M A T E R I A L S

A N D

METHODS

MATERIALS

The life cycle of PallJsentis nagpuren~Js was tirst stud1ed

by George and Nadakal 11978). They described Ophiocephalus

stri.atu:::; as th" definitiv" host a11d the copepod Cyclaps

strennu~ as the intermediate host. Eggs were described to

float in water and when ir>gested by the intermed1ate host,

hatched in the gut of cyclops ... Jithin B-10 hours. The newly

hatched larva, kno ... rn as acanthor, penetrated the gut "'all and

escaped into the hemocoel "'ithin 30-48 hours. It lost its

characteristic shape and rostellar hooks in about 7-10 days

in the hemocoel of cyclops and gradually metamorphosed into

acanthella. The acanthor ... ras said to develop into infect1ve

acanthellae in about 15-20 days and the acanthellae trans-

formed into immature juveniles in about 11 days 1n the hemoc-

oel of cyclops. On ingestion of infected cyclops by the

definitive host, the juveniles reached the intestine, the

proboscis became everted and the worms grew to se:<ual maturi-

ty in about 50 days. On several instances the immature "'orms

penetrated the intestine and encysted in the peripheral lobes

of the liver. Cannibalism among the fish species helped in

establishment of encysted worms for normal development in the

intestine. Ho~.tJever, transport hosts, namely, Hacropodus

cupanus, Aplocheilus melastig•a, Barbus spp., and Ophiocepha-

lus gachua have also been reported for P. nagpuren:sis by

George and Nadakal 11978).

This author has studied the life cycle of P. nagpurensi$ at

Raipur. Basically, the pattern i~ similar; however, some

differences are pretty obvious, namely, that the juveniles

were never found to penetrate the intestine of Channa puncta-

tus and that they were never found encysted in its liver;

Coli$& lalia was found to harbour encysted juveniles. Obvi-

OUIOly, juveniles reached the intestine of c. lalia after

ingestion of cyclops along •rith the phytoplankton and aquatic

I>Jeeds and penetrated the intestinal wall, reached the coelom­

ic cavity and became encysted in the liver tissue. Usually in

each cyst 3-6 juveniles were found. C. punctatus feeds upon

C. lalia and thus gets infected. ~ is apparent that ~order

!.Q. have ~ continuous ready supply of the worms and the in­

fected hosts, it ~necessary to plan ~strategy to maintain

cyclops, ~ punctatus and ~ lalia in the laboratory. Mainte­

nance of these were as follows:

~Collection, Maintenance and Infection of the Intermediate

Host, Cyclops strennus:

E:<tensive survey of the local ponds and tanks ~oras done for

incidence of acanthocephalan infection in Cyclops spp. Water

of the ponds and tanks, found positive for infected Cyclops

spp.' ••as agitated 1>1i th a •rood en stick; the concentrated

organisms on the surface were collected through a fine mesh

and transported to the laboratory. The micro-organisms

(ostracods, copepods, etc.) thus concentrated were observed

23

for Cyclops spp. lwh1ch have charact1trist1c cyclopo1d move-

ment). Cyclops spp. were isolated and transferred to a pe-

tridish by a fine glass dropper and were then identified

following Wilson and Harry 11959). The species identified

were, C. bicuspidatu~, C. vernali~, C. navu~ and C. strennuJ.

All these species of cylops were o.:<amined for infection of

acanthars, acanthellae, and juvenil,.s. The acanthellae or the

juvenile worms can be observed superficially wh"n viewed from

the dorsal surface und"r a binocular microscope {:<20). Only

C. :>trennu:> was found to harbour this infection. Taking cue,

experiments were planned to infect C. :>trennus in the labora-

tory. Uninfected and infected C. strennu:> were maintained

separately in small aquaria, 1' x 1.5' x 1'. Phytoplankton

~•ere made available throughout the rearing period. Also, the

aquaria "'ere e:<posed to full light to allo"' ample thriving of

the planktons. At 4-6 days interval, the water in the aquaria

was replaced. For infection experiments, 15-20 uninfected c.

:>trennu:> were transferred to small beakers of 50 ml capacity

containing filtered pond water. Eggs of P. nagpuren:>is pro-

cured from gravid females, collected from naturally infected

C. punctatu:>, "'ere released in these beakers. Phytoplanktons

were supplemented in these beakers. C. st-rennu:i' ingested

these embryonated eggs and acquired infection. They were

e:<amined everyday and the first acanthors could be observed

in their hemocoel in about one "'eek. The acanthors changed

into acanthellae in the hemocoel itself during th" second

24

w~~k. Exper1m~ntally 1nfect~d C. strennus were then used to

inf~ct C. punctatus and c. lalia.

lLL Cgllectipn. Maintenance AQ2 lnfrctign oL ~ Drfinitivp

Host, Channa pynctaty~a

Channa punctatus wer@ procured live from fishin~ sites and

hatcheries during breeding period (July-October). They wer@

stocked in large aquaria, 3' X 1.5' x 1.5". Food, of minced

goat liver, ~oas made available to fishes ad Iibitu•. As the

young C. punctatus were prone to a number of other infections

(corynebacteriosis, epizootic ulcerative syndrome, fin rot

et•:.) proper care was taken to give the fishes antibiotic

bath prior to stocking. Aside this, the water of the aquaria

was also changed at ~•eekly intervals. C. pun•:tatus were

dissected and if infected, the mature P. nagpurensis "'•arms

~•ere recovered. Gravid females were transferred to cavity

blocks containing tap water. Worms e:<pelled eggs in about 2-4

hours and a dense ,mass of eggs "'ere observed to accumulate a.t

the bottom of the cavity blocks. The eggs were fully embryon-

a ted. These ""re used to infect C. strennus a.s described

under the subhead I. Young C. punctatus, after a.cclimatiza-

tion, were used for infection in experiments. About 3-6

infected Cyclops strennus were fed to these C. punctatus with

a. glass cannula for 5 consecutive days. Daily e:<a.mina.tion of

these fishes, commencing day 6 post infect ion, revealed

juveniles in the intestinal wall of C. punctatus in about 5

25

weeks.. However, gravid females were noted after 7 weeks.

S1multaneously, in parallel e~periments, 3-5 cysts recovered

from C. lalia were fed to another set of C. punctatus with a

glass dropper for 5 consecutive days. Examination of these

fishes, commencing 6 days post infection yielded, immature

worms attached to the intestinal wall of C. punctatus. Thus,

infection of P. nagpurensis in C. punctatus could occur

either through ingestion of infected C. strennus or through

ingestion of C. lalia.

