t t in g paper sh e e ts under p ressu re.

g. Clear in Eycleshm yer's f l u i d and mount i n balsam.

3. FREE HAND: How t o make fr e e hand s e c t io n s .

a. Place t is s u e between two s p l i t p ie c e s o f e ld e r p ith .

b. Wrap th e two p ie c e s o f pith"' togeth er w ith th e t is s u e

between.

c. Hold a razor a s in shaving by th e r ig h t hand and draw the

blade in lo n g sweeping stro k es from th e h e e l t o th e t i p
o f th e specim en.

d. Keep th e k n ife and the t i s s u e flood ed with water or

a lc o h o l. ($0 - 70%.)

e. F loat the t is s u e s a s cut in water or 70$ a lc o h o l.

f. F ix and k i l l i f th e t is s u e i s fr e s h .

g. Proceed im m ediately t o the d esir ed s t a in . (See Chap. 9*)

4. HAND MICROTOME: How t o make hand microtome s e c tio n s .

a. P lace th e t is s u e i n th e hand microtome.

b. Turn th e screw so th a t th e t is s u e appears on to p .

c. S e le c t a sharp k n ife , razor, or c h i s e l.

 32

d. Draw th e c u ttin g t o o l across th e t i s s u e , each time

a d ju stin g th e screw fo r th e d esired th ic k n e ss.

e. Keep th e k n ife and th e s e c tio n s flood ed w ith water or

w ith a lc o h o l. (50 - 10%.)

f. F loat th e t is s u e i n water or 10% a lc o h o l.

g. F ix and k i l l t i s s u e . (See Chap. 2 .)

h. Proceed im m ediately to th e d esired s t a i n . (See Chap. 9*)

5. SLIDING MICROTOME: How to make s lid in g microtome s e c t io n s .

a. P lace th e specimen i n the microtome holder jaw s.

b. Use s p l i t p ie c e s o f e ld er or sunflower p ith t o brace th e

tis s u e .

e» Set th e k n ife a t an oblique p o s itio n so a s t o g iv e a long

sla n tin g str o k e .

d. Keep th e k n ife and t is s u e flood ed with water or w ith

a lc o h o l. (50 - 7056.)

e. Adjust th e k n ife and t is s u e so th a t th ey are in c o n ta c t.

f. Adjust th e blade fo r th e d esired th ic k n e ss .

g. Draw th e k n ife through by a lon g h o rizo n ta l str o k e .

h. C o llect th e se c tio n s as th ey gather on th e blade w ith a

camels h a ir brush.

i. Continue as in th e case o f th e hand microtome.

SOURCES

1. BOCK SHELF: Some refer e n c es which may a id you.

a. Chamberlain, pages (154-153.)

b. Johansen, pages (102-104)J (1 0 6 .)

c. McClung, pages (174-178.)

d. S a s s, pages (9 4 -9 8 .)
 33

2. SPECIAL REFERENCES: R elated r e fe r e n c e s.

a* C row ell, Ivan , H .: "Cutting M icroscopic S ectio n s o f Wood

Without Previous Treatment i n Ifydrofluoric Acid". S ta in
Technology. 5: 149-150. 1930.

b. Richards, Oscar, W.: The E ffe c tiv e Uses and Proper Care
o f th e Microtome.

c. Walton, J . : "Improvement in th e Reel Method o f Preparing

S e c tio n s o f F o s s il P la n ts"• Mature. October 1 3 , 1930 and
March 1 5 , 19 30.

d. H oskins, J . H.: "Transfer Method fo r Thin Rock Sections"*

B otan ical G azette. 89: 414-415. 1930.

C. ACTIVITIES: P ro je cts fo r learn in g t o properly produce s l i d e s o f

u n in filtr a te d t i s s u e *

1. TRYING: Attempt t o do as many o f th e su ggested u n in filtr a te d

techniques a s p o s s ib le . C r it ic iz e your r e s u lt s and ask your
in s tr u c to r t o g iv e you h e lp fu l su ggestion s*

2* EXAMINING: P er fec t your technique w ith th e variou s c u ttin g

in stru m en ts. Ask your in str u c to r fo r added help and
su g g e stio n s.

3. READING: Refer to the variou s refer e n c es fo r other h e lp fu l

h in ts which you could incorporate in your tech n iq u e.

4. AIDING: Exchange c r itic is m s w ith your fe llo w stu dents and

attempt t o e lim in a te your own f a u lt s as w e ll as making use
o f th e p r a c tic a l su ggestion s o f other stu d e n ts.
 34

P. EVALUATION: Sample e v a lu a tio n instrum ents fo r cheeking your

technique on unembedded t i s s u e .

1* TRU&-FALSE: Place X i n th e co rrect space fo r time or f a l s e ,

T F

a* ( ) ( ) You should use th e grinding or p e e lin g method on

f o s s i l p la n ts ,

b. ( ) ( ) You should use some kind o f support in c u ttin g

fr e e hand, and th e s lid in g microtome,

c. ( ) ( ) Your blade should always be sharp, when c u ttin g

se ctio n s*

d. ( ) ( ) T issu es should always be kept w et,

e. ( ) ( ) Free hand se c tio n in g should g iv e an in s ig h t in t o

th e variou s microtome se c tio n in g methods,

f• ( ) ( ) You. should not consid er th e s la n t o f th e k n ife in

c u ttin g se c tio n s*

g, ( ) ( ) Free hand, hand microtome, and s lid in g microtome

se c tio n s should be handled more or l e s s a lik e
a f te r se ctio n in g *

h, ( ) ( ) Alder and sunflower p it h should be used as a

supporting t is s u e fo r c u ttin g se ctio n s*

i, ( ) ( ) This experience should bring a wider knowledge o f

s lid e making p o s s i b i l it i e s *

2* BEST ANSWER: P lace th e number o f th e co rrect answer i n th e

parentheses*

a* () F o s s il p la n ts should be prepared by: (1) The fr e e

hand method* (2) S lid in g microtome, (3) Hand
microtome, (4) Grinding*

b. () For the most, r e fin e d r e s u lt s you should use :

( l ) The grinding method. (2) S lid in g microtome.
v3) Hand microtome* (4) F eelin g method*

c* () In a l l th ree c u ttin g methods you should proceed

im m ediately a f t e r c u ttin g th e t i s s u e to : ( l ) S ta in ­
in g . (2) F ixing* (3) Mounting, (4) Blocking*

d. () Having k i l l e d and fix e d th e t is s u e s you should go

d ir e c t ly t o : ( l ) S ta in in g . (2) Embedding. ( 3 )
S to r in g . ( 4 ) Grinding*
 35

CHAPTER 6 . EMBEDDED TISSUE

HGW TO MAKE SLIDES OF INFILTRATED TISSUE

A. MOTIVATION: Rewards th a t w i l l be yours I f you le a r n t o make

s lid e s o f i n f i lt r a t e d t i s s u e .

1. ACADEMIC STANDING: . Good grades mean more r ec o g n itio n a ft e r

graduation.

