Appendix

A. REAGENTS/SOLUTIONS
1. Diazo reagent
Stock solution (a):
Sulfanilic acid
1.0 gm
Distilled water
200 ml
Concentrated hydrochloric acid
1.0 ml
Stock solution (b):
Sodium nitrite
0.5 gm
Distilled water
100 ml
Working reagent (c):
Stock solution (a)
10 ml mix well
Stock solution (b)
0.1 ml
before use
2. RBC diluting fluid
a. Gowers solution:
Sodium sulfate
Glacial acetic acid
Distilled water
b. Hayems fluid:
Sodium sulfate
Sodium chloride
Mercuric chloride
DW

6.25 gm
16.6 ml
100 ml
2.5 gm
0.5 gm
0.5 gm
100 ml

3. WBC diluting fluid

a. For animals:
Glacial acetic acid
3.0 ml
DW
97.0 ml
Add few drops of aqueous methylene blue
to give colour to the solution.
b. For birds:
Sodium citrate
3.8 gm
Formalin (neutral)
0.2 ml
Brilliant cresyl blue
0.5 gm
DW
100 ml
4. Benedicts reagent
Solution (a):
Sodium carbonate
DW

Dissolve by heating
Sodium citrate
200.0 gm
Potassium thiocyanate
125.0 gm
Mix well and dissolve
Solution (b):
Copper sulfate
18.0 gm
DW
100 ml
Dissolve by shaking
Solution (c):
Potassium ferrocyanide
0.25 gm
DW
5 ml
Dissolve by shaking.
Mix the solution (a), (b) and (c) at room temperature and make up the volume to 1000 ml with
distilled water. Keep the solution in a dark
coloured bottle.
5. Roberts reagent
Solution (a):
Magnesium sulfate saturated solution in
distilled water
Solution (b):
Concentrated nitric acid
Working reagent:
Solution (a)
5 parts
Solution (b)
1 parts
6. Pandeys reagent
Phenol crystals
DW
Mix to dissolve

10.0 gm
100 ml

7. Sodium nitroprusside solutions

Sodium nitroprusside 10.0 gm
DW
20.0 ml
Dissolve by heating.
8. Trichloroacetic acid-ferric chloride solution

100.00 gm
800 ml

Trichloroacetic acid
Ferric chloride
DW

25.0
0.9 gm
100 ml

322

Textbook of Veterinary Clinical and Laboratory Diagnosis

9. Alkaline copper solution

Solution (a):
Sodium carbonate
Sodium potassium tartarate
Sodium bicarbonate
Sodium sulfate
DW to
Solution (b):
Copper sulfate
Concentrated sulfuric acid
DW
Working reagent:
Solution (a)
Solution (b)

25.0 gm
25.0 gm
20.0 gm
2.0 gm
1000 ml
15.0 gm
0.l ml
100 ml
24.0 ml
1.0 ml

10. Phosphomolybdic acid solution

Solution (a):
Sodium hydroxide
40.0 gm
DW
400 ml
Solution (b):
Molybdic acid
70.0 gm
Sodium tungstate
10.0 gm
DW
300 ml
Mix by boiling.
Solution (c):
Phosphoric acid
250 ml
Mix solution (a), (b) and (c) and to make
it 1000 ml add DW
11. Biuret reagent
(Benedicts qualitative reagent)
Solution (a):
Cupric sulfate pentahydrate
17.3 gm
DW
100.0 ml
Solution (b):
Sodium citrate
173.0 gm
Sodium carbonate
100.0 gm
DW
800.0 ml
Dissolve by heating. Mix solution (a) and
solution (b) and make it 1000 ml by DW.
12. Sulfate-sulfite solution
Sodium sulfate
Sodium sulfite
Concentrated sulfuric acid
Dissolve in DW to