III. Collection, Maintenance and lnftctian 21 the Paratenic Hgst,

Colisa lalia:

Colisa lalia were procured form local fishing sites. During

the breeding season (July-September) fingerlings of C. lalia

were procured in bulk and stocked in large aquaria,

1.5. x 1.5'. In other seasons adult C. lalia were procured

from fishing sites and were also stocked in separate large

aquaria. Artificial fish food (a local brand • containing

crude protein and fat) "'as made available to the fishes ad

libitWtl. Fishes collected form nature invariably harboured

encysted juveniles of P. nagpurensis in the liver tissue. The

cysts were ruptured and the number of infective juveniles in

each cyst were counted and the average number computed.

Intact cysts "'ere used to teed C. punctatus, the definitive

host. Care was taken to segregate fishes with other infec-

tions such as, Senga visakhapatna»ensis, Clinostaaua coapla-

natua or other microbial infections. C. coaplanatua infection

26

is vtsible superficially as it encysts in the muscle tissue

of C. lal1a and infected ftshes can be sorted and e:<cluded

easily. S. visakhapatna•ensis juvenile infecttons form cysts

1n the liver but these are, however, smaller and can be made

out by naked eye. Infection e:<periments in C. lalia included

feeding of 3-6 infected C. ~trennus through a glass cannula

for 5 consecutive days. Daily e:<amination of these fishes,

commencing from day 6 post infection, revealed occurrence of

encysted juveniles in about 4 weeks time. It can be interred

that infection of C. lalia occurred through ingestion of

infected C. strennus.

METHODS

The intestine of C. punctatus ~•as removed and transferred to

a petridish containing physiological saline. It ~•as opened by

a vertical slit. Worms usually occupied the posterior

portion, 3-6 em. below the pyloric valve. This portion of the

intestine was removed and cut into about 1 em. pieces, and

were either fi:<ed for paraffin embedding or rapidly frozen in

the Cryostat cabinet. These 'sections were used for different

pathological and histochemical determinations. c. !alia

were likewise dissected to expose the liver of both infected

and uninfected fish. The liv"r \>laS removed and washed in

physiological saline. Infected liver usually carried two

k1nds of cysts, smaller Senga V>~akhapatnaaen~•~ cysts and

larger P. nagpuren~is cysts. Liver harbouring only P. nagpu­

rensi~ cysts were processed for pathological and hlstochemt­

cal studies. After dissection of C. punctatus, P. nagpuren~is

were r·ecovered, washed in physiolog1cal saline, f i :«•d 1n

various fi:<atives/rapidly frozen in the cryostat cabinet,

serially sectioned and processed for histochemtcal and histo­

enzymological studies. Methods to study pathology are de­

scribed under subhead <I) and to study Histochemistry and

Histoenzymology under the subhead (11).

~ TECHNIQUES USED IN STUDY OF PATHOLOGY

A. EXPLORATIVE PATHOLOGY

The epithelial cell proliferacy, alterations in the chemical

composition of mucins in the goblet cells and the morphologi­

cal alterations in the gut mucosa of C. punctatus were stud­

ied in separate e:<periments at various periods fallowing

e:<perimental infection. A resume of these methods are de-

scribed as under:

1. The Metaphase Arrest Technique <Wright and Appleton, 1980)

Normally, the intestinal epithelial cells originate in the

crypts, migrate onto the villi, and move to~1ards its ape:< and

finally are extruded at a specific site called the extrusion

zone. Attempts were made to study the intestinal epithelial

28

cell ~1net1cs in the gut of C. punctatus during PalllsentlS

nagpurensi:; infect1on. For studying th& intestinal cellular

turnover the important parameter appeared to be the 1nflu:<

of cells onto the villi. Hence the incoming cells on to the

epithelial layer was calculated by the product of numbers of

crypt/villus (C/V) ratio X the cell production rate/crypt.

To appreciate the cell production rate per crypt, a meta-

phase-arresting substance <stathmokinetic agent) was used.

21 C. punctatus were selected for e:<perimental infection and

21 others served as control. Optimal dosage of colchicine for

the intestinal epithelium was determined by a preliminary

dose-response curve e:<periment <Wright and Appleton, 1980).

All fishes were injected intraperitoneally at 18 days post

infection with 1.5 mg of colchicine/Kg body weight, between

10.00 to 10.30 hrs. to minimize the effects of diurnal varia-

tions in the proliferative rate <Al Dewachi et al., 1976;

Haste, 1989). Three fishes from each group were dissected

every 30, 60 1 90 1 120 1 150 and 180 minutes, after colchicine

injection, and about b em. long intestinal segment descending

from the pylorus 1>1as cut out~ The segment ~•as cut into 4

smaller segments each of 1.5 em, fi:<ed in acetic-alcohol

( 1:3)

tion

for 24 h and stained in bulk "'ith the Feulgen's reac-

<Feulgen and Rossenbec~:, 1924 >. Crypts were dissected

out under a dissecting microscope, squashed 1n 45% acetic

ac>d and the squashes were observed for metaphase arrested

cells. Also, total number of cells per crypt and total number

of metaphases/crypt were determtned by counts at a x640

magnlficatiorl. Metaphase indices were determtn&d after count­

ing 3000 cells/fish, and were expressed as the number of

blocked metaphases/1000 cells and per hour <Haste, 1989).

The slope of the blocked metaphases accumulation line gives

an estimate of the cell production rate/1000 cells and per

hour (kb). As the arrest of mitosis between 0 and 30 mins

after colchicine injection is apparent the slope of metaphase

accumulation 1>1as only measured bet1>1een 30 and 180 mins af.ter

infection. The mean crypt cell production rate <kbc) ~>las

calculated as kb X mean cell number per crypt. The influx of

cells on villi (kbv) per hour was estimated as kbc X C/V

ratio <Wright, 1980). Statistical analysis of metaphase

accumulation rates was done by drawing straight lines by the

method of least squares. Differences in the slopes of these

regression 1 ines 1>1ere analyzed by a test for comparison of

slopes of regression lines <Dagnelie, 1070; Symons, 1965; Da

Costa, 1971).

2. Assessment of Changes in the Goblet Cell Mucins

Male c. punctatus, 12-24 ems long and weighing 100-150

g were selected for treatment.

