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APPENDIX 2 Biochemical Test Reagents Barritt’s reagent, for detection of acetylmethylcarbinol Solution A Alpha-naphthol 5.0 g Ethanol, absolute 95.0 ml Note: Dissolve the alpha-naphthol in the ethanol with constant stirring. Solution B Potassium hydroxide 40.0 g Creatine 0.3 g Distilled water 100.0 ml Note: Dissolve the potassium hydroxide in 75 ml of distilled water. The solution will become warm. Allow to cool to room temperature. Add the creatine and stir to dissolve. Add the remaining water. Store in a refrigerator.

Biotin-histidine solution, for Ames test L-histidine

HCl Biotin Distilled water

0.5 mM 0.5 mM 10.0 ml

Buffered glycerol (pH 7.2), for immunofluorescence Glycerin Phosphate buffered saline

90.0 ml 10.0 ml

Diphenylamine reagent, for detection of nitrates Dissolve 0.7 g diphenylamine in a mixture of 60 ml concentrated sulfuric acid and 28.8 ml of distilled water. Cool and slowly add 11.3 ml of concentrated hydrochloric acid. Allow to stand for 12 hours. Sedimentation indicates that the reagent is saturated.

Ferric chloride reagent Ferric chloride Distilled water

10.0 g 100.0 ml

Gram’s iodine, for detection of starch As in Gram stain

Hydrogen peroxide, 3%, for detection of catalase activity Note: Refrigerate when not in use.

Kovac’s reagent, for detection of indole p-Dimethylaminobenzaldehyde 5.0 g Amyl alcohol 75.0 ml Hydrochloric acid (concentrated) 25.0 ml Note: Dissolve the p-dimethylaminobenzaldehyde in the amyl alcohol. Add the hydrochloric acid.

McFarland Barium Sulfate Standards, for STAPH-IDENT procedure Prepare 1% aqueous barium chloride and 1% sulfuric acid solutions. Using the table below, add the amounts of barium chloride and sulfuric acid to clean 15-  150-mm screw-cap test tubes. Label the tubes 1 through 10.

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Barium chloride 1% (ml)

Sulfuric acid 1% (ml)

1 2 3 4 5 6 7 8 9 10

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

9.9 9.8 9.7 9.6 9.5 9.4 9.3 9.2 9.1 9.0

Corresponding approximate density of bacteria (million/ml) 300 600 900 1200 1500 1800 2100 2400 2700 3000

Methyl cellulose, for microscopic observation of protozoa Methyl cellulose Distilled water

10.0 g 90.0 ml

Methyl red solution, for detection of acid Methyl red 0.1 g 95% ethyl alcohol 300.0 ml Distilled water 200.0 ml Note: Dissolve the methyl red in the 95% ethyl alcohol. Dilute to 500 ml with distilled water.

Nessler’s reagent, for detection of ammonia Potassium iodide 50.0 g Distilled water (ammonia-free) 35.0 ml Add saturated aqueous solution of mercuric chloride until a slight precipitate persists. Potassium hydroxide (50% aqueous) 400.0 ml Note: Dilute to 1000 ml with ammonia-free distilled water. Let stand for 1 week, decant supernatant liquid, and store in a tightly capped amber bottle.

Nitrate test solution, for detection of nitrites Solution A, Sulfanilic acid Sulfanilic acid Acetic acid, 5 N: 1 part glacial acetic acid to 2.5 parts distilled water

1000.0 ml

Solution B, Alpha-naphthylamine Alpha-naphthylamine Acetic acid, 5 N

5.0 g 1000.0 ml

8.0 g

Orthonitrophenyl-ß-D-galactoside (ONPG), for enzyme induction 0.1 M sodium phosphate buffer (pH 7.0) ONPG (8  10–4 M)

50.0 ml 12.5 mg

p-Aminodimethylaniline oxalate, for detection of oxidase activity p-Aminodimethylaniline oxalate 0.5 g Distilled water 50.0 ml Note: To dissolve fully, gently warm the solution.

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Full file at Phosphate-buffered saline, 1% (pH 7.2–7.4), for immunofluorescence Solution A Disodium phosphate Distilled water

1.4 g 100.0 ml

Solution B Sodium dihydrogen phosphate 1.4 g Distilled water 100.0 ml Note: Add 84.1 ml of Solution A to 15.9 ml of Solution B. Add 8.5 g of sodium chloride and q.s. to 1 liter.

Rabbit plasma, for detection of coagulase activity Note: Store vials at 2°C to 8°C. Reconstitute by the addition of 7.5 ml of sterile water.

Sodium barbital buffer, for immunofluorescence Sodium barbital Sodium chloride 1 N hydrochloric acid Distilled water, q.s. to 1000 ml

6.98 g 6.0 g 27.0 ml

Toluidine blue solution, 0.1%, for detection of DNase activity 1% toluidine blue solution Distilled water

0.1 ml 99.9 ml

Tris-acetate buffer 10 Tris base 48.4 g Glacial acetic acid 11 g EDTA (0.5 M) 20 ml Distilled water 1000 ml Note: Add ingredients to 1-liter volumetric flask and then add water to volume.

Tris-acetate buffer 1 Tris-acetate buffer 10 (see previous entry) 100 ml Distilled water 900 ml Note: Buffer can be