208.0 gm
70.0 gm
2.0 ml
100 ml

13. Chloranilic acid solution

Chloranilic acid

1.0 gm

1 N Sodium hydroxide
DW to
Filter before use. pH 3.5-7.0.

7.0 ml
100 ml

14. EDTA solution

Tetra sodium EDTA
DW

5.0 gm
100 ml

15. Trichloroacetic acid solution

Trichloroacetic acid
DW

10.0 gm
100 ml

16. Elon reducing solution

Sodium bisulphite
DW
Mix to dissolve and add elon

3.0 gm
100 ml
1.0 gm

17. Polyvinyl alcohol

Polyvinyl alcohol
DW

0.20 gm
100 ml

18. Titan yellow

Stock solution:
Titan yellow
DW
Working solution:
Stock solution
DW

0.050 gm
100 ml
3.0 ml
7.0 ml

19. Sodium hydroxide solution

Sodium hydroxide
DW

20.0 gm
100.0 ml

20. Colour development reagent

Stock solution:
Acetic anhydride
300 ml
Glacial acetic acid
200 ml
Mix well.
Working solution:
Stock solution
200 ml
Concentrated sulfuric acid
20 ml
This solution is stable for 24 hours.
21. Alkaline picrate solution
Stock solution (a):
Picric acid
DW

11.7 gm
900 ml

Appendix 323
Adjust pH 2.0 by adding 2 N sodium hydroxide
and dilute to 1000 ml with DW.
Working solution (b):
Solution (a)
36 ml
10% NaOH
4.0 ml
Make fresh, just before use.
22. Urease solution
Glycerine
DW
Urease

67.0 ml
33 ml
2.0 gm

23. Barium hydroxide solution

Barium hydroxide
45.0 gm
[Ba(OH)2 8H2O]
Boiling distilled water 600 ml
Boil and cool filter and make it to 1000 ml.
24. Zinc sulfate solution
Zinc sulfate
DW

50.0 gm
1000 ml

2, 4-dinitrophenyl hydrazine
1NHCI

0.0198 gm
100 ml

30. P-nitrophenol phosphate

P-nitrophenol phosphate
DW

100 mg
25 ml

31. Normal saline solution (NSS)

Sodium chloride
DW

0.85 gm
100 ml

32. Phosphate buffer saline (PBS) (pH 7.2)

Sodium chloride
Potassium chloride
Potassium dihydrogen
orthophosphate
Disodium hydrogen
orthophosphate
DW

8.00 gm
0.20 gm
0.2 gm
2.312 gm
1000 ml

33. Lugols iodine

25. Iodine solution

Iodine crystals
Potassium iodide
DW

29. 2, 4-Dinitrophenyl hydrazine

2.0 gm
3.0 gm
100 ml

26. Nesslers solution

Potassium iodide
30.0 gm
Iodine crystals
22.5 gm
DW
20.0 ml
Mix to dissolve. Add metallic mercury
30.0 gm. Dilute to 200 ml with DW and
mix it with 975 ml of 10% NaOH.
27. Aspartate glutarate substrate
a-Ketoglutaric acid
0.0292 gm
dl-aspartic acid
2.66 gm
Add 1 N sodium hydroxide to make it in
solution. Adjust pH 7.5. Dilute to 100 ml with
PBS.
28. Alpha-ketoglutarate-dl-alanine substrate:
Alpha ketoglutaric acid
0.0292 gm
dl-alanine
1.78 gm
Add 1 N NaOH till solution. Adjust pH 7.5
dilute to 100 ml with PBS.

Iodine crystals
Potassium iodide
DW

1.0 gm
2.0 gm
100 ml

34. Alsevers solution

D-glucose
Sodium chloride
Citric acid
Sodium citrate
DW

2.050 gm
0.420 gm
0.055 gm
0.800 gm
100 ml

35. Carbol saline

(a) 0.5%:
Sodium chloride
Phenol crystals
DW
(b) 5%:
Sodium chloride
Phenol crystals
DW

0.85 gm
0.50 gm
100 ml
5.0 gm
0.50 gm
100 ml

36. Kovacs reagent

Amyl alcohol
Paradimethyl amino benzaldehyde
Concentrated hydrochloric acid

75 ml
5.0 gm
25 ml

324

Textbook of Veterinary Clinical and Laboratory Diagnosis

37. Methyl red reagent

Methyl red
95% ethanol

0.02 gm
100 ml

38. Formol saline

35-40% formaldehyde
Normal saline solution to

10 ml
100 ml

39. Formol sublimate

35-40% formaldehyde
Saturated mercuric chloride to

10 ml
100 ml

40. Buffered formalin

35-40% formaldehyde
PBS to
(pH 7.2)

