Appendix

A. REAGENTS/SOLUTIONS

1. Diazo reagentStock solution (a):

Sulfanilic acid 1.0 gmDistilled water 200 mlConcentrated hydrochloric acid 1.0 ml

Stock solution (b):Sodium nitrite 0.5 gmDistilled water 100 ml

Working reagent (c):Stock solution (a) 10 ml mix wellStock solution (b) 0.1 ml before use

2. RBC diluting fluid

a. Gowers solution:Sodium sulfate 6.25 gmGlacial acetic acid 16.6 mlDistilled water 100 ml

b. Hayem’s fluid:Sodium sulfate 2.5 gmSodium chloride 0.5 gmMercuric chloride 0.5 gmDW 100 ml

3. WBC diluting fluida. For animals:

Glacial acetic acid 3.0 mlDW 97.0 ml

Add few drops of aqueous methylene blueto give colour to the solution.

b. For birds:Sodium citrate 3.8 gmFormalin (neutral) 0.2 mlBrilliant cresyl blue 0.5 gmDW 100 ml

4. Benedicts reagentSolution (a):

Sodium carbonate 100.00 gmDW 800 ml

Dissolve by heatingSodium citrate 200.0 gmPotassium thiocyanate 125.0 gmMix well and dissolve

Solution (b):Copper sulfate 18.0 gmDW 100 mlDissolve by shaking

Solution (c):Potassium ferrocyanide 0.25 gmDW 5 mlDissolve by shaking.

Mix the solution (a), (b) and (c) at room tem-perature and make up the volume to 1000 ml withdistilled water. Keep the solution in a darkcoloured bottle.

5. Robert’s reagentSolution (a):

Magnesium sulfate saturated solution indistilled water

Solution (b):Concentrated nitric acid

Working reagent:Solution (a) 5 partsSolution (b) 1 parts

6. Pandey’s reagent

Phenol crystals 10.0 gmDW 100 mlMix to dissolve

7. Sodium nitroprusside solutionsSodium nitroprusside 10.0 gmDW 20.0 mlDissolve by heating.

8. Trichloroacetic acid-ferric chloride solutionTrichloroacetic acid 25.0Ferric chloride 0.9 gmDW 100 ml

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9. Alkaline copper solutionSolution (a):

Sodium carbonate 25.0 gmSodium potassium tartarate 25.0 gmSodium bicarbonate 20.0 gmSodium sulfate 2.0 gmDW to 1000 ml

Solution (b):Copper sulfate 15.0 gmConcentrated sulfuric acid 0.l mlDW 100 ml

Working reagent:Solution (a) 24.0 mlSolution (b) 1.0 ml

10. Phosphomolybdic acid solution

Solution (a):Sodium hydroxide 40.0 gmDW 400 ml

Solution (b):Molybdic acid 70.0 gmSodium tungstate 10.0 gmDW 300 mlMix by boiling.

Solution (c):Phosphoric acid 250 mlMix solution (a), (b) and (c) and to makeit 1000 ml add DW

11. Biuret reagent(Benedicts qualitative reagent)Solution (a):

Cupric sulfate pentahydrate 17.3 gmDW 100.0 ml

Solution (b):Sodium citrate 173.0 gmSodium carbonate 100.0 gmDW 800.0 ml

Dissolve by heating. Mix solution (a) andsolution (b) and make it 1000 ml by DW.

12. Sulfate-sulfite solutionSodium sulfate 208.0 gmSodium sulfite 70.0 gmConcentrated sulfuric acid 2.0 mlDissolve in DW to 100 ml

13. Chloranilic acid solution

Chloranilic acid 1.0 gm

1 N Sodium hydroxide 7.0 mlDW to 100 mlFilter before use. pH 3.5-7.0.

14. EDTA solutionTetra sodium EDTA 5.0 gmDW 100 ml

15. Trichloroacetic acid solution

Trichloroacetic acid 10.0 gmDW 100 ml

16. Elon reducing solutionSodium bisulphite 3.0 gmDW 100 mlMix to dissolve and add elon 1.0 gm

17. Polyvinyl alcoholPolyvinyl alcohol 0.20 gmDW 100 ml

18. Titan yellow

Stock solution:Titan yellow 0.050 gmDW 100 ml

Working solution:Stock solution 3.0 mlDW 7.0 ml

19. Sodium hydroxide solutionSodium hydroxide 20.0 gmDW 100.0 ml

20. Colour development reagentStock solution:

Acetic anhydride 300 mlGlacial acetic acid 200 mlMix well.