30

Experiaental De$iqn

Fout· groups at 12 ftstles were ted 3-6 JUVenll~ cysts eact1 and

one g~oup of 12 control f1shes were used in th1s study. On

6, 12, 18 and 29 days post tnfect1on, 12 1ntected and 3

cont~ol f1shes were dissected after demob1l1zing the f1shes

by a sharp blo~1 on the head bet~Jeen 8.00 to 9.01) Hrs. The

intestinal segment, 6 ems long, descending from the pylorus

was removed from each fish. It ~•as cut open longitudinally

and four segments, 1.5 ems long, ~Jere prepared. Frozen sec­

tions of thickness 8 fill" ~•ere obtained at -15°C cabinet tem­

pe~atuPe. Sections wePe o~iented along the longitudinal a:<is

of the intestine and cut at right angles to the surface of

the mucosa. Average intensity of infection "''as 7-12 bJorms per

1.5 ems long segment. Goblet cells were counted 1n un1nfected

and infected samples, after staining with periodic acid­

Schiff's method <PAS). Histochemical alterations in the mucin

composition of the goblet cells "'as assessed after staining

with alcian blue-periodic acid Schiff's method <AB-PAS) for

acid (blue) and neutral mucins (redl, high iron diamine

method-alcian blue <HID-ABl for differentiating sulphated

(puPple black) and sialomucins (blue). The number of goblet

cells positive for each technique were recorded. Statistical

significance, at different time periods following infection,

t>Jas analyzed by ANOVA and Duncan· s multiple-range test

<Duncan, 1955; Brunning and Kintz, 1977).

3. Assessment of Morphological Alterations in th& Gut Mucosa

Male C. punctatus, 12-24 ems long and •ungh1ng 100-1~1) g

were selected for this study.

£xperiaental Ds~ign

Four groups of 12 fishes were fed with 3-6 infective juve-

niles of P. nagpuren~is and one group of 12 control fishes

were used for this study. On day 6, 12, 18 and 29 post

infection 12 infected and 3 control fishes •1ere dissected

after demobilizing the fishes by a sharp blo•1 on the head,

between 8.00 to 9.00 Hrs. 6 ems long portion of the intes-

tine descending from the pylorus "'as removed. The intestine

were cut open longitudinally and four segments, 1.5 ems long,

•1ere prepared. Frozen sections of thickness B ,rwm were ob-

tained at -15"'c cabinet temperature. Using hemato:<ylin and

eosin stained sections, 5 well oriented villi and

crypts/fish/time interval •1ere measured using a micrometer.

Villus/crypt ratio <Konink:< et al., 1988) •1as calculated to

indicate the extent of abnormality.

B. G£H£RAL TISSU£ PATHOLOGY

Tissue Processing: '

Pathological study of the infected organs v•s-a-vJs control

was done after fixation of the infected/control tissues in

Cornoy"s fixative, Bouir\·s (alcoholic and aqueous), 10"

.,,.., •,..I..;

formal1n, formol-calclum, 90% alcohol and acet1c alcohol.

Fi:<ed ttsc:;ues were Pmbeodded tn pat•afttn '-'J·l.:< and sectioned In

a rotator-y microtome at 6 }'JlT1 thickness. F01· frozen sect1ons,

tissue, immediately after dissection, were rinsed in

phys1ological s•l1ne followed by distilled water and were

rapidly frozen in the Histostat cab1net 1 sect1oned at various

temper-atur-es <-SOC to -35°Cl depending upon the type of

tissue <Bancroft and Stevens, 1977). Usually the tissues

wer-e pr-ocessed unfixed, but post fixation, in acetic-alcohol

or- for-mol-saline, was done wherever- necessary. Sections lBpm

Both paraffin and fr-ozen sections

were used for var-ious staining. The techniques involved in

pathological studies ar-e descr-ibed as under:

Standar-d Hematoxylin and Eosin Stain for- Paraffin Sections

<Bancroft and Stevens, 19771

Fixation: Cornoy·s fluid, Bouins fluid or- 10~ Formalin

Sections: Paraffin as well as Frozen.

Reagents Used:

L Harris alum haematoxylin: 2.5 9 haemato:<ylin was dissolved

in 25 ml of absolute alcohol; 50 g of potassium alum was

dissolved in 500 ml of distilled water in another- beaker.

Both were mi:<ed together and 20 ml glac1al acet1c acid and

1.25 g of mercuric a:<ide were added to it. The solution bias

ripened for- 1 month and filtered before use.

2. 1~ Eosin: 1g of eosin. yellow was dissolved in 100 ml of

d1sti lled ~•ater.

Procedure: Paraffin sect1ons were dewaKed, hydrated through

descending ser1es of alcohol to water. Frozen sections were

postfi:<ed in lOY. Formalin for 20 'iiecond"' or processed unflKed

and rinsed in tap water. Paraffin/frozen sections were

stained in alum hemato:<ylin for 1-2 minutes, rinsed in tap,

d1fferentiated in lY. acid alcohol for 5-10 seconds, washed

again in tap water for 5 minutes or less, sta1ned in lY. eosin

Y for 1-5 minutes, washed 1n running tap water, dehydrated

through graded alcohols, cleared in :<ylene and mounted 1n

DPX.

Results: Nuclei - blue-black; Cytoplasm - varying shades of

pink; Muscle f1bres deep pinkish red; Collagen

pinkish red; Red blood cells - orange/red;

pink.

,

Fibrin

Basic Fuchsin-Ferric Chloride Stain for Elastic

<Horobin et al., 1975)

Fi:<ation: lOY. Formalin

Sections: Frozen as well as Paraffin Sections.

Reagents Used:

pale

deep

Tissu&

1. Basic fuchsin-ferric chloride solution: 2g of basic fuch-

sin ~•as dissolved in 200 ml of distilled water and it was

boiled. 25 ml of 30Y. ~dv ferric chloride ~•as added to thts

boiling solution, it was cooled, filtered and the precipitate

was dried in an oven at 6o0C. Finally, the dried precipitate

34

was dtssolved in 200 ml of 95% ethanol; 4 ml of concentrated

HCl ••as added and the solution \>las made to 200 rnl •nth 9511.

ethanol. This solution was stored at 4°C.

Procedure: Sections were brought to water tt1rough graded

alcohols, stained in the staining solution for 30 minutes,

differentiated in 1% acid alcohol for 5 mtnutes, dehydrated,

cleared in :<ylene and mounted in DPX.

Result: Elastic tissues, mast cell granules purple.

~ HISTOCHEHICAL AHD HISTOEHZYHOLOGICAL HETHODS

CARBOHYDRATES

Periodic acid-Schiff"s method for poly~accharides

<Mcmanus, 1946}

Fixation: Post fi~ation 1n Ptcrate-formalin for ~ minutes.

Sections: Frozen

Reagents used:

1. Picrate-formalin fi:<.ative: 0.18 9 sodium chloride and 0.15

9 picric acid were dissolved in 25 ml of distilled water; 56

ml absolute alcohol and 10 ml of 4011. formaldehyde "'as added

to it and the "'hole •ras made to 100 ml with distilled water.