10 ml
100 ml

41. PBS-glycerine (FAT mounting media)

PBS (pH 7.2)
glycerine
42. Glycine HCL buffer

10 ml
90 ml

53.65 gm
1000
X
Y
100
as follows

ml
ml
ml
ml
at

Y
5.4 ml
10.2 ml
14.1 ml
17.7 ml
20.6 ml
23.3 ml
25.7 ml
27.8 ml
30.3 ml
33.1 ml
36.4 ml
43.5 ml

44. Phosphate buffer (pH 7.2)

(pH 2.2-3.6)

Solution (a):
Glycine
15.01 gm
DW to
1000 ml
Solution (b):
Hydrochloric acid
0.2 m
Working solution
Solution A
50 ml
Solution B
X ml
DW
200 ml
The values of X is as follows at different pH
levels:
pH
X
2.2
44.0 ml
2.4
32.4 ml
2.6
24.2 ml
2.8
16.8 ml
3.0
11.4 ml
3.2
8.2 ml
3.4
6.4 ml
3.6
5.0 ml
43. Citrate phosphate buffer (pH 2.6-7.0)
Solution (a):
Citric acid
DW to

Solution (b):
Dibasic sodium phosphate
(Na2HPO4.7H2O)
DW to
Solution (a)
Solution (b)
DW
The values of X and Y are
different pH levels.
pH
X
2.6
44.6 ml
3.0
39.8 ml
3.4
35.9 ml
3.8
32.3 ml
4.2
29.4 ml
4.6
26.7 ml
5.0
24.3 ml
5.4
22.2 ml
5.8
19.7 ml
6.2
16.9 ml
6.6
13.6 ml
7.0
6.5 ml

19.21 gm
1000 ml

Solution (a)
Na2HPO4
DW
Solution (b):
NaH2PO4
DW
Solution (a)
Solution (b)
DW to

35.61 gm
100 ml
27.67 gm
1000 ml
28.0 ml
72.0 ml
200 ml

45. Tris-HCl Buffer (pH 7.2)

Solution (a):
Tris-(hydroxymethyl)
aminomethane
DW to
Solution (b):
Hydrochloric acid
Solution (a)
Solution(b)
DW to

24.2 gm
1000 ml
0.2 M
50.0 ml
44.2 ml
200 ml

46. Coating buffer (pH 9.6)

Sodium carbonate (Na2CO3)
Sodium bicarbonate (NaHCO3)
DW to

1.59 gm
2.93 gm
1000 ml

Appendix 325

47. Washing solution (pH 7.2)

Sodium chloride
Potassium dihydrogen phosphate
(KH2PO4)
Disodium hydrogen phosphate
(Na2HPO4.2H2O)
Tween-20
DW to

20.20 gm
0.2 gm
1.15 gm
0.5 ml
1000 ml

48. Citric acid-phosphate buffer (pH 5.0)

Citric acid
Disodium hydrogen phosphate
(Na2HPO4.2H2O)
DW

7.30 gm
11.86 gm
1000 ml

49. Sticking material

Egg white (egg albumin)
1 part
glycerine
1 part
Mix well and store in tightly stoppered container
at room temperature.
50. Buffered glutaraldehyde
25% glytaraldehyde
PBS (pH 7.2)

10.0 ml
100 ml

51. Sorensens buffer

Phosphate buffer (pH 7.2)
Sucrose

100 ml
4.5 gm

52. Em embedding media

Solution (a):
Epon 812
Dodecenyl succinic anhydride
Solution (b):
Epon 812
Methyl nadic anhydride
Equal parts of Solution a and b should

62.0 ml
100 ml
100 ml
89.0 ml
be used.

53. Veronal buffer (pH 8.6)

Solution (a):
Sodium barbitone
DW to
Solution (b):
0.2 M hydrochloric acid

20.60 gm
500 ml

Buffer solution:
Solution A
Solution B
DW to
Adjust pH 8.6.