Working solution:Stock solution 200 mlConcentrated sulfuric acid 20 mlThis solution is stable for 24 hours.

21. Alkaline picrate solution

Stock solution (a):Picric acid 11.7 gmDW 900 ml

Appendix 323

Adjust pH 2.0 by adding 2 N sodium hydroxideand dilute to 1000 ml with DW.Working solution (b):

Solution (a) 36 ml10% NaOH 4.0 ml

Make fresh, just before use.

22. Urease solutionGlycerine 67.0 mlDW 33 mlUrease 2.0 gm

23. Barium hydroxide solution

Barium hydroxide 45.0 gm[Ba(OH)2 8H2O]Boiling distilled water 600 mlBoil and cool filter and make it to 1000 ml.

24. Zinc sulfate solutionZinc sulfate 50.0 gmDW 1000 ml

25. Iodine solutionIodine crystals 2.0 gmPotassium iodide 3.0 gmDW 100 ml

26. Nessler’s solution

Potassium iodide 30.0 gmIodine crystals 22.5 gmDW 20.0 mlMix to dissolve. Add metallic mercury30.0 gm. Dilute to 200 ml with DW andmix it with 975 ml of 10% NaOH.

27. Aspartate — glutarate substratea-Ketoglutaric acid 0.0292 gmdl-aspartic acid 2.66 gmAdd 1 N sodium hydroxide to make it insolution. Adjust pH 7.5. Dilute to 100 ml withPBS.

28. Alpha-ketoglutarate-dl-alanine substrate:Alpha ketoglutaric acid 0.0292 gmdl-alanine 1.78 gmAdd 1 N NaOH till solution. Adjust pH 7.5dilute to 100 ml with PBS.

29. 2, 4-Dinitrophenyl hydrazine2, 4-dinitrophenyl hydrazine 0.0198 gm1–NHCI 100 ml

30. P-nitrophenol phosphate

P-nitrophenol phosphate 100 mgDW 25 ml

31. Normal saline solution (NSS)

Sodium chloride 0.85 gmDW 100 ml

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

Sodium chloride 8.00 gmPotassium chloride 0.20 gmPotassium dihydrogen

orthophosphate 0.2 gmDisodium hydrogen

orthophosphate 2.312 gmDW 1000 ml

33. Lugol’s iodine

Iodine crystals 1.0 gmPotassium iodide 2.0 gmDW 100 ml

34. Alsever’s solutionD-glucose 2.050 gmSodium chloride 0.420 gmCitric acid 0.055 gmSodium citrate 0.800 gmDW 100 ml

35. Carbol saline(a) 0.5%:

Sodium chloride 0.85 gmPhenol crystals 0.50 gmDW 100 ml

(b) 5%:Sodium chloride 5.0 gmPhenol crystals 0.50 gmDW 100 ml

36. Kovac’s reagent

Amyl alcohol 75 mlParadimethyl amino benzaldehyde 5.0 gmConcentrated hydrochloric acid 25 ml

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37. Methyl red reagentMethyl red 0.02 gm95% ethanol 100 ml