2. GOOD RESULTS: Good s l i d e s o f i n f i lt r a t e d t is s u e are th e

r e s u lt o f a p e r fe cte d tech n iq u e.

3. EXCELLENT EXPERIENCE: The a b i l i t y to perform a wide v a r ie ty

o f s p e c ia liz e d tech n iq u es g iv e s you s a t is f a c t io n and r e a l
su c c e s s.

B. PRESENTATION: Key p oin ts th a t w i l l a s s i s t you i n making fin e

s lid e s of in filtr a te d t is s u e .

DIRECTIONS

1. SOAP SECTIONS: How to make s e c tio n s o f t i s s u e embedded in

soap.

a. Use t h i s method fo r a lg a e th a t cannot s a f e ly be dehydrated.

b. Saponify 70 c c . o f hot cocoanut o i l w ith 3 8 .5 c c . o f 28$

aqueous KOK.

c. P u lverize when th e product becomes firm .

d. P lace algae i n warm water t o which th e soap i s gradually

added u n t il i t becomes q u ite concen trated .

e. Dry the m ixture u n t il firm .

f. Attach th e blocks formed* with the t is s u e in sid e* to

wooden b locks which have been taken from a s lid in g micro­
tom e.

g. Cut s e c tio n s on th e s lid in g microtome. (See Chapter 5 .)

h. Attach th e s e c tio n s t o th e s l i d e w ith Mayer's a d h esiv e.

(Use a l i t t l e x y l o l .) (See Chapter 7 .)

i. lfesh away th e soap by using warm w ater.

 36

4. Immerse th e s l i d e i n 95$ e th y l a lc o h o l to f in is h the

washing.

k. S ta in as th e p a r a ffin s e c t io n . (See Chapter 1 0 .)

2* FREEZING SECTION: How to make se c tio n s by th e fr e e z in g

method*

a* Use t h i s method w ith f r a g i l e s o f t or g ela tin o u s algae*

b. Transfer t i s s u e s from th e storage b o t t le t o 50$, then t o

35$ a lc o h o l, and then i n su c c e ssiv e waterbaths fo r about
12 hours*

c. Prepare s gum arabic syrup o f a th ic k c o n sisten cy by

taking 60 grams o f th e b e s t gum i n SO c c . o f d i s t i l l e d
water*

d. P lace a few c r y s ta ls o f c a r b o lic a c id t o th e gum*

e. Dip th e specimen in t o th e gum*

f• P lace a th in la y e r o f gum on th e d isk o f th e fr e e z in g

microtome* Freeze a 2-3 mm* la y e r o f t h i s gum on th e d is k ,
by turnin g on th e C02g a s.

g. P lace th e specimen on th e d is k , wrapping a generous

q u a n tity o f gum around th e tis s u e *

h« Turn on th e fr e e z e r and a s th e gum b egin s to con geal,

wrap more gum on th e specimen u n t il th e m aterial i s w e ll
supported.

i* Proceed w ith se c tio n in g by drawing th e moving blade across

th e t i s s u e in quiek c u t s . (Chapter 5«)

(1) Work th e microtome screw w ith one hand and plane o f f

s e c tio n s (15 to 20 microns th ic k ) w ith th e o th e r .

( 2) Keep th e blade co ld by r e s tin g i t on th e specimens

now and then*

(3) Work r a p id ly so a s t o cut s e c tio n s i n quick su c c e ss­

ion*

( 4) Pick up th e s e c tio n s w ith a camel h a ir 's brush.

J. F lo a t th e t i s s u e s i n 70$ alcoh ol*

k* Hake a g e la tin s o lu tio n th a t i s e m u lsified a t room

temperature*
 37

1. Add 0 .1 $ c a rb o lic a c id as a p r e se r v a tiv e .

m. Warm th e above s o lu tio n i n a water bath . Use i n th e manner

d escrib ed fo r gum a r a b ic .

a. Transfer s e c tio n t o d i s t i l l e d w ater, ffesh thoroughly.

o. Mount i n drop o f water and g ly c e r in fo r a temporary s l i d e .

(See Chapter 7 .)

p. Proceed to s ta in in g . (See Chapter 9 .)

3. CELLOIDIN SECTION: How t o make s l i d e s o f t is s u e embedded in

c e l lo i d in .

a. Use fo r hard firm t is s u e s ( b r i t t l e or f r ia b le t is s u e s )

th a t would be hard to keep in a fix e d p o s itio n .

b. Method 1 .

(1) Transfer dehydrated t is s u e t o ab so lu te e th y l a lc o h o l.

(2) Leave t i s s u e here fo r about 12 to 24 hours.

(3) Transfer th e m aterial t o equal p arts o f ab so lu te

a lc o h o l and eth er fo r 12 t o 24 hours.

(4) Transfer t o a corked b o t t le o f 2$ c e llo id in s o lu tio n .

(a ) Soak 15 grams o f dry c e llo id in overnight i n a b so lu te

a lc o h o l. (Thin m a teria l w ith e th e r -a lc o h o l s o lu t io n .)

(5) F asten th e cork to th e b o t t le by means o f a wire lo o p .

(6) Transfer th e b o t t le to th e oven a t 53°C. fo r 12 t o 24

h ours.

(7) Cool th e b o t t l e , remove stop p er, and pour th e c e llo id in

in t o a dry pan. (Beware o f flam es or sp ark s.)

(8) Cover th e t i s s u e immediately w ith !& c e llo id in and re­

s e a l th e b o t t le and repeat th e in te r v a l under p ressu re.

(9 ) Bepeat t h i s op eration w ith 6 $ , 8$, and 10$ c e l lo i d in .

(10) Thicken th e c e l lo i d in by adding chip o f dry c e llo id in

every 24 hours.
 38

(11) Poor th e s o lu tio n when i t J u st flow s a t room.

temperature*

(a) Remove an i n f i lt r a t e d p ie c e o f m a teria l and a

mass o f enveloping c e llo id in and immerse in
chloroform*

(b) Leave th e m aterial i n th e chloroform for 12

hours to harden.

(c) Transfer th e m a teria l i n a m ixture o f equal

p a rts o f 95/6 e th y l a lc o h o l and glycerin *

(d) Block s e c tio n by removing from th e storage

f lu id o f 95% a lco h o l and g ly c e r in and soak in
anhydrous e th y l a lc o h o l.

(e) Change th e a lc o h o l tw ice a t 4 to 8 hour

i n t e r v a ls ,

(f) Transfer th e p ie c e s t o th ic k c e llo id in of a

c o n siste n c y fo r castin g*

(g) P lace a supply o f mounting blocks in to t h i s

so lu tio n a l s o .

(h) A fter a t l e a s t 24 hours in th e th ic k so lu tio n

mount and harden as d escrib ed above.

(i) Trim th e p ie c e s and p la c e in the 95% e th y l

a lco h o l and g ly c e r in u n t i l ready fo r u se .

(12) Make a c a r e fu l study o f th e autom atic c e llo id in

microtome*

(a) P lace the block o f embedded t i s s u e in the proper

p la in i n th e o b ject c a r r ie r o f th e microtome a t
th e proper l e v e l .