50 ml
6 ml
200 ml

54. Ringers solution

Sodium chloride
Potassium chloride
Sodium bicarbonate
Calcium chloride
DW to

0.7 gm
0.026 gm
0.03 gm
0.003 gm
1000 ml

B. STAINS
1. Sellers stain
Solution A:
Methylene blue
1.0 gm
Methanol (acetone free)
100 ml
Mix by using pestle and mortar and store in a
tightly stoppered bottle.
Solution B:
Basic fuchsin
1.0 gm
Methanol (acetone free)
100 ml
Working stain:
Solution A
2 parts
Solution B
1 parts
2. Methylene blue stain
Methylene blue
95% ethanol
DW to

0.3 gm
30.0 ml
100 ml

3. Giemsa stain
Stock solution:
Azure II - eosin
Azure II
Glycerol
Acetone free methanol
Working stain:
Stock solution
DW

3.0 gm
0.8 gm
250 ml
250 ml
1 part
9 part

4. Lactophenol cotton blue stain

Phenol crystals
Lactic acid syrup
Glycerol
DW to
Aniline blue/cotton blue stain

10 gm
10 ml
20 ml
10 ml
50 mg

326

Textbook of Veterinary Clinical and Laboratory Diagnosis

Mix properly and filter before use. Decolorize
with 0.5% citric acid, counter stain with 0.1%
aqueous methylene blue.

5. Wrights stain
Solution A:
Methylene blue
0.9 gm
0.5% aqueous sodium
bicarbonate
100 ml
Heat in water bath to boiling for 1 hour cool
and filter. Add following solution:
Solution B:
Eosin Y
1.0 gm
DW
500 ml
Mix both solutions and filter. Collect precipitate
and dry at 37C.
Working stain:
Dried precipitate
0.1 gm
Acetone free methanol
50 ml

11. Kinyouns carbol fuchsin (tissues)

Basic fuchsin
Phenol
Ethanol
Heat to dissolve
DW to
Prepare fresh

Basic fuchsin
1.0 gm
Ethanol
10 ml
5% phenol aqueous
100 ml
Decolorizing agent:
Concentrated hydrochloric acid
3.0 ml
70% ethanol
97.0 ml
Counter stain:
0.1% aqueous methylene blue.

Leishmans stain powder

0.15 gm
Methanol
100.0 ml
Mix in pestle and mortar. Filter before use.

Methylene blue
95% ethanol
Tetrachloroethane
Glacial acetic acid

1.12 gm
54.0 ml
40.0 ml
6.0 ml

II. Smears:
(a) Carbol fuchsin:
Basic fuchsin
Ethanol
Phenol
DW to
(b) Decolourizer:

8. May Grunwald stain

Stock solution:
May Grunwald stain powder
3.0 gm
Methanol
1000 ml
Warm to 50C and put on stirring for few hours.
Working solution:
Stock solution
2 parts
DW to
1 part
9. Manns methylene blue-eosin stain (tissues)
1.0% aqueous solution of methylene
blue
1.0% aqueous solution of eosin
DW

35 ml
45 ml
100 ml

10. Macchiavello stain (tissues)

Basic fuchsin
0.1 M disodium phosphate
0.1 M sodium acid phosphate
(NaH2PO4.H2O)
DW to

0.5 gm
3.6 ml
1.4 ml
95 ml

100 ml

12. Zeihl Neelsen carbol fuchsin (tissue)

6. Leishmans stain

7. Newmans stain

4.0 gm
8.0 gm
20.0 ml

(c) Counter stain:

0.3 gm
10 ml
5 ml
95 ml
3% hydrochloric acid
in 95% ethanol
0.3% methylene blue
(aqueous)

13. Grams staining

I.

Tissues
(a) Crystal violet:
1.0% crystal violet (aqueous)
40 ml
5% sodium bicarbonate
10 ml
(b) Grams iodine
Iodine crystals
1.0 gm
Potassium iodide
2.0 gm
DW
300 ml
(c) Counter stain
Basic fuchsin
25 mg
DW
1000 ml
II. Smears:
(a) Crystal violet:
Stock solution A: 1% crystal violet in
ethanol
Stock solution B: 1% ammonium oxalate

Appendix 327
Working solution:
Stock solution A
20 ml
Stock solution B
80 ml
(b) Grams iodine: Same as for tissues
(c) Decolorizer:
Ethanol (95%)
250 ml
Acetone
250 ml
(d) Counter stain
Safranin
2.5 gm
95% ethanol
100 ml
Working stain:
Safranin 1:4 diluted with
DW.
14. Silver nitrate for spirochaete in tissues
Silver nitrate
DW

2.0 gm
100 ml

15. Grocatts methenamine silver nitrate

Solution A:
Silver nitrate
DW
Solution B:.
Methenamine
DW
Stock solution
Solution A
Solution B
Working solution
Stock solution
DW
5% borax