38. Formol saline

35-40% formaldehyde 10 mlNormal saline solution to 100 ml

39. Formol sublimate35-40% formaldehyde 10 mlSaturated mercuric chloride to 100 ml

40. Buffered formalin35-40% formaldehyde 10 mlPBS to 100 ml(pH 7.2)

41. PBS-glycerine (FAT mounting media)

PBS (pH 7.2) 10 mlglycerine 90 ml

42. Glycine HCL buffer (pH 2.2-3.6)Solution (a):

Glycine 15.01 gmDW to 1000 ml

Solution (b):Hydrochloric acid 0.2 m

Working solutionSolution A 50 mlSolution B X mlDW 200 ml

The values of “X” is as follows at different pHlevels:

pH X2.2 44.0 ml2.4 32.4 ml2.6 24.2 ml2.8 16.8 ml3.0 11.4 ml3.2 8.2 ml3.4 6.4 ml3.6 5.0 ml

43. Citrate phosphate buffer (pH 2.6-7.0)

Solution (a):Citric acid 19.21 gmDW to 1000 ml

Solution (b):Dibasic sodium phosphate 53.65 gm(Na2HPO4.7H2O)DW to 1000 ml

Solution (a) X mlSolution (b) Y mlDW 100 ml

The values of X and Y are as follows atdifferent pH levels.

pH X Y2.6 44.6 ml 5.4 ml3.0 39.8 ml 10.2 ml3.4 35.9 ml 14.1 ml3.8 32.3 ml 17.7 ml4.2 29.4 ml 20.6 ml4.6 26.7 ml 23.3 ml5.0 24.3 ml 25.7 ml5.4 22.2 ml 27.8 ml5.8 19.7 ml 30.3 ml6.2 16.9 ml 33.1 ml6.6 13.6 ml 36.4 ml7.0 6.5 ml 43.5 ml

44. Phosphate buffer (pH 7.2)Solution (a)

Na2HPO4 35.61 gmDW 100 ml

Solution (b):NaH2PO4 27.67 gmDW 1000 ml

Solution (a) 28.0 mlSolution (b) 72.0 mlDW to 200 ml

45. Tris-HCl Buffer (pH 7.2)

Solution (a):Tris-(hydroxymethyl)

aminomethane 24.2 gmDW to 1000 ml

Solution (b):Hydrochloric acid 0.2 M

Solution (a) 50.0 mlSolution(b) 44.2 mlDW to 200 ml

46. Coating buffer (pH 9.6)Sodium carbonate (Na2CO3) 1.59 gmSodium bicarbonate (NaHCO3) 2.93 gmDW to 1000 ml

Appendix 325

47. Washing solution (pH 7.2)Sodium chloride 20.20 gmPotassium dihydrogen phosphate

(KH2PO4) 0.2 gmDisodium hydrogen phosphate

(Na2HPO4.2H2O) 1.15 gmTween-20 0.5 mlDW to 1000 ml

48. Citric acid-phosphate buffer (pH 5.0)

Citric acid 7.30 gmDisodium hydrogen phosphate

(Na2HPO4.2H2O) 11.86 gmDW 1000 ml

49. Sticking materialEgg white (egg albumin) 1 partglycerine 1 partMix well and store in tightly stoppered containerat room temperature.

50. Buffered glutaraldehyde25% glytaraldehyde 10.0 mlPBS (pH 7.2) 100 ml

51. Sorensen’s buffer

Phosphate buffer (pH 7.2) 100 mlSucrose 4.5 gm

52. Em embedding mediaSolution (a):

Epon 812 62.0 mlDodecenyl succinic anhydride 100 ml

Solution (b):Epon 812 100 ml

Methyl nadic anhydride 89.0 mlEqual parts of Solution a and b should be used.

53. Veronal buffer (pH 8.6)Solution (a):

Sodium barbitone 20.60 gmDW to 500 ml

Solution (b):0.2 M hydrochloric acid

Buffer solution:Solution A 50 mlSolution B 6 mlDW to 200 mlAdjust pH 8.6.

