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 ..