5.0 gm
100 ml
3.0 gm
100 ml
5 ml
100 ml
25 ml
25 ml
2 ml

16. Gold chloride

Gold chloride
DW

0.10 gm
100 ml

17. Sodium thiosulfate

Sodium thiosulfate
DW

2.0 gm
100 ml

18. Lendrums phloxine tetrazine

A. Phloxine B
0.045 m calcium chloride
B. Saturated tetrazine in cellosolve

0.5 gm
100 ml

19. Harris haematoxylin

Hematoxylin
Absolute ethanol

2.5 gm
50 ml

Potassium alum
DW
Mercuric oxide
Glacial acetic acid
Filter before use

50 gm
500 ml
1.5 gm
20 ml

20. Meyers hematoxylin

Hematoxylin
DW
Potassium alum
Sodium iodate
Citric acid
Chloral hydrate

0.5 gm
500 ml
25.0 gm
0.1 gm
0.5 gm
25.0 gm

21. Acid alcohol

Concentrated hydrochloric acid
70.1% ethanol

3.0 ml
97.0 ml

22. Ammonia water

Concentrated ammonia
DW

2-3 drops
250 ml

23. Eosin
(a) Aqueous: Eosin
DW
(b) Alcoholic: Eosin
70% ethanol

1.0 gm
100 ml
1.0 gm
100 ml

24. Staining for electronmicroscopic examination

(a) Lead citrate:
Lead nitrate
Sodium citrate
DW
(b) Uranyl acetate:
Uranyl acetate
DW
(c) Lead hydroxide:
Lead oxide
1 N NaOH
Heat to dissolve, cool and filter. Make
with DW.

1.33 gm
1.76 gm
30.0 ml
1.0 gm
100 ml
0.5 gm
20 ml
it 100 ml

25. Periodic acid-Schiffs staining

(a) Periodic acid:
Periodic acid
DW
2.72% sodium acetate solution
ethanol

0.4 gm
10.0 ml
5.0 ml
35.0 ml

328

Textbook of Veterinary Clinical and Laboratory Diagnosis

(b) Reducing solution:

Potassium iodide
1.0 gm
Sodium thiosulfate
1.0 gm
DW
20 ml
Ethanol
30 ml
1 N HCl
1.0 ml
(c) Schiffs reagent:
Basic fuchsin
1.0 gm
DW
200 ml
Boil and cool to 50C, filter add 20 ml 1 N HCl
cool to 25C and add sodium metabisulfite
1.0 gm, store for 24 hours in dark and add
2.0 gm activated charcol. Shake, filter and keep
in dark stoppered bottle at 4C.
C. CULTURE MEDIA
1. Nutrient broth
Bacto beef extract
0.3 gm
Bacto peptone
0.5 gm
DW
100 ml
Adjust pH 7.2 sterilize at 121C for 15 minute in
autoclave.
2. Nutrient agar
Nutrient broth
Agar
Sterilize by autoclave.

100 ml
1.5 gm

3. Brain-Heart Infusion broth (BHI broth)

Infusion from calf brain
Infusion from beef heart
Proteose peptone
Glucose
Sodium chloride
Disodium hydrogen phosphate
DW
Sterilize by autoclave

20.0 gm
25.0 gm
1.0 gm
0.2 gm
0.5 gm
0.25 gm
100 ml

100 ml
1.5 gm

5. Blood agar
Bacto-tryptose
Beef extract
Sodium chloride
Glucose
Dextrin

6. Trypticase soy agar

Trypticase
Phytone
Sodium chloride
Dipotassium hydrogen
phosphate (K2HPO4)
Glucose
Para-aminobenzoic acid
Agar
DW

8.5
1.5
2.5
1.25

gm
gm
gm
gm

1.25 gm
10.0 gm
7.5 gm
500 ml

7. Cooked meat medium

Lean beef
250 gm
Proteose peptone
10.0 gm
Glucose
1.0 gm
Sodium chloride
2.5 gm
DW
500 ml
Autoclave at 121C, 15 lbs for 15 min.
8. Brilliant green agar
Bacto-yeast extract
Proteose peptone
Sodium chloride
Bacto-lactose
Saccharose
Bacto-phenol red
Bacto-brilliant green
Agar
DW
Autoclave at 121C for

1.5 gm
5.0 gm
2.5 gm
5.0 gm
5.0 gm
0.04 gm
0.006 gm
10.0 gm
500 ml
15 min

9. Eosin-methylene blue agar (EMB agar)

4. BHI agar
BHI broth
Agar

Para-aminobenzoic acid
10.0 gm
Agar
7.5 gm
DW
500 ml
Adjust pH 7.4. Autoclave at 121C and 15 lbs
pressure for 15 min. Cool to 45C and add 50 ml
blood (defibrinated).