(b) Arrange the microtome k n ife o b liq u e ly so th a t i t

w i l l s l i c e through th e o b ject w ith a lon g drawing
cut fo r a t l e a s t h a lf th e le n g th o f th e blade*

(c) Keep both th e k n ife and th e ob ject flood ed w ith

the mixture o f 95% e th y l a lc o h o l and g ly c e r in .

(d) Draw th e k n ife through th e o b ject w ith stra ig h t

stea d y p u l ls . (Avoid p u llin g down on or l i f t i n g
th e k n if e - c a r r ie r .)
 39

(e ) I f the fe e d i s not autom atic push the k n ife

back to p o s itio n always before turning th e screw
which r a is e s the o b je c t.

(f) Cut se c tio n s 1 $ to 20 microns th ic k .

(g ) Use a cam el's h air brush to pick up the s e c tio n s .

(h ) F lo a t the s e c tio n s i n a 95% a lc o h o l s o lu tio n .

(i) Proceed im m ediately to s ta in in g . (Chapter 1 0 .)

c. Method 2 .

(1) Give th e m a teria l i±8 hours in 2 % c e llo id in in a

se a le d b o t t le in the oven.

(2 ) Cool and unseal a t in te r v a ls o f 1*8 hours to sev era l

days and add chips o f dry c e l lo i d in .

(3) Add c e llo id in i n a ch eeseclo th bag when t is s u e s are

d e lic a t e .

(U) Continue the p e rio d ic ad d ition s o f c e llo id in u n t il

thickened to the degree describ ed in the preceding
method.

( 5) Continue on from (12) in Method 1 .

1*. PARAFFIN SECTIONS: How t o make s l i d e s o f t i s s u e embedded i n

p a ra ffin .

a. S e le c t t i s s u e s from s to ra g e m a te ria l o r d i r e c t from th e

d e h y d ra tio n p ro c e ss .

b . From the h ig h est a lc o h o l tra n sfer ihe t is s u e to the next

h ig h est a lc o h o l bath fo r about an hour. (Be sure to keep
an accurate record o f the tim e. )

c. Immerse in 9$% a lco h o l for 30 to 1*5 minutes o n ly .

d. Transfer to absolute a lco h o l for 1 hour.

(1) Remove e x cess 95% a lco h o l from the o b je ct before

tr a n sfe r , by means of a b lo t t e r or clean c lo th .
(Do not l e t the tis s u e d ry .)

e. Place i n equal parts of x y lo l and ab solu te a lco h o l fo r 30

m inutes•
f. Transfer to pure x y lo l fo r if- hours or u n t i l c le a r .
 Uo

g. Remove a l l e x c e ssiv e x y lo l but do not allow t is s u e to dry*

h. Place in m elted p a r a ffin for 2 hours. (50°C .)

(1) S h if t the s e c tio n in the p a r a ffin many tim es during

th e two hours.

(2 ) Duration of bath v a r ie s w ith the s iz e o f the o b je c t.

(a) S e c tio n s of 3 to 5 l e s s than an hour.

(b) S e c tio n s o f la r g e r -than 5 mm. about 2 hours.

(c ) Do n o t have the bath too h o t. (Cooked t i s s u e s

are no good .)

(d) Keep the m aterial clean by having a f a l s e

bottom in th e p a r a ffin con tain er.

(3 ) Prepare sm all paper boxes for b lock in g. (Boxes 1 $ x -

20 mm. are v ery handy-. )

(ll) Transfer s u f f ic i e n t m elted p a r a ffin by means of a

warmed p ip e tte to one o f th e paper boxes.

(5) With wanned fo rc ep s renave th e tis s u e to th e box.

(6) Arrange th e t is s u e as d e sir ed in the bottom of th e

box w ith heated n e e d le s.

(7) Pour m elted p a r a ffin over the to p .

(8 ) Plunge th e box w ith th e p a r a ffin i n co ld w ater as

soon as the p a r a ffin has congealed s u f f ic i e n t ly . ( I f
W hitish -look in g patches are present rem elt and r e c a s t .)

(9 ) Be sure t o mark a l l blocks properly.

(a) Mark by scratching the number o f the record card

in p a r a ffin or by stic k in g w ith p a ra ffin a sm all
paper marker on th e b lo c k .

(ID) Remove the paper boxes and trim the p a r a ffin blocks
so th a t the t is s u e s are properly o r ie n te d . (See
F igures 1 and 3*)

(11) Leave a margin o f about 2 mm. around the o b je c t.

(12) Proceed to the rotary microtome f o r s e c tio n in g .

 41

(a) Mount th e p a r a ffin block containing th e specimen

on the o b ject c a r r ie r . (S lid e a hot sp atula
between the c a r r ie r and th e b lo c k , press th e two
m elted p a r a ffin su rfaces to g eth er and p lace in
cold water to harden q u ic k ly .)

(b) Trim the block w ith a razor b lad e, have the two
edges that, w i l l be h o r izo n ta l in th e microtome
a s c lo s e to p a r a lle l a s p o s s ib le . (See Figure 3 . )

(c) Disengage the r a tc h e t o f the feed in g mechanism

and b ring the o b ject clamp back from the k n ife
as fa r a s i t vd.ll g o .

(d) F ix the block i n d ie microtome and ad ju st i t

u n t i l the o b ject i s c o r r e c tly o rien ted with
r esp ec t t o the k n ife ed ge. (Make su re the micro­
tome i s lock ed w h ile o r ie n ta tin g the b lo c k .)

(e) S et th e feed in g mechanism a t 10 m icrons.

(f ) Set the k n ife a t a s lig h t an gle from th e perpen­

d ic u la r toward th e b lock . ( I f s e c tio n s s t i c k to
the k n ife in c re a se the a n g le .)

(g) Turn th e crank o f the microtome a t a steady

moderate r a te .

(h) L if t the ribbon o f s e c tio n s w ith a n eedle and

hold th e ribbon out from th e k n ife edge w h ile
c u ttin g the e n tir e b lo c k . (See Figure 3 . )

(i) Do not bring a needle or any other hard object

near the edge o f th e k n ife . Remove se ctio n s
with a s o f t camel*s hair brush.

(j) With a n eedle arrange the ribbons on clean paper.

(13) Cut th e ribbon in to d esir ed len g th s w ith a s in g le

rocking motion o f the curved s c a lp e l.

(14) P lace a th in film o f an a f f ix a t iv e on the s l i d e .

(Mayer*s Albumen i s su g g ested .) (See Chapter 7 .)

(15) Remove water w ith f i l t e r paper and arrange ribbons

with a n eedle i f n e c essa ry .

(16) Cover s l i d e s to p r o te c t them from dust and lea v e them

dry over n igh t or se v er a l hours in a moderate temper­
atu re oven. (When th e s l i d e s are dry they w i l l keep
fo r y e a r s .)

(17) Proceed to s ta in in g of p a r a ffin i n f i l t r a t e d t i s s u e .