54. Ringer’s solutionSodium chloride 0.7 gmPotassium chloride 0.026 gmSodium bicarbonate 0.03 gmCalcium chloride 0.003 gmDW to 1000 ml

B. STAINS

1. Seller’s stainSolution A:

Methylene blue 1.0 gmMethanol (acetone free) 100 ml

Mix by using pestle and mortar and store in atightly stoppered bottle.Solution B:

Basic fuchsin 1.0 gmMethanol (acetone free) 100 mlWorking stain:

Solution A 2 partsSolution B 1 parts

2. Methylene blue stainMethylene blue 0.3 gm95% ethanol 30.0 mlDW to 100 ml

3. Giemsa stain

Stock solution:Azure II - eosin 3.0 gmAzure II 0.8 gmGlycerol 250 mlAcetone free methanol 250 ml

Working stain: Stock solution 1 partDW 9 part

4. Lactophenol cotton blue stainPhenol crystals 10 gmLactic acid syrup 10 mlGlycerol 20 mlDW to 10 mlAniline blue/cotton blue stain 50 mg

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5. Wright’s stainSolution A:

Methylene blue 0.9 gm0.5% aqueous sodium

bicarbonate 100 mlHeat in water bath to boiling for 1 hour cooland filter. Add following solution:Solution B:

Eosin Y 1.0 gmDW 500 ml

Mix both solutions and filter. Collect precipitateand dry at 37°C.

Working stain:Dried precipitate 0.1 gmAcetone free methanol 50 ml

6. Leishman’s stain

Leishman’s stain powder 0.15 gmMethanol 100.0 mlMix in pestle and mortar. Filter before use.

7. Newman’s stainMethylene blue 1.12 gm95% ethanol 54.0 mlTetrachloroethane 40.0 mlGlacial acetic acid 6.0 ml

8. May Grunwald stainStock solution:

May Grunwald stain powder 3.0 gmMethanol 1000 ml

Warm to 50°C and put on stirring for few hours.Working solution:Stock solution 2 partsDW to 1 part

9. Mann’s methylene blue-eosin stain (tissues)

1.0% aqueous solution of methyleneblue 35 ml1.0% aqueous solution of eosin 45 mlDW 100 ml

10. Macchiavello stain (tissues)Basic fuchsin 0.5 gm0.1 M disodium phosphate 3.6 ml0.1 M sodium acid phosphate 1.4 ml

(NaH2PO4.H2O)DW to 95 ml

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

11. Kinyoun’s carbol fuchsin (tissues)Basic fuchsin 4.0 gmPhenol 8.0 gmEthanol 20.0 mlHeat to dissolveDW to 100 mlPrepare fresh

12. Zeihl Neelsen carbol fuchsin (tissue)

Basic fuchsin 1.0 gmEthanol 10 ml5% phenol aqueous 100 mlDecolorizing agent:

Concentrated hydrochloric acid 3.0 ml70% ethanol 97.0 mlCounter stain:

0.1% aqueous methylene blue.

II. Smears:(a) Carbol fuchsin:

Basic fuchsin 0.3 gmEthanol 10 mlPhenol 5 mlDW to 95 ml

(b) Decolourizer: 3% hydrochloric acidin 95% ethanol

(c) Counter stain: 0.3% methylene blue(aqueous)

13. Gram’s stainingI. Tissues

(a) Crystal violet:1.0% crystal violet (aqueous) 40 ml5% sodium bicarbonate 10 ml

(b) Gram’s iodineIodine crystals 1.0 gmPotassium iodide 2.0 gmDW 300 ml

(c) Counter stainBasic fuchsin 25 mgDW 1000 ml

II. Smears:(a) Crystal violet:

Stock solution A: 1% crystal violet inethanol

Stock solution B: 1% ammonium oxalate

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Working solution:Stock solution A 20 mlStock solution B 80 ml

(b) Gram’s iodine: Same as for tissues(c) Decolorizer:

Ethanol (95%) 250 mlAcetone 250 ml

(d) Counter stainSafranin 2.5 gm95% ethanol 100 mlWorking stain: Safranin 1:4 diluted with

DW.