10.0
1.5
2.5
0.15
0.25

gm
gm
gm
gm
gm

Bacto-peptone
5.0 gm
Bacto-lactose
2.5 gm
Saccharose
2.5 gm
Dipotassium phosphate
1.0 gm
Agar
7.0 gm
Eosin Y
0.2 gm
Methylene blue
0.035 gm
DW
500 ml
Autoclave it for 15 min at 15 lbs pressure and
121C.

Appendix 329
10. Mac Conkey agar
Peptone
4.4 gm
Proteose peptone
0.75 gm
Lactose
2.5 gm
Bile salts
0.4 gm
Sodium chloride
1.25 gm
Agar
4.0 gm
Neutral red
7.5 mg
Crystal violet
0.25 mg
DW
250 ml
Autoclave it for 15 min at 15 lbs pressure and at
121C.
11. Tripple sugar iron agar (TSI)
Beef extract
3.0 gm
Yeast extract
3.0 gm
Proteose peptone
5.0 gm
Peptone
15.0 gm
D-glucose
1.0 gm
Sucrose
10.0 gm
Lactose
10.0 gm
Sodium chloride
5.0 gm
Sodium thiosulfate
300 mg
Ferrous sulfate
200 mg
Agar
2.0 gm
1% solution of phenol red
2.5 ml
DW
1000 ml
Autoclave at 115C for 20 min and pour in tubes
to make the slants.
12. Tetrathionate broth
Proteose peptone
2.5 gm
Bile salts
0.5 gm
Calcium carbonate
5.0 gm
Sodium thiosulfate
15.0 gm
DW
500 ml
Autoclave and pour/distribute in tubes.
13. Selenite broth
Bacto-peptone
Bacto-lactose
Sodium acid selenite
Disodium phosphate
DW
Autoclave it and dispense in tubes.

2.0 gm
2.0 gm
2.5 gm
5.0 gm
500 ml

14. Urea agar

A. Dextrose
Peptone
Sodium chloride
Monopotassium phosphate
Urea

10
1.0
5.0
2.0
20

gm
gm
gm
gm
gm

Phenol red
0.012 gm
DW
100 ml
Filter through seitz filter.
B. Agar
15 gm
DW
900 ml
Autoclave at 121C for 15 min.
Cool and mix A and B and pour in tubes.
15. Nitrate agar
Beef extract
Potassium nitrate
Peptone
Agar
DW
pH 6.8. Autoclave at 121C for 15 min.

1.5 gm
0.5 gm
2.5 gm
6.0 gm
500 ml

16. Simmons citrate agar

Sodium chloride
2.5 gm
Sodium citrate
1.0 gm
Magnesium sulfate
0.1 gm
Monoammonium phosphate
0.5 gm
Dipotassium phosphate
0.5 gm
Bacto-agar
7.5 gm
Bromothymol blue
0.04 gm
DW
500 ml
pH 6.8 autoclave at 121C PC for 15 min.
17. PPLO Broth
Infusion from beef heart
25.0 gm
Sodium chloride
2.5 gm
Peptone
5.0 gm
Crystal violet
0.005 gm
Potassium tellurite
0.15 gm
pH 7.8. Autoclave it at 121C for 15 min.
18. Sabourauds dextrose agar
Peptone
10.0 gm
Dextrose
40.0 gm
Agar
18.0 gm
DW
1000 ml
pH 5.6. Autoclave at 121C for 15 min.
19. Sugar fermentation media
Sodium chloride
5.0 gm
Tryptone
2.0 gm
1% phenol red
2.0 ml
DW
1000 ml
Autoclave at 121C for 15 min. Pour in tubes and
add sugar 0.2 ml of 10% sugar solution (sterilized
by filtration).