(See Chapter 1 0 .)
 42

\ \\ \ \ \ \\
\ \ \ \ \ \\N
vk->vxV

ORIENTATION OF KNIFE AND TISSUE

i
5 D.

3 CORRECTION OF A
CURVED RIBBON
J
5

C. E

CORRECT RIBBON INCORRECT RIBBON

FIGURE 3 .

ORIENTATION OF KNIFE AND TISSUE

PARAFFIN RIBBONS

* Adapted from S a s s. (se e b ib liography fo r complete referen ce)

 43

5. CELLULOSE ACETATE: How t o make s lid e s o f t is s u e embedded in

c e llu lo s e a c eta te*

a. Use t h i s method fo r hard veg eta b le stru ctu res*

b* Cut th e m a teria l in sm all h a lf in ch cubes. (See Chapter 1 .)

c. Transfer blocks t o water and remove th e a ir from them*

(When blocks sin k they are ready t o p r o c e ss. (See Chapter 1 .)

d* Put th e soaked blocks in t o pure acetone fo r 1 to 2 hours*

e* Transfer t is s u e t o a s o lu tio n d£ c e llu lo s e a c e t a te .

(12$ so lu tio n of c e llu lo s e a c e ta te * ) (12 g . o f c e llu lo s e
a c e ta te i n 100 c c . o f pure a c eto n e .)

(1) Keep s o f t t i s s u e i n t h i s s o lu tio n fo r tiro days*

(2) Keep hard t is s u e i n t h i s so lu tio n fo r 14 days*

f* Cut s e c tio n s e it h e r by hand or microtome. (See Chapter 5 .)

g. Proceed im m ediately t o s ta in in g . (See Chapter 1 0 .)

 44

A.

C.

D.

FIGURE 4 .

STEPS IN SHARPENING A MICROTOME KNIFE

* Adapted from Richards (See bibliography for complete referen ce)

 45

SOURCES

1. BOOKS: R eferences which could g iv e you added inform ation .

a. Chamberlain, pages (112-139.)

b. Guyer, pages (3 6 -7 0 .)

c. Johansen, pages (126-154.) (121-125.)

d. MeClung, pages (178-183.)

e. S a s s, pages (3 2 , 53, 7 9 -8 9 .)

2. SPECIAL REFERENCES: A d ditional r e fe r e n c e s.

a. Church, Margaret: "C elloidin P a ra ffin Method." Scien ce

N.S. . 47: 640. 1918.

b. Davenport, H. A. and R. L ., Swank: "Embedding w ith Low

V iso c ity N it r o c e llu lo s e . M S ta in Technology. 9: 137-139. 1934.

c. De Zeeuw, R .: "The Value o f Double I n f il t r a t io n in B otanical

M icrotechnique." Papers Michigan Academy o f S c ien ce , 1: 8 3 -8 4 .
1923.
d. Dufrency, J .: "A Method of Imbedding Plant T issues Without
Dehydration." S cien ce. 82: 335-336. 1935.

e. Hance, R. T ,: "A New P a ra ffin Embedding M ixture."

S c ie n c e . 77: 3 5 3 . 1933.
f. Lang, A. G.: “The Use of N-butyl A lcohol in the P ara ffin
Method." S ta in Technology. 12: 113-119. 1937.

g. M ettler, F. A .; C. C. H e ttle r , and F. C. Strong: "The

C e llo so lv e T echnic." S ta in Technology. 11: 165.

h. Plowman, A. B .: " C elloid in Method with Hard T issu es."

B otan ical G a zette. 37: 456-461. 1904.

i. Richards, Oscar W.: The E ffe c tiv e Uses of and Proper Care
o f the Microtome. Spencer Lens Company. B u ffa lo , N.Y., 1942.
 j. W alls, G. L .: "A Rapid C e llo id in Method fo r the Rotary
Microtome. 11 S ta in Technology, 11: 89-93.

k. Wetmore, R. H .: "The Use o f C e llo id in in B otanical Technic

S ta in Technology. 7: 3 7 -6 2 . 1932.

C. ACTIVITIES: P ro jects to p e r fe c t your a b i l i t y in preparing s l i d e s

o f i n f i lt r a t e d t i s s u e .

1. TRYING: S e le c t a few o f your processed t i s s u e s and use th e

methods su g g ested . Try as many o f the d iffe r e n t methods as
tim e w i l l perm it. Compare your r e s u lt s w ith those presented
by th e in s tr u c to r fo r exam ination.

2. PERFECTING: Having t r ie d th e methods su ggested , s e l e c t th e

one which you l i k e b e s t and which you are planning to u se .
P erfect t h i s technique u n t il you f e e l a t ease in every phase.

3. PRACTICING: Use the variou s c u ttin g instrum ents on some

p r a c tic e t i s s u e u n t i l you are sure your technique i s p o lish e d .
Proceed to your prepared t is s u e s and use th e exp erien ce gain ed .
Have your in str u c to r c r i t i c i z e your r e s u lt s .

D. EVALUATION: Samples of e v a lu a tio n instrum ents th a t may be em­

ployed in r e la tio n to your use of i n f i l t r a t i o n methods.

1. RATING SCALE: P lace X in each space in which your

i n f i l t r a t i n g technique q u a l if i e s .

Yes No

a. ) Soap s e c tio n s should be made of a lg a e which

cannot s a f e ly be dehydrated.

b ) F reezing s e c tio n s should be made o f t is s u e s

which are f r a g i l e , s o f t , g e la tin o u s.

c. ) C e llo id in s e c tio n s should be made o f hard firm

tis s u e s .

d. ) You should use th e s lid in g microtome in c e l l ­

o id in s e c tio n in g .

) You should g iv e great care and time t o embedding

tis s u e .
 47

f. ( ) You should use the rotary microtome in p a r a ffin

s e c tio n in g .

g. ( ) C e llu lo se a c e ta te se c tio n s should be cu t cm the

hand or s lid in g microtome.

h. ( ) B ayer's albumen should be used fo r p a r a ffin

s e c tio n a f f ix a t io n .

i. ( ) You should u se C02 in fr e e z in g s e c tio n s .

J. ( ) You should use a gum arabic t o a id you in

fr e e z in g s e c t io n s .

2. BEST ANSWER: P lace th e number of th e b est answer in th e

p a ren th eses.

a. () You should have good p a r a ffin s e c tio n s :

(1) I f vrh itish -look in g patches a re v is a b le . (2) I f
hard p a r a ffin i s u sed . (3) I f th e t i s s u e has been
g iv en tim e t o be com pletely i n f i l t r a t e d . (4) I f care
i s given during th e c u ttin g .

b. () C e llo id in s e c t io n s should be cut on th e : (1) Hand

microtome. (2) S lid in g microtome. (3) Rotary micro­
tome. (4) By hand.
 48

PART IV. ATTACHMENT AND MOUNTING

HOW TO ATTACH AND MOUNT TISSUES ON THE SLIDES

Once a good job has been done th ere e x i s t s th e d e sir e t o preserve

th e r e s u lt s o f hard la b o r . To do t h i s experim entation and p r a c tic e are

req u ired . The two chapters t o f o llo w are s e t a sid e fo r you t o acquire

a f in e and smooth running technique th a t w i l l keep your s l i d e s fo r

fu tu re r e fe r e n c e .
 49

CHAPTER 7 . ADHESIVES

HOW TO AFFIX TISSUE TO THE SLIDE

A. MOTIVATION: Rewards th a t w i l l be gained i f you a f f i x your t i s s u e

t o th e s l i d e p rop erly ,

1, PERMANENT SLIDES: S lid e s made good once show th e se q u a lit ie s

and remain th a t way over a lon g p erio d o f tim e.