14. Silver nitrate for spirochaete in tissuesSilver nitrate 2.0 gmDW 100 ml

15. Grocatt’s methenamine silver nitrate

Solution A:Silver nitrate 5.0 gmDW 100 ml

Solution B:.Methenamine 3.0 gmDW 100 ml

Stock solutionSolution A 5 mlSolution B 100 ml

Working solutionStock solution 25 mlDW 25 ml5% borax 2 ml

16. Gold chlorideGold chloride 0.10 gmDW 100 ml

17. Sodium thiosulfateSodium thiosulfate 2.0 gmDW 100 ml

18. Lendrum’s phloxine tetrazine

A. Phloxine B 0.5 gm0.045 m calcium chloride 100 ml

B. Saturated tetrazine in cellosolve

19. Harris haematoxylinHematoxylin 2.5 gmAbsolute ethanol 50 ml

Potassium alum 50 gmDW 500 mlMercuric oxide 1.5 gmGlacial acetic acid 20 mlFilter before use

20. Meyer’s hematoxylinHematoxylin 0.5 gmDW 500 mlPotassium alum 25.0 gmSodium iodate 0.1 gmCitric acid 0.5 gmChloral hydrate 25.0 gm

21. Acid alcohol

Concentrated hydrochloric acid 3.0 ml70.1% ethanol 97.0 ml

22. Ammonia waterConcentrated ammonia 2-3 dropsDW 250 ml

23. Eosin(a) Aqueous: Eosin 1.0 gm

DW 100 ml(b) Alcoholic: Eosin 1.0 gm

70% ethanol 100 ml

24. Staining for electronmicroscopic examination

(a) Lead citrate:Lead nitrate 1.33 gmSodium citrate 1.76 gmDW 30.0 ml

(b) Uranyl acetate:Uranyl acetate 1.0 gmDW 100 ml

(c) Lead hydroxide:Lead oxide 0.5 gm1 N NaOH 20 ml

Heat to dissolve, cool and filter. Make it 100 mlwith DW.

25. Periodic acid-Schiff’s staining(a) Periodic acid:

Periodic acid 0.4 gmDW 10.0 ml2.72% sodium acetate solution 5.0 mlethanol 35.0 ml

328 Textbook of Veterinary Clinical and Laboratory Diagnosis

(b) Reducing solution:Potassium iodide 1.0 gmSodium thiosulfate 1.0 gmDW 20 mlEthanol 30 ml1 N HCl 1.0 ml

(c) Schiff’s reagent:Basic fuchsin 1.0 gmDW 200 ml

Boil and cool to 50°C, filter add 20 ml 1 N HClcool to 25°C and add sodium metabisulfite1.0 gm, store for 24 hours in dark and add2.0 gm activated charcol. Shake, filter and keepin dark stoppered bottle at 4°C.

C. CULTURE MEDIA

1. Nutrient brothBacto beef extract 0.3 gmBacto peptone 0.5 gmDW 100 mlAdjust pH 7.2 sterilize at 121°C for 15 minute inautoclave.

2. Nutrient agar

Nutrient broth 100 mlAgar 1.5 gmSterilize by autoclave.

3. Brain-Heart Infusion broth (BHI broth)Infusion from calf brain 20.0 gmInfusion from beef heart 25.0 gmProteose peptone 1.0 gmGlucose 0.2 gmSodium chloride 0.5 gmDisodium hydrogen phosphate 0.25 gmDW 100 mlSterilize by autoclave

4. BHI agarBHI broth 100 mlAgar 1.5 gm

5. Blood agar

Bacto-tryptose 10.0 gmBeef extract 1.5 gmSodium chloride 2.5 gmGlucose 0.15 gmDextrin 0.25 gm

Para-aminobenzoic acid 10.0 gmAgar 7.5 gmDW 500 mlAdjust pH 7.4. Autoclave at 121°C and 15 lbspressure for 15 min. Cool to 45°C and add 50 mlblood (defibrinated).

6. Trypticase soy agarTrypticase 8.5 gmPhytone 1.5 gmSodium chloride 2.5 gmDipotassium hydrogen 1.25 gmphosphate (K2HPO4)Glucose 1.25 gmPara-aminobenzoic acid 10.0 gmAgar 7.5 gmDW 500 ml

7. Cooked meat medium

Lean beef 250 gmProteose peptone 10.0 gmGlucose 1.0 gmSodium chloride 2.5 gmDW 500 mlAutoclave at 121°C, 15 lbs for 15 min.