2. Periodate solution: 400 mg of periodic acid ~>ras dissolved

in 15 ml of distilled water; 135 mg of crystalline sodium

acetate 4>tas dissolved in 35 ml of absolute ethanol separately

and both mixed just before use.

3. Reducing rinse: 1 9 each of potassium iodide and sodium

thiosulphate ••ere dissolved in 20 ml distilled water; 30 ml

of ethanol and 0.5 ml of 2N HCl were added to it.

4. Sch1ff'<o reagent: 1 g bas1c fuchsin was d1ssolved tn 'LOO

ml bo1ling distilled water. After cooling to 50°C, the solu­

tion was filtered and 30 ml IN HCl and 1 g potasstum metabi­

sulphide was added to it. The resulting solutton was allowed

to stand in dark for 24 h. 2 g activated charcoal was added

with stirring to it, filtered and stored in brown bottle.

Procedure: Secttons after fiMation tn picrate formalin tor ~

minutes, were washed with 70% alcohol; treated with periodate

solution for 5 minutes; ~tashed again in 70 % alcohol and im-

mersed in the reducing rinse for 5 minutes. Sections "-Jere

stained in Schiff's reagent for 30 minutes, washed in three

changes of S~ water, dehydrated and mounted in DPX.

Result:

tion.

Red or purple colour indicates a PAS pos1tive reac-

Best Carmine Method for Glycogen <Best, 1906)

FiMation: 90% alcohol

Sections: Paraffin or Frozen

Reagents used:

1. Carmine stock solution: 2g.carmine, 1g potassium carbonate

and 5g potassium chloride ~tere dissolved in 60 ml of dis-

tilled ~>tater. Boiled for 5 minutes, cooled and 20 ml of

concentrated ammonia solution was added. Filtered and stored

at 4°C. in bro~tn bottle.

2. Carmine working solution: 15 ml of the stock solution,

12.5 ml of concentrated ~mmon1a solut1on and 2.5 ml of metha-

nol ~1ere mt:<ed together.

Best's difterentiator: 40 ml methanol, BU ml ethanol and

100 ml of distilled water were m1xed tagPther.

Procedure: Sections were brought to water through descend1ng

series of alcohol, immersed in carmine wor~tng solution for

15 m1nutes, differenttated in Best's difterenttator, r1nsed

in alcohol, cleared in :<ylene and mounted in DPX.

Results: Glycogen granules deep red.

Lead Tetracetate-Schiff's Method (Shimizu and Kumanoto, 1952)

Fi:<ation: Not critical; Various fi1<atives

Sections: Paraffin or Frozen

Reagents used:

1. Tetracetate solut ian: lg lead acetate was dissolved 1n 30

ml glacial acetic acid; 70 ml of saturated sodium acetate

solution .. ras added to it just before use.

2. 1 M sodium acetate solution: 8.2g sodium acetate ~oras

dissolved in 100 ml of distilled ~orater.

3. Schiff's reagent: As above <pp.36l.

Procedure: Sections "'ere brought to water, immersed in 1 M

sodium acetate solution for 5 minutes, washed in running tap

water for 10 minutes, treated with Schift's reagent for 15

minutes, "lashed in three changes of bisulphide water follo .. red

by one change 1n distilled water, dehydrated in graded alco-

hols, cleared in :<ylene and mounted in DPX.

Hesul t: Glycogen and vartous mucosubstances reddtsh-puq.Jle.

Re agen.t used:

disttlled water.

Enzyme E~traction of Glycogen

lg U1astase was dissolved 10 100 ml of

Procedure: Control sections were brought to d1st1lled ~ater,

treated with diastase solution for 1 hour at 37°C, washed in

running tap water tor 3-5 minutes and stained by Best ~armine

or Lead Tetracetate-Schiff's method alongwith test sections.

HUCOPOLYSACCHARIDES

Alcian b lue-·PAS method <Mowry, 1956)

Sections: Frozen

Reagent used:

1. 3 % acetic acid: 3 ml glacial acetic a.c1d "'as made.a to 100

ml ~ith distilled water.

2. 1 % Alcian blue: 1 9 alcian blue B GX was dissolved 1n lOU

ml of 3 % acetic acid.

3. 1 % periodic acid:

ml of distilled water.

1 g periodic acid ~•as dissolved in 100

4. 0.5 % sodium bisulphide: 0.5 9 sodium b1sulphide was

dissolved in 100 ml distilled water.

5. Schiff's reagent: Prepared as for PAS react1on (pp.3b ).

Procedure: Sections "'ere brought to water, briefly rinsed in

3% acetic acid, stained in alcian blue solution for 2 hours,

sections were oKidized in 1% aqueous periodic acid for 10

38

minutes and 1mmersed 1n Schiff's reagent for 10 m1nutes.

Finally, the sections were rinsed In three changes at U.5~

sodtum bisulphtde, washed tn running water,

cleared and mounted xn DPX.

dehydrated,

Hesult:

blue,

Alcinophillic (perlodate-unreactlve) mucosubstances

alc1nophillic <periodate-reactive) mucosubstances

blulsh-purple, and periodate reactive non-alc1nophill>c

components <Neutral mucins) red.

Low Iron / High Iron Diamine-Alcian Blue Method <Spicer, 1965)

Fixation: 10~ Formalin

Sections: Paraffin Sections

Reagents used:

1. lX Periodic acid: lg periodic acid was dissolved in 100 ml

of distilled water.

2. Diamine solution (for LID>: 30 mg N,N-dimethyl-m-phneyle­

nediamine dihydrochloride and 5 mg N,N-p-phenylenediamine

dihydrochloride ~Jere dissolved in 50 ml distilled water. 0.5

ml lOX ferric chloride 1>1as added to it.

3. Diamine solution <for HID): 120 mg metadiamine and 20 mg

paradiamine were dissolved in 50 ml of distilled water. 1.4

ml of lOX ferric chloride was added to it.

4. Alcian blue solution: 1g alcian blue stain ~1as dissolved

in 100 ml of 3% acetic acid.

Procedure: Duplicate sections ~>Jere brought to water, one

section ~>Jas o:<idized in 1% periodic acid for 10 min, r1nsed

39

1n runn1ng tap water, both sectlOflS WPre lmmPrSPd 1n diamine

<separate for LIU and HIU) tor 18 hours, r1nsed solution

•·ap idly in \oJater, stained 1n al(~1a11 bluP sta1n for· ~0

m•nutes, dehydrated 1n graded alcohols, cleared 1n ~ylene and

mounted in DPX.