2* MORE SUCCESS IN STAINING: T issu es th a t remain i n p la ce w hile

sta in in g in su re f i n e s lid e s *

3* LESS WASTE OF TIME: Once th e s e le c t io n o f th e a f f ix a t iv e i s

s e t , going ahead i s th e only d ir e c tio n *

B* PRESENTATION: Some p o in te r s to help you g e t th e very b e st a f f i x ­

a tio n o f t i s s u e *

DIRECTIONS

1* SELECTION: How t o s e l e c t th e b e st a f f ix in g m aterial*

(Use th e em p ircia l method*)

2* HAUPT*S: How t o use Haupt1s adhesive*

a* P lace a drop o f th e adhesive on th e c le a n s lid e *

b* Smear ev en ly so as t o le a v e a b a rely p e r c e p tib le layer*

c* Add im m ediately a form alin s o lu tio n on th e s l i d e by means

o f a p ip e tte *

d* P lace th e s e c tio n s on th e form alin so lu tio n *

e* Put th e s l i d e on a warming ta b le (temperature 40 t o 43°C)

u n t il s e c tio n s str a ig h te n out*

f * Remove the s l i d e from th e warming ta b le and s e t i n a c o o l

place*

g* Drain o f f th e e x c e ss water*

h* Arrange th e s e c tio n s a s d esired and s e t them a sid e t o dry*

(See fig u r e 5*)

i« Proceed t o th e sta in in g * (See Chapter 1 0 .)

 50

3* MAYER’S: How t o use Mayer’s adhesive*

a* P lace a drop o f th e adhesive on th e c le a n slid e *

b* Smearth e s o lu tio n evenly* (Too ouch w i l l g iv e your s lid e s

a m isty appearance*}

c* Allow th e s o lu tio n t o s l i g h t l y dry fo r a few hours*

d* P lace th e s e c tio n on th e s l i d e i n th e presence o f a l i t t l e

water* (See Figure 5*)

e* P lace the s lid e s i n a warm sp ot so as t o s l i g h t l y warm

p a r a ffin and not enough t o m elt i t *

f• Remove th e e x cess water and p la c e i n a c o o l sp ot t o dry*

g* Proceed t o th e sta in in g * (See Chapter 10*)

4. CELLOIDIN: How to use th e c e llo id in adhesive*

a* A ffix th e t i s s u e s t o th e s lid e w ith Haupt's adhesive*

(See Figure 5*)

b* Coat th ic k woody s e c tio n s and s e r i a l s e c tio n s o f marine

alg a e w ith 1 t o 2 % s o lu tio n o f c e l l o i d i n . (In equal p a rts
o f a b so lu te a lco h o l and eth er*)

c* Remove th e p a r a ffin w ith carb on -xylol*

d* Vfeish th e s l i d e w ith 95% e th y l alcoh ol*

e« Proceed t o th e sta in in g * (See Chapter 10*)

5* ULLRICH’S: How t o use U llr ic h ’s adhesive*

a* Mix a s o lu tio n o f lOOcc. o f d i s t i l l e d water* Add lc c * o f

standard water g la s s so lu tio n and lc c * o f concentrated
ammonia*

b* F lace a drop o f th e m ixture on a s l i d e and spread out e v e n ly ,

c* P lace a s e e tio n on th e s lid e * (See Figure 5*)

 51

d. Allow th e m a teria l t o dry in th e a ir a f t e r str e tc h in g the

s e c tio n s w ith th e a id o f heat*

e* D isso lv e th e p a r a ffin w ith x y lo l a s usual*

f* Bring down the s e r ie s o f a lc o h o ls t o 10%, t o whieh has

been added a tra c e o f hydrochloric acid*

g* Proceed to th e sta in in g * (See Chapter 1 0 .)

 52

LABEL LABEL

A. B.

LABEL LABEL

C. ■D.

LABEL LABEL

E. F.

FIGURE 5 .

SPACING AND ARRANGEMENT OF SECTIONS

* Adapted from S a s s . (See b ib liograp h y far complete referen ce)

 53

SOURCES

1. BOOK SHELF: A d ditional h elp fo r you,

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

b, McClung, pages (1 8 3 -1 8 4 .)

C, ACTIVITIES: Performances th a t w i l l a s s i s t you i n using th e

variou s a f f i x a t i v e s ,

1, TRYING: S e le c t your se c tio n e d m a teria l and t r y th e vario u s

adh esives u n t i l th e most e f f e c t i v e one has been used fo r th e
p a r tic u la r t i s s u e . Check your r e s u lt s w ith th e samples your
in s tr u c to r has prepared,

2, READING: Refer t o th e lit e r a t u r e fo r modern advances i n th e

use o f a f f i x a t i v e s . Try th e se id ea s and make use o f th e v a l­
uable a d d itio n s in your tech n iq u e. Get your fe llo w stu d en ts
t o t r y a few of th e se methods and share th e g a in s .

D. EVALUATION: Some samples t o e v a lu a te your knowledge o f th e proper

a f f i x a t iv e to use w ith your t i s s u e s .

TRUE-FALSE: P lace an X i n th e co rrect space fo r tru e or f a l s e ,

T F

a, ( ) ( ) You should a f f i x your t is s u e s properly to insure

su cc ess in s ta in in g ,

b, ( ) ( ) I f you s e l e c t the r ig h t a f f i x a t i v e , you should have

more time fo r oth er s t e p s ,

c, ( ) ( ) You should proceed t o s ta in in g a f t e r a f f ix in g th e

s e c tio n s t o th e s l i d e ,

d, ( ) ( ) You should always spread the adhesive e v en ly on th e

s lid e ,

•. ( ) . ( ) You should use th e c e llo id in ad h esive w ith n arin e a lg a e .

f• ( ) ( ) You should use th e em p irical method in s e le c t in g your

a f f ix in g s o lu tio n .

g. ( ) ( ) You should m elt a l l th e p a r a ffin from th e s l i d e a s soon

as th e t is s u e has been a f f ix e d ,

h. ( ) ( ) You should use water i n Mayer's ad h esive t o wash away

th e d ir t from th e s l i d e .
 CHAPTER 8 . MOUNTING TISSUE

HOW TO MAKE SLIDES PERMANENT

A. MOTIVATION: Rewards th a t w i l l be yours i f you s e l e c t and use

Pr°P er ly a good mounting f l u i d l

1* SUPERIOR SLIDES: Everyone d e s ir e s th e b e s t product a f t e r long

hours o f p a tie n t e f f o r t .