8. Brilliant green agarBacto-yeast extract 1.5 gmProteose peptone 5.0 gmSodium chloride 2.5 gmBacto-lactose 5.0 gmSaccharose 5.0 gmBacto-phenol red 0.04 gmBacto-brilliant green 0.006 gmAgar 10.0 gmDW 500 mlAutoclave at 121°C for 15 min

9. Eosin-methylene blue agar (EMB agar)Bacto-peptone 5.0 gmBacto-lactose 2.5 gmSaccharose 2.5 gmDipotassium phosphate 1.0 gmAgar 7.0 gmEosin Y 0.2 gmMethylene blue 0.035 gmDW 500 mlAutoclave it for 15 min at 15 lbs pressure and121°C.

Appendix 329

10. Mac Conkey agarPeptone 4.4 gmProteose peptone 0.75 gmLactose 2.5 gmBile salts 0.4 gmSodium chloride 1.25 gmAgar 4.0 gmNeutral red 7.5 mgCrystal violet 0.25 mgDW 250 mlAutoclave it for 15 min at 15 lbs pressure and at121°C.

11. Tripple sugar iron agar (TSI)

Beef extract 3.0 gmYeast extract 3.0 gmProteose peptone 5.0 gmPeptone 15.0 gmD-glucose 1.0 gmSucrose 10.0 gmLactose 10.0 gmSodium chloride 5.0 gmSodium thiosulfate 300 mgFerrous sulfate 200 mgAgar 2.0 gm1% solution of phenol red 2.5 mlDW 1000 mlAutoclave at 115°C for 20 min and pour in tubesto make the slants.

12. Tetrathionate brothProteose peptone 2.5 gmBile salts 0.5 gmCalcium carbonate 5.0 gmSodium thiosulfate 15.0 gmDW 500 mlAutoclave and pour/distribute in tubes.

13. Selenite brothBacto-peptone 2.0 gmBacto-lactose 2.0 gmSodium acid selenite 2.5 gmDisodium phosphate 5.0 gmDW 500 mlAutoclave it and dispense in tubes.

14. Urea agarA. Dextrose 10 gm

Peptone 1.0 gmSodium chloride 5.0 gmMonopotassium phosphate 2.0 gmUrea 20 gm

Phenol red 0.012 gmDW 100 mlFilter through seitz filter.

B. Agar 15 gmDW 900 mlAutoclave at 121°C for 15 min.Cool and mix A and B and pour in tubes.

15. Nitrate agarBeef extract 1.5 gmPotassium nitrate 0.5 gmPeptone 2.5 gmAgar 6.0 gmDW 500 mlpH 6.8. Autoclave at 121°C for 15 min.

16. Simmon’s citrate agarSodium chloride 2.5 gmSodium citrate 1.0 gmMagnesium sulfate 0.1 gmMonoammonium phosphate 0.5 gmDipotassium phosphate 0.5 gmBacto-agar 7.5 gmBromothymol blue 0.04 gmDW 500 mlpH 6.8 autoclave at 121°C PC for 15 min.

17. PPLO BrothInfusion from beef heart 25.0 gmSodium chloride 2.5 gmPeptone 5.0 gmCrystal violet 0.005 gmPotassium tellurite 0.15 gmpH 7.8. Autoclave it at 121°C for 15 min.

18. Sabouraud’s dextrose agarPeptone 10.0 gmDextrose 40.0 gmAgar 18.0 gmDW 1000 mlpH 5.6. Autoclave at 121°C for 15 min.

19. Sugar fermentation mediaSodium chloride 5.0 gmTryptone 2.0 gm1% phenol red 2.0 mlDW 1000 mlAutoclave at 121°C for 15 min. Pour in tubes andadd sugar 0.2 ml of 10% sugar solution (sterilizedby filtration).