Result:

LIU: Sulphated mucins gray to purple black.

HID: Sulphated mucins purple-black; sialomucins blue.

Colloidal Iron Method for Acid Mucopolysaccharide&

<Hale, 1946)

Fi:<ation: Frozen or Cornoy's ti:<ed Paraffin sections

Reagents used:

1. 0.2M acetic acid: 1.2 ml of glacial acetic acid dissolved

in 100 ml of distilled water.

2. Colloidal Iron: 75g ferr1c chlor1de was dissolved 1n 250

ml distilled oJater, 100 ml of glycerol and 55 ml of concen­

trated ammonia were added to it.

3. Working colloidal iron-acetic acid solution: Equal volumes

of solution 1 and 2 were mi:<ed together.

4. Ferrocyanide solution: Equal parts of 0.02 M-potassium

ferrocyanide and 0.14 M-HCl were mi~ed together.

Procedure: Sections were brought to ••ater, f loaded "'i th

solution 3 for 10 minutes, washed in distilled 1>1ater, flooded

w1th ferrocyanide solution for 10 minutes, dehydrated,

cleared in :<ylene and mounted in DPX.

Hesult: Strongly ac1d1c muc1n blue.

PROTEIHS

Mercury -Bromophenol Blue Method for Proteins <Chapman, 1975)

Fixation: Corney's f~xat1ve

Sections: Paraff1n or Frozen

Reagents used:

1. 2X glacial acetic acid: 2 ml glac1al acat1c acid was made

to 100 ml with distilled water.

2. 0.5X glacial acetic acid: 0.5 ml glacial acetic acid 1>1as

made to 100 ml with distilled water.

3. Bromophenol blue stain: 0.4 g mercuric chloride was dis­

solved in 40 ml of 2X glacial acetic acid 25 mg of bromophe­

nol blue was later dissolved in it.

Procedure: Sections 1>1ere brought to <>later and stained in

bromophenol blue solution for 15 min at room temperature,

washed twice in 0.5X glacial acetic acid and transferred to

butyl alcohol for bluing. They were cleared in

mounted in DPX.

:<ylene and

Result: Proteins appear deep blue or grayish blue.

Fi:<ation:

Ninhydrin-Schiff Method for Amino Groups

<Vasuma and Itchikawa, 1953)

Neutral formal saline; formaldehyde vapours

freeze dried sections).

Sections: Paraffin, Frozen or Freeze dried

41

<for

Heagents used:

1. 0.5% Ninhydrtn solut1on: 500 mg ntnhydrtn was dissolved 1n

100 ml absolute alcohol.

2. Gchiff"s rGagent: Prepared as for PAS reaction (pp.36>.

Procedure: Sections were brought to 70% alcohol, treated in

ninhydrin solution for 12 hours~ ~ashed in runntng tap water~

1mmersed in Schiff"s reagent for 45 minutes, washed in run-

ning tap water, dehydrated, cleared in 1<ylene and mounted 1n

DPX.

Result: Amino groups p1nkish purple.

Performic Acid-Alcian Blue MPthnrl for Disulphide Linked

Proteins (Adams and Sloper, 1955)

Fi:<ation: Neutral formal saline; Formaldehyde vapoul' (for

freeze dried sections)

Sections: Paraffin or Frozen.

Reagents used:

1. Performic acid: 40 ml 98% formic acid, 4 ml 100 val.

hydrogen peroxide and 0.5 ml concentrated sulphuric acid were

mi:<ed together.

2. Alcian blue solution: lg alcian blue stain was dissolved

in 2.7 ml 98% sulphuric acid and 47.3 ml of distilled water

~•as added to it.

ProcedurP: Sections were brought to water, blotted to remove

surplus water, treated with performic acid for 5 minutes,

4,, L

washed tn tap water and drted tn an oven at 60°C unttl

dry, dehydratPd, cl<•ar·t>d, and mounted '" UPX.

Result: Dtsulphtde lin~ed protetns appear blue.

Alkaline Fast Green Method for Basic Protetns

(Alfert and Geschwind, 19531

Fi:<ation: 10~ Neutral formaltn <3-6 Hours).

Secttons: Paraffin

Reagents used:

JUSt

1. 5~ Trichloroacetic acid llCAI: 5g TCA was dtssolved 1n 100

ml of distilled water.

2. 0.1~ Fast green "FCF" stain: 100 mg fast green "FCF" was

dissolved in 95 ml distilled water·, pH was adjusted to 8.0-

8.1 "'ith dilute NaOH and the solut1on "'as made up to 100 ml

with distilled water.

Procedure: Sections were brought to water, 1mmersed tn 5~ TCA

at 95Dc for 15 minutes, ~>rashed in three changes of 70~ alco­

hol, rinsed in distilled water, stained in 0.1~ aqueous fast

green "FCF" sta1n for 30 minutes, washed in distilled "rater,

dehydrated quickly in 95~ alcohol, cleared

mounted in DPX.

in :<ylene and

Result: Basic proteins of the nuclei stain in a bright green

colour.

Blocking Methods for Protein End Groups <Pearse, 19851

Reagents Used:

1. 10~ acetic anhydride: lOg of acetic anhydride was dis-

solved 1n lOtJ ml pyrtd1ne.

2. 0.1 N Hydrochlortc actd: 0.85 ml hydrochlortc

made up to 100 ml with dtstilled water.

actd was

Acetylation <drastic): Control sections were upgraded to

absolute alcohol, immersed in 10% acetic anhydride 1n solu-

tion 1 in reflu:< condenser at 100°C for 4-8 hours.

Acetylation (mild); Control sections ~•erE! upgraded to 'bso-

lute alcohol, immersed in solution 1 for 3 minutes, treated

with a mixture containing 4 ml acetic anhydride, 36 ml ethyl

acetate and 0.05 ml perchloric acid for 36 hours and rinsed

in alcohol.

Methylation <Drastic): Control se'-"" .. <"'ere treated with 0.1

N-HCl in absolute methanol at 60°C for 8-96 h.

Methylation (mild); Control sections were treated with 0.1

N-HCl in absolute methanol at 37°C for 8-96 hours.

Acetylated/methylated sect ions ~•ere processed ~•i th

sections for various techniques.

HUCLEIC ACIDS

The Feulgen Reaction <Feulgen and Rossenbeck, 1924)

Fi:<ation: Not critical but not Bouins fluid

Sections: Paraffin and Frozen

Reagents used:

test

1. 1 N-Hydrochloric acid: 8.5 ml concentrated hydrochloric

44

acid was made to 100 ml with distilled water.