2. PERMANENT COLLECTION: A ll r e a l s c i e n t i s t s want a c o lle c t io n

o f s lid e s th a t can be used over and over w ithout fe a r o f de­
s tr u c tio n .

3* FEWER DIFFICULTIES: Constant rep a ir o f improperly mounted

s l i d e s means a lo s s o f time and unbearable d i f f i c u l t i e s .

B. PRESENTATION: H elpful h in ts which w i l l a id you in makingyour

s lid e s l a s t .

DIRECTIONS

1. SELECTIONS: How to s e l e c t the b e s t mounting medium.

a. Decide what use i s going to be made o f th e s l i d e .

b. Try to determine how lon g th e s l i d e i s going to be k ep t.

c. Decide how va lu a b le the m aterial i s .

d. Determine which t is s u e i s going to be mounted.

e. F ollow some o f th e su g g estio n s l i s t e d below .

2. AQUEOUS: How t o make aqueous mounts.

a. Use on p la n t epiderm is, h a ir s , f i b e r s , p o lle n , sp ores,

u n c e llu la r or filam entous algae or oth er sm all o b je c ts ,
(g e n e r a lly g iv e s s lid e s o f a f a i r degree o f permanence.)

b. Place sta in e d or u n stained t is s u e on a s l i d e and p lace a

few drops o f water on th e o b je ct and in s e r t the cover g la s s .

c. Make w ater mounts l a s t by adding a 10% g ly c e r in s o lu tio n .

(1) As w ater evaporates introduce g ly c e r in under the

cover g la s s .
 (2 ) Continue u n t il no more evaporation takes place*

(3) These s l i d e s can be stored fo r an almost in d e fin it e

tim e i f sto red f l a t and handled w ith c a r e .

d. Make durable s l i d e s of a lg a e , fu n g i, and fer n s by

mounting in the same manner as above in Amann’ s
Lactophenol.

(1) Phenol 20 c c . , l a c t i c a cid 20 c c . , g ly c e r in 1*0 c c . ,

and water 20 c c .

e. Label and s to r e . (See Figure 5»)

NON-DRYING OIL: How to prepare mounts w ith non-drying o i l s .

a. Use fo r th e t is s u e su ggested above.

b. Use fo r p reserving s lid e s over a lon g p eriod o f tim e,

c* Dehydrate t is s u e and c le a r through x y l o l, or b en zo l.

d. P lace sta in e d or unstained t is s u e on th e s l i d e as above.

e. Introduce one of th e many o i l s . (Castor o i l , lin s e e d o i l ,

o liv e o i l , w hite m ineral o i l , or p a r a ffin o i l . )

f. S ea l edges o f the s l i d e w ith e ith e r p la in glue or w ith

g ly c e r in j e l l y .

g. Label and s to r e . (See Figure 5.)

GLYCERIN JELLY; How to make mount in g ly c e r in j e l l y .

a. Use fo r permanent mounts o f algae and fu n g i.

b. Prepare th e j e l l y by d is s o lv in g 5 g* o f g e la tin in 30 c c .
o f w ater a t 35>°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 so lu tio n

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 .

(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

(19) S e a l the cover g la ss -with balsam or a quick-

drying lacq u er.

(20) P lace in an undisturbed p lace and l e t dry.

(21) Clean th e s l i d e s .

(22) Label and s t o r e . (See Figure 5*)

5» RESINOUS MOUNTING: 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 f o r a few days.

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 . (See F igure £ .)

6. VENETIAN TURPENTINE: 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 . (See Chapter 9 & 1 0 .)

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 ) Use an e x sic c a to r f o r evaporation.
 58

(a ) 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. (See F igure $ .)

7. HYGROBUTOL: 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 ) 3 parts a lco h o l to 1 p a rt hygrobutol.

(2 ) 2 p a rts a lco h o l to 2 p arts hygrobutol.
(3) 1 p art a lco h o l to 3 p arts hygrobutol*
(U) 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 .

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. (See Figure £ .)

8. DIOXAN-BALSAM: 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* 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 .)

2. 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.

C. 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. 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 medium 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 evaporation,

2. 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 ,

b. ) 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*
 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 . Your su ccess w i l l

*

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

 63

CHAPTER 9 . UNEMBEDDED TISSUE

HOW TO STAIN UNINFILTRATED TISSUE

A. 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 .

B. 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. G enerally n ot sta in e d .

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. G enerally n o t sta in e d .

3. 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: 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 . (See Chapter 1 0 .)

7* DISSOCIATION: 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. (See Chapter 1 2 .)

8, SMEARS: How to s t a in sm ears.

a, 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

(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 .)

b. 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 .)

e. 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. 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 .)

2. SPECIAL REFERENCES: A d ditional a id s fo r you.

a. 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.

bi 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". S ta in Technology.

4: 9 7 . 1929.
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. ACTIVITIES: Pro.jects t o help you p e r fe c t your technique o f

s ta in in g .

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.

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

T 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. ( ) { ) Safranin and D e la fie ld , s hematoxylin should be used

to s ta in freehand s e c t io n s .

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 .

2. 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. 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.

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

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

2. FREEZING: 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 . (S ta r t a t # 4 -d .)

b. The freehand method o f sta in in g i s a ls o recommended. (See

Chapter 9 .)

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

3* CELLOIDIN: 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 of a

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 .)

 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. (See Chapter 7 & 8 .)

_h. 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 .)

i. 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.

4. 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) 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.

d. 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) 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 .

5. 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 . (See Chapter 12 fo r s e l e c t io n s .)

e. Dehydrate as i n the p a r a ffin s e c t io n s . (See # 4 - e .)

f. Clear a s in p a r a ffin s e c t io n s . (See # 4 - f .)

g. Mount as i n th e p a r a ffin s e c t io n s . (See # 4 -g .)

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

(1) 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. 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 .)

2. SPECIAL REFERENCES: Added a s s is ta n c e ,

a. See Chapter 9*

C. 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 .

P. 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. 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 .

B. PRESENTATION: Su ggestion s on the u ses and r e s u lts o f s p e c if ic

s t a in s .

DIRECTIONS

1. BULK STAINS: How to s t a in t is s u e s in bulk*

a. 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 .)

2. CELLULOSE CELL WALLS: How to s t a in c e llu lo s e c e l l w a lls .

a. 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 .)

3. CHITIN: How to s t a in e h it in .

a. Use sa fr a n in . (See Chapter 12 fo r in s tr u c tio n s in the use

o f sa fr a n in .)

b. LIGNIFIED CELL WALLS: How to s ta in l i g n i f i e d c e l l w a lls .

a. 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 .)

5. CUTINIZED CELL WALLS: How to s ta in c u tin iz e d c e l l w a lls .

a. 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 How to s ta in the middle lam ella*

a* 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 .)

7. CHROMOSOMES: How to s ta in chromosomes.

a. 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*)

8. MITOCHRQNDRIA: How to s ta in m itochrondria.

a. Use iro n haem atoxylin. (See in s tr u c tio n in Chapter 1 2 .)

9. ACHROMATIC FIGURE: How to s t a in achromatic f ig u r e s .

a. Use c r y s ta l v i o l e t or f a s t green FGF. (See Chapter 12 fo r

in s t r u c t io n s .)

10 . FILAMENTOUS FUNGI IN HOST TISSUES: How to s ta in filam entous

fu n g i in h o st tissu e s *

a. 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 .)

11 . CYTOPLASM: How to s ta in cytoplasm .

a. Use e o s in , e ry th r o sin , f a s t green FCF, or orange G. (See

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

12 . PECTINS: How to s t a in p e c tin s .

a. Use iod in e s t a in . (G ives y e llo w c o lo r .) (See Chapter 1 2 .)

13. PROTEINS: How to s ta in p r o te in s.

a. Use sa fr a n in . (See in s tr u c tio n s in Chapter 1 2 .)

1U. PLASTIDS: How to s ta in p l a s t i d s .

a. Use c r y s t a l or methyl v i o l e t , or iro n haem atoxylin. (See

in s tr u c tio n s in Chapter 1 2 .)
 76

SOURCES

1. 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 .)

2. SPECIAL REFERENCES: A d d ition al a id s for you.

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

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

T 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 .

2. BEST ANSWER: P lace th e number of the b e st answer in the

p aren th eses.

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 .

3. COMPLETION: Supply th e m issin g word i n th e statem ents below .

a. You should use ________________ fo r s ta in in g m ito-

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. 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 o f s t a in 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 .

B. 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. 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.

2. HEMATOXYLIN: How t o use hem atoxylin s t a in s .

a. 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. (See Chapter 8 .)

b. 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

(4) Pass through 50$, 70$, and 95$ a lc o h o ls . (A few

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 ).

c. Mayer*s haem-alum. (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. (4 or 5 m inutes

i s u s u a lly p le n ty .)

(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) 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. (See Chapter 8 .)

2. CARMINES: How to use carmine s t a in s .

a. 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 . (This may

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. (See Chapter 8 .)

 ai

b. 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. ( 2 . , a . - ( 2 ) , (3)> (4)> ($)»

and (6) . )

c. 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.

3. ANILINS: How t o use a n ilin s t a i n s .

a. 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) Mount. (See Chapter 8 .)

b. 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 fo r

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 . (See Chapter 8 .)

e. E rythrosin (Cytoplasm s t a in s p in k .) (Use as double s t a i n .)

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 .)

(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 .)

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

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

h. 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: How t o use double s t a i n s ,

a* 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, (se e Chapter 8 .)

b. 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 . (Safranin and D ela-

F ie ld * s hem atoxylin.)

(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

(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 ) (Safranin and

D e la fie ld 's hem atoxylin.)

c. 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, (In clo v e o i l ) fo r 3 to 30

minutes*

(6) Proceed t o c le a r and mount i n balsam . (See Chapter 8 .)

d. 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 . (See

Chapter 8*)

e. 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* (See

Chapter 8*)

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) D iffe r e n tia te and proceed t o mount i n balsam* (See

Chapter 8 .)

h* 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) 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 .)

i, 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, (See,Chapter 8 ,)

j, Safran in and a n ilin b lu e ,

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

k, Safranin and Orange G.

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

1, Auramine and a n ilin b lu e ,

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

m. 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

(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 mount i n balsam . (See Chapter 8 .)

n. 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 mount in balsam . (See Chapter 8 .)

5. TRIPLE STAINS: How to use t r i p le s t a i n s .

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

2. SPECIAL REFERENCES: (See Chapter 9 .)

C. 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.

D. 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

A. BOOKS

1, Beck, C .: The M icroscope, Beck. London, 1938*

2. B e llin g , John: The Use of* +.he Microscope. McGraw-Hill. Hew York,
1930.

3* Bower, F. The Ferns. Cambridge U n iv ersity P r e s s . 1926.

4. B a llard , C.W.: The Elements o f V egetable H isto lo g y . John W iley

and Sons, I n c ., 19257

5. Chamberlain, C harles, J .: Methods i n P lant H isto lo g y . The

U n iv e r sity o f Chicago P r e ss. Chicago, I l l i n o i s , 1928. (Referred
t o as Chamberlain.)

6. Campbell, D. H.: Mosses and Ferns. MacMillan. 1918.

7. Conn, H. J .: B io lo g ic a l S ta in s . 3rd Ed. Commission on

S tan dardization o f B io lo g ic a l S ta in s . Geneva, New York. 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. Ehmes, Arthur, J .: Morphology o f Vascular P la n ts. McGraw-Hill.

Hew York, 1936.

11. Earnes, Arthur J .: An In trod u ction t o P lant Anatomy. McGraw-Hill.

Hew York, 1941.

12. Gage, S . H.: The M icroscope. 16th Ed. Comstock P ub lish in g Co. I n c .
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. Gwynne, Vaughan, H. C. L. and Barnes, B .: The Structure and

Development o f th e Fungi. Cambridge U n iv e r sity P r e ss. 1927.

15. Johansen, D. A .: P lan t M icrotechnique. McGraw-Hill. New York,

1940. (Referred t o as Johansen.)
 91

16. Loomis, W. E. and C. k i S h u ll.: Methods i n P lant P h ysiology.

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 . 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. R och ester, New York.

20. S a s s, J . E.s Elements o f B otanical M icrotechnique. McGraw-Hill.

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.; Cryptogamic Botany. McGraw-Hill. New York,

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.
 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 ." Journal o f

B a c ter io lo g y . 7: 127-148. 1922.

5. . Conn, H. J . : "Haematin and Acid Fusion." S ta in Technology. 4: 97.

1929.
6. C r a fts, A. S .: "A Technique fo r Demonstrating Plasmodesma." S ta in
Technology. 6: 127-129. 1931.
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. 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•

9. 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. Dufrency, J .: "A Method o f Imbedding Plant T issu es Without De­

hydration, " S c ie n c e . 82: 335 -3 3 6 .

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. G ourley, J . H.: "Basic Fuchsin for S ta in in g Vascular Bundles."

S ta in Technology. 5: 99-100. 1930.

13. Hance, R. T .: "A New P a ra ffin Embedding M ixture." S c ie n c e .

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." S ta in
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.
 93

16. 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.

17* 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.** B o ta n ica l

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. Stoughton, R. H .s "Thionin and Orange fo r th e D if f e r e n t ia l

S ta in in g of B a cteria and Fungi in P lan t T is s u e s .” Annals o f
A pplied B io lo g y , 17s 162-I6U . 1930.

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. W alton, John: ”Improvements in the P ee l Method o f Preparing
S e c tio n s of F o s s i l P la n t s .” Nature, October 1 3 , 1928j and March
1 5 , 1930.
29* Wetmore, R. H .s ”The Use o f C e llo id in in B o ta n ica l Technic."
S ta in Technology, 7 t 37 -6 2 . 1932*

30. W illiam son, H. S . s "A New Method o f Preparing S e c tio n s o f Hard

V egetable Stru ctu res.® Ann. B o t ., January 1921.
APPENDIX
 95

APPENDIX

FORMULAE FOR REAGENTS .