2. Sch1ff's reagent·: As for the PAS react1on lpp.3b).

3. Bisulphite solution: 5 ml each of 10% potassium metabisul­

phite and 1 N-HCl was made to 100 ml with distilled water.

Procedure: Sections were, brought to water, rinsed in 1N-HC1

for 1 minute, hydrolyzed in 1 N-HCl at 6oPC for various

periods 18-20 minutes) depending upon the type of fi:<ative

used !Pearse, 1985>, rinsed again in 1 N-HCl for 1 minute,

immersed in Schiff's reagent for 45 minutes, rinsed in bisul-

phite solution (three changes of 2 minutes each), rinsed in

distilled "'ater, dehydrated, cleared and mounted in DPX.

Result: DNA red-purple.

Methyl Green-Pyronin V Method for DNA and RNA

<Kurnick and Mirsky, 1949)

Fi:<ation: Corney's fluid

Sections: Paraffin and Frozen

Reagents used:

1. 2Y. Methyl green: 200 mg methyl green stain "'as dissolved

in 10 ml distilled water.

2. 2Y. Pyronin Y: 200 mg of pyronin Y was dissolved in 10 ml

distilled water.

3. Methyl green-Pyronin Y stain: 2Y. aqueous solution of

pyronin V was extracted with chloroform in a separating

funnel until chloroform layer became colourless .. 2~ a.queou$

45

solut ton a1 methyl Ill tt:~ s<tmP.

manner. 7.5 ml each of th~se eKtracted stain~ were mlKed

•ntlo ~0 ml thstlll"d ••ater.

Procedure: Sect1ons were brought to water, sta1ned in methyl

green-pyronin V for 6 minutes, blotted Wlth filter paper, im-

mersed in two changes of n-bytyl alcohol of 5 m1nutes each,

cleared 1n :<ylene for 5 minutes and mounted in euparol.

Result: Chromatin blue-green, nucleoli red, cytoplasmic RNA

bright red.

EKtraction Methods for Nucleic Acids

Trichloroacetic acid <TCA): Control sections were brought to

••ater, treated ••ith 4% TCA (4g TCA dissolved in 100 ml dis­

tilled "'ater) at 90"'1: for 15 minutes. They were washed "'ith

distilled water and processed "'ith test sections.

Hydrochloric acl.d (HCl}: Control sections "'ere brought to

water, treated "'ith N-HCl at 37"'1: for ~ hours. They were

••ashed ~>lith distilled ~o1ater and processed ••ith test sections.

LIPIDS

Sudan Black B Method <Lillie, 1944)

Fi:<ation: Various fiKatives

Sections: Paraffin and Frozen

Reagents used:

1. Sudan Black staining solution: A saturated

Sudan Black B in 70% absolute alcohol "'as made.

46

solution of

ProLPdurP: Sect1ons we1'e bt'ought to water·., stauu~d 1n 8udan

!Hac!< sta1n1ng solut1on for 30 m1nutes, r1nsed 1n 7<>14 alcohol

to ren,ove e:<cess sta1n, ~•ashed 1n r'unntng tap water and

mounted in glycerin jelly.

Result: Lip1ds black or blue.

Oil Red 0 Method (Lillie and Ashburn, 1943)

Fixation: Formal-calcium for 5 min.

Sections: Frozen

Reagents used:

1. Oil Red Stock Solution: A saturated solution of o1l red in

isopropanol ~•as made.

2. Working Oil Red solution: This solution is prepared just

before use by mixing three parts of a stock solution with two

parts of distilled water and filtered if requ1red.

Procedure: Frozen sections were air dried, stained in the

~•orking solution for 15 minutes, differentiated 1n 6014 iso-

propanol, "'ashed in tap ~•ater, rinsed in distilled "'ater and

mounted in glycerine jelly.

Result: Unsaturated hydrophobic lipids red.

Acid Haematin Method for Choline-Containing Lipids

<Baker, 1946>

Fixation: 10% Formalin post fixation for 3 min.

Sections: Frozen

" . ' '-t·,·

Reagents used:

1. 0.1% haematoxyl1n: 100 mg haemato~yl1n was d1fiSolved tn

100 ml of d1stilled water.

2. 1% so<t..i.J.!m periodate: 100 g sodium pertodate 1o1as dist;olved

in 100 ml of distilled water.

3. Acid Haematin solution: 50 ml of Q.lX haematoxylin <sol.

and 1 ml of sodium periodate (sol. 2) were mixed togeth-

er, and boiled, cooled and 1 ml of glacial acettc acid was

added to it.

4. Dichromate-Calcium Solutton: 5g potassium dichromate and

lg calcium chloride were dissolved in 100 ml distilled water.

Procedure: Sections ~Jere air dried, treated l>li th

dichromate-calcium at 22Dc for 18 hours and later at 600C tor

2 hours, washed in distilled water, stained with acid haema­

tin solution for 5 hours, washed in distilled 1o1ater and

mounted in glyceriC' 1 ly.

Results: Choline-L .~aining lipids blue-black.

Plasmal Reaction for Phospholipids <Hayes, 1949)

Fi:<ation ~ Sections: Fresh Frozen sections

'Ceagents used:

1. 2% mercuric chloride: 2g mercuric chloride was dissolved

1n 100 ml of distilled water.

2. Schiff's reagent: Described as earlier <pp.36) •

..:.. Bisulphite water: 500 mg potassium bisulphite was· dis-

solved an 100 ml of u.05 N HCl.

Procedure: A1r drted secttons were hydrolyzed 111 2 % mercur1c

chlor1de solutton for 10 a'1r1ut~s, r1r1sed 1n btsulphlte water,

staaned in Schaff's reagent for 10 mtnutes, ~•ashed tn t<~p

water and mounted 1n glycerine jelly.

Results: Phospholipids dtffuse magenta colour.

Extraction Method for Lipids

Bakers Pyridine E•traction: Bouin fixed tissues are washed in

alcohol to remove picric acid, immersed in pyridine at 20°C

for 30 minutes followed by another 24 hours at b0°C, washed

in running tap ~•ater for 2 hours and transferred to dichro­

mate-calcium mordant (same as used for actd haematin method).

Such delipidized tissues ~•ere used as control.

ENZYHES

Phosphatase:;

Alkaline Phosphatase <Gomori, 1952 a)

Fi:<ation & Sections: For-mol-Calcium at 4°C; Frozen Sections.

Reagents used:

1. 2Y. sodium B glycerophosphate: 2 g sodium B glycerophos­

phate was dissolved in 100 ml distilled ~•ater.

2. 2X sodium veronal: 2 9 sodium barbiturate ~1as dissolved in

100 ml distilled water.

3. 2% calcium chloride: 2 g calcium chloride was dissolved in

100 ml distilled water.

4. lY. magnesium sulphate: lg of magnesium chloride was

49

dtssolved in 100 ml of dtsttlled water.

~. 2% Cobalt nttrate: 2g cobalt nttrate was dtssalved 1n 100

ml distilled water.

6. 1% Ammontum sulphide: 1 ml of yellow ammontum sulphide was

made to 100 ml with distilled water.

Preparation of Incubattnq Medtum: For preparation of tncubat­

ing medium, 2.5 ml each of solution 1 and solution 2, 5.0 ml

of solution 3, 0.25 ml of solutton 4 and 1.25 ml of dts­

tilled water were mixed together and final pH adjusted to

9.0 using 0.1 N NaOH.

Procedure: Sections were atr dried in incubating medium at

37'4::: for 1 hour, washed thrtce in distilled ~tater and treat-

ed with 2% cobalt nitrate for 3 minutes, washed again with

distilled water and transferred to 1% ammonium sulphide,

after development of precipitate again ~•ashed with distilled

~tater and mounted in glycerine jelly.

Results: Sites of enzyme activity brownish-black.

Acid phosphatases <Gomori, 1950)

Fi:<ation: Formal-Calcium post fixation at 4°C for 2h.

Sections: Frozen.

Reagents used:

1. 0.05 M acetate buffer <pH 5.011 70 ml 0.2 t1 sodium acetate

~tas added to 30 ml of 0.2 M acetic acid.

2. 3% sodium B glycerophosphate: 3 g sodium B glycerophos-

pl1ate was d1ssolved 1n 100 ml of dtstillPd water.

3. 1% Ammon1um sulphide: 1 ml at yellow ammon1um •ulphtde •••s

made to lOU ml w1th dlst1lled water.

Preparation at Incubating Medium: 40 ml of solut1on 1 '

0.053 9 of lead nitrate and 4 ml of solut1on 2

together.

Procedure: Sections were freeze dried and tncubated 1n the

medium at 37° C for 1 hour, washed w1th distilled water and

immersed in 1% ammonium sulphide for 2 minutes, 1>1a,.;hed aga•n

mounted in glycerine jelly.

Results: Sites of enzyme activity black.

Adenosine triphosphatase <Wachstein et al., 1960)

Fi:<ation: Formal-Calcium at 4°C.

Se•tions: Frozen

Reagents used:

1. 0.125% ATP: 125 mg ATP was dissolved in 100 ml of dis-

tilled water.

2. 2% lead nitrate: 2 9 lead nitrate "'as dissolved in 100 ml

at distilled water.

3. 2.5% magnesium nitrate: 2.5 g magnes1um n1trat~ was d1s-

solved in 100 ml of distilled' water.

4. Tris butter <pH 7.2): 25 ml 0.2 M Tris was added to 44 ml

of 0.1 N HCl and "'hole was made to 100 ml "'ith distilled

water.

5. 1% Ammonium sulphide: 1 ml at yella•• ammonium sul-

51

I ~lliilllllmfr i!III/Ui !1!6 ffi~ ml ~~ T 12730

T..L//.JJ

phide was mad" to 100 ml with distilled ••ater.

Preparation ot lncubating Medium: 4 ml each of solution 1

and solution 4, 0.6 ml of solution 2, 1 ml of solut1on 3 and

0.4 ml of distilled water were mixed together.

Procedure: Secttons were incubated 1n the medtum far 1 Hour

at 37°. After washing in distilled water they were immersed

in lX anunontum sulphtde for 2 mtnutes .. lhey were was.hed agatn

and mounted in glycerine jelly.

Result: ATPase activity - Black.

Lead Method for 5-Nucleotidase (Wachstein and Meisel, 1957)

Fi:<ation: Formal-Calcium at 4°C

Sections: Frozen

Reagents used:

1. 1.25~ Adenosine-5-Phosphate: 50 mg adenosine-5-phosphate

was dissolved in 4 ml of distilled water.

2. 0.2 M Tris Buffer: Prepared as for above method (pp. 51).

3. 0.1 M Magnesium Sulphate: 12.057g magnesium sulphate ••as

dissolved in 100 ml of distilled water.

4. 2Y. Lead Nitrate: 2g lead nitrate dissolved in 100 ml of

distilled water.

5. lY. Ammonium sulphide: 1 ml of yello•• ammonium sulph1de ••as

made to 100 ml with distilled water.

Preparation of Incubating Medium: 4 ml each at solution l and

solution 2, 0.6 ml of solution 4, 1 ml ot solution 3 and 0.5

ml of d1st1lled water were mtxPd toq~thet·.

Procedure: Frozen sect1ons were incubated 1n the med1um at

:':7"'' f l I -~ -L. or 1our, washed 1n dist1lled water, treated 1n

ammonium sulphide for 3 minutes, washed again in distilled

water and mounted in glycerine jelly.

Results: Blackish-brown deposits show sites of enzyme activi-

ty.

Lead method for Glucose-6-Phosphatase

<Wachstein and Meisel, 1956)

Fi~ation & Sections: Fresh Frozen Sections.

Reagents used:

1. 0. 125:t, Glucose-6-Phasphate<: 125 mg glucose-b-phosphate< was

dissolve<d in 100 ml of distille<d wate<r.

2. Tris-Male<ate Buffer <pH. 6.7):

AJ 0.2 n Tris Acid naleate: 2.42g Tris and 2.32g Male<ic acid

were< dissolve<d in 100 ml of distilled water. J

BJ 0.2 H Sodiua Hydroxide: 0.8g sodium hydroxide "'as dis-

solved in 100 ml of distilled "'ater.

25 ml of solution A was added to 21.8 ml of solution B aod

whole "'as made to 100 ml "'ith distilled water.

3. 2Y. Lead Nitrate: As described earlier (pp. 52).

Preparation of Incubating Medium: 4 ml each of solution 1 and

solution 2, 0.6 ml of solution 3 and 1.4 ml of distilled

wate<r were mi:<ed together.

Procedure: Sections were freeze dried and incubated in the

53

medtum at .!./De fur~ ::~0 mtnutes, washed 111 dtst1ll(!d ~•ater

(three changes of 2 minutes eachl, immersed in lX ammon1um

sulphide for 2 m1nutes, t.Jashed again 1n d1sttlled water and

mounted in glycerine jelly.

Results: Glucose-6-phosphate acttvity - browntsh-black.