( As adapted from Chamberlain. )

A. FIXING AGENTS.

1. Absolute a lc o h o l. (Used alon e w ithout any m ix tu res.)

2* Bouin*s S o lu tio n , A llen M odification !

a. P ic r ic a c id , aqueous s o lu tio n *•••«•••• 75 p a rts

b. Form alin, (C. P .) 15 p arts
e. G la c ia l a c e t ic a c id 10 p a rts
d. Urea . . • • • .......... .. 1 part

3. Carnoy*s flu id s

a. A bsolute a lco h o l 3 p a rts

b. Chloroform 3 p arts
c. G la c ia l a c e t ic a c id 1 part

4* Farmer*s f lu id :

a. A bsolute a lc o h o l • • • • . • • « • • • • • • • • . • • • • • 3 p arts
b. G la c ia l a c e t ic a c id 1 part

5. Formalin:

a. Commercial form alin • • • • • • • • • • • . . . . . • • • 3 t o 10 c c .

b. Vfoter .......... .. 100 c c .

6. Strong chrom o-acetic so lu tio n :

a. Chromic a c id ........ ................................ 1 g.

b. G la c ia l a c e t ic a c id ................................. .. 1 cc.
c. Water ........ ......................... .. 100 c c .

7. Weak chrom o-acetic s o lu tio n :

a. Chromic a c id 10$............................................. .. 2 .5 c c .
b. A cetic a c id ...................................... 10$ so lu tio n
c. Water 5 c c . t o 100 c c .

8. Medium chromic a c id :

a. Chromic a c id 10$... .......................... .. 7*0 c c .

b. A cetic a cid .......... ................... 10$ s o lu tio n
c. Water .................................... 10 c c . to 100 c c ,

1
Chamberlain, C harles, J .: Methods in P lant H isto lo g y . The
U n iv ersity of Chicago Press"! Chicago, I l l i n o i s , 1928.
 96

9. Chrom-osmo-acetic f lu id :

a* Flemming1s f l u i d (weak.)
(1) 1% chromic a c id (in water) .......... 25c c .
(2) 1% g l a c i a l a c e t ic a c id (in water) ..... 10 c c .
(3) l a t e r 55 c c .
(4) 1% osmic a c id (in water) 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. Flemmingt s f lu id (Stronger):
(1) 1% chromic a c id 45 c c .
(2) 2J6 osmic a c id 12 c c .
(3 ) G la c ia l a c e t ic a c id • • • • 3 cc.

10. Naswaschin's flu id :

a. Chromic a c id 10^ 1 .5 cc.

b. G la c ia l a c e t ic a c id ................ 1 0 .0 cc.
c. D i s t i l l e d water .......................................... 90.0 cc.
d. Commercial form alin 4 0 .0 cc.
e. D i s t i l l e d water 6 0 .0 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. 95% a lc o h o l .............................. 42 cc.

b. la t e r 60 cc.
c. G la c ia l a c e t ic a c id IS cc.
d. Concentrated n i t r i c a c id 2 cc.
e. Corrosive sublim ate
(Saturated s o lu tio n in water ) . . . . . . . . . . . . . U cc.

12. Schaudinn's f lu id :

a. D i s t i l le d water 200 cc.

b. Sodium c h lo r id e 1 .2 gm.
c. Mercuric c h lo rid e ......1 0 .0 gm.
d. A bsolute a lc o h o l 100.0 cc.

13. Strom sten, s f lu id :

a. Chromic a c id 10% 16 cc*

b. A cetic a c id 10$ 100 c c .
c. lite r 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. D i s t i l le d w a t e r .50 c . c .
b. Absolute a l c o h o l ................................................ 50 c . c .
c. G ly c e r i n ... 50 c . c .
d. G la c ia l a c e t ic a c i d . . ....................................... 5 c . c .
e. 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 . If

w e ll stop pered, i t w i l l keep i n d e f in it e ly .

3* 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. C arm ine............. 3 g.
b. Borax.................... 4 g.
c. D i s t i l le d water 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* 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)*

7. Alum c o c h in e a l.

a* Powdered co ch in ea l ............................................. 50 g .
b* A lu m 5 g*
c* 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. Carminic a c i d .......................................................... 1 g .
b. A lu m 10 g*
c. D i s t i l le d water .....2 0 0 c.c.

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* 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 .

10. E osin .

a. E o s i n ...................................................... 1 g.
b. Water or 70^ a l c o h o l ...................... . . . . 1 0 0 c*c«
 99

11. A n ilin blue*

a* A n ilin blue .......................................................... 1 g .

b. &5% i r 90% a lc o h o l 100 c.c*

12. Methyl g reen .

a* Methyl green 1 g.
b. G la c ia l a c e t ic a c id ......................................... 1 c . c .
c* Viater ..1 0 0 c* c .

13. Light green (A).

a. Light green 1 g.
b* 90% a lc o h o l 100 c . c .

14 . Light green (B).

a. Light green ............................................................... 1 g .

b. Glove o i l .100 c . c .

15. Light green (C).

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

16. Acid fu c h sin .

a. Acid fU chsin ............................... 1 g.

b. T fe te r 100 c . c .

17. Safranin (A).

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

IS . Safranin (B) •

a. Safranin 1 g.
b. Water 100 c . c .

19. Safranin (C)•

a. S a f r a n in 1 g.
b. 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. Gentian v i o l e t .................................................... l g .
b. 95% a lc o h o l 20 c . c .
c. W a te r ............... 80 c . c .
d. A n ilin o i l ............................................................... .. 3 c . c .

21. Orange G.

a. Orange G. 1 g.
b. Water .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. Bismark brown .................. 2 g.

b. 70% a lc o h o l 100 c . c .

23* Congo r e d .

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

24. E r y th r o s in .

a* E ry th ro s in .................... 1 g.
b. 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 . (Same a s gen tian v i o l e t ) .

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

C. MISCELLANEOUS.

1. Mayerl s albumen f i x a t iv e .

a. White o f egg (a c tiv e p r in c ip le ) 50 c . c .

b. G lycerin (to keep i t from drying u p ) 50 c . c .
c. S a lic y la t e o f soda or c a rb o lic a c i d 1 g.

Shake w e ll and f i l t e r through lin e n .

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

a. Guta arab ic .............. 1 g.

b. Potassium dichromate 1 g.
c. W ater . . . . . . . ............. 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. G e la tin 1 g.
b. Phenol .............................................. 2 g.
c. G ly c e r in .................................................................... 15 c . c .
d. 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. C e llu lo se a c e ta te . . 1 2 g.
b. Pdre acetone ........................... 100 c .c .
lis-'ir ■r.r*'
G lycerin j e lly *

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

H8IVERSITY OF SOUTHERN CALIFORNIA UERAR?