Ea:.; terases

Indo~yl Acetate Method for Non-Specific Esterase

<Holt and Withers, 1952)

Fi:<at1on: Formal-Calcium at 4°C.

Sections: Frozen

Reagents used:

1. Tris Buffer <pH. 7.2): As described earlier lpp. 511.

Preparation of Incubating medium: 1 mg of 5 Bromo-4-chloro­

indo:<ylacetate was dissolved in 0.1 ml of ethanol 1 2 ml Tr1s

buffer added to it, 17 mg potassium ferricyanide, 21 mg

potassium ferrocyanide and 11 mg calcium chloridl! ~•ere

dissolved in 7.9 ml of distilled water sl!parately and mi~l!d

~lith the above solution.

Procedure: Sections incubated in the medium at 37°C for 20

minutes, rinsed in tap "'ater, dehydrated in graded alcohols,

cleared in :<ylene and mounted in DPX.

Result: Sites of esterase activity retains blue deposits.

Tween Method for Lipase (6omorl, 1952 bl

F1:<at1on: Formol-Calclum at 4°C.

Sections: Frozen

Reagents used:

1. Tr1s butter CpH 7.21: 25 ml 0.2 M lr1~ was added to 44 ml

of 0.1 N HCl and whole ~la5 made to 100 ml ~•ith d15t1lle-d

water ..

2. 5lo: T~1een: 5 g Tween 60 ~•as dissolved in 100 ml at Tris

buffer.

3. 2lo: Calcium chloride: 2 g Calcium chloride was dlfiSolved 1n

100 ml of distilled water.

4. 2% lead nitrate: 2 g lead nitrate "'as dissolved in 100 ml

of distilled water.

Preparation of Incubating Medium: 9 ml of solution 1, 0.6 ml

of solution 2 and 0.3 ml of solution 3 we~e mixed together.

Procedure: Sections "'ere freeze dried and immersed in the

incubating medium at 370C for 2-8 hours, rinsed in distilled

water and placed in preheated lead nitrate solution at 55° C

for 10 minutes, "'ashed in distilled water and treated ... tth 111.

ammonium sulphide for 3 minu,tes, "'ashed again in

bJater and mounted in glycerine jelly.

Results: Sites of lipase activity yellobl to brown.

55

distilled

Thiochol ine Method for Acetylcholinesterase

(Gerebtzoff, 19591

F1xat1on: Formol-Calclum at 4°C.

Sect1ons: Frozen

Reagents used:

1. Solution A (0.5 M acetate buffer; pH 3.0): Prepared as de-

scribed earlier (pp. 50).

2 .. Solution B: 150 mg acetylth>ocholine 1odide and 70 mg

cupr1c sulphate were dissolved in 14 ml of dist>lled water.

It was centrifuged at 4000 rpm for 15 minutes and suparnatent

used.

Solution C: 375 mg glycine ~•as dissolved in 10 ml of

distilled water.

4. Solution D: 250 mg cupric sulphate was dissolved in 10 ml

of distilled water.

5. 2% ammon1um sulphide: 2g ammonium sulphide was d1ssolved

in 100 ml of distilled water.

Preparation of Incubating Medium: 5 ml solution A, o.e ml

solution B, 0.2 ml solution C, 0.2 ml solut1on D and 3.8 ml

distilled water were mixed together.

Procedure: Sections 1>1ere freeze dried and incubated in medium

at 37°c for 1 hour, rinsed in t1>10 changes of distilled water

and treated ~>lith 2% ammonium sulphide solution foP 2 minutes,

washed again in distilled 1>1ater and mounted 1n glycer1ne

jelly.

H~.:::;ults: Sltt!s at choltnestttrase act1v1ty bt~own.

Dehydrogenase:;

Standard Dehydrogenases Procedure <Bancroft, 1977)

Fi:<at1on and Sections: Fresh Frozen sect1ons.

Reagents used:

1. 0.2 M Tris buffer <pH 7.4): 25 ml 0.2 M Tr1s was added to

42.0 ml at 0.1 N HCl and whole made to 100 ml with distilled

~.>Jater.

2. 1.0 M Sodium lactate: 1.25 ml sodium DL-lactate made to 10

ml with distilled water.

3. 1.0 M Glucose-6-phosphate: 3 9 glucose-6-phosphate was

dissolved in 10 ml of distilled water.

4. 2.5 M Sodium succinate: 6.75 9 sodium succinate wag

dissolved in 10 ml of distilled water.

5. 1.0 M L-Glutamic acid <Na salt): 1.87 g L-glutarnic acid

was dissolved in 10 ml of distilled water.

6. 2.7 g DL-Isocitric acid (tri Na salt) ~•as dissolved in 10

ml of distilled water.

7. 1.0 M Sodium hydrogen malate: 1.55 g sodium hydrogen

malate was dissolved in 10 ml of distilled water.

8. NBT solu.t ion: 10 mg Nitro Blue fetrazol iu.m salt <NBT) was

dissolved in 2.5 ml of distilled water. 2.5 ml of 0.2 M Tris

buffer <pH 7.4), 1.0 ml of 0.05 M magnesium chloride and 3.0

ml of distilled water ~>tere added to it.

Preparation of the Incubating t1edium:

Lactate Dehydrogenase: 0.9 ml solution B and 0.1 ml solution

:-~ t.olt>l'e 1111 :<ed tuy(_•ther·. 2 mg ot Nf\U Wd~ •. u.Jd~ll a•. L()L'I'.•yme.

Glucose-6-Phosphate Dehydrogenase: 0.9 ml at sotut1on tJ and

0.1 ml of solut1on 3 were m1Med together. 2 rug of NAUP was

added as coenzyme.

Succinate Dehydrogenase: 0.9 ml of solut1on B and 0.1 ml of

solution 4 were miMed together.

Glutamate Dehydroganaser 0.9 ml of solution U &lid <J.l ml at­

solution 5 were miMed together.

Isocitrate Dehydrogenase: 0.9 cnl o1' solut1on tl and 0.1 ml of

solution 6 "'ere mixed together. 2 mg of NAU "'as added as

coenzyme.

Malate Dehydrogenase: 0.9 ml of solution 8 and 0.1 ml of

solution 7 were mi:<ed together. 2 mg of NADP "'as added as

coenzyme.

Procedure: Sections were freeze dried and incubated rn the

appropriate incub"ating solution at 37°C far 30 minutes,

transferred to 15% formal saline for 15 minutes to stop the

reaction, "'ashed in distilled water and mounted in glycerine

jelly.

Result:. Enzyme activity indicated by purple formazan depos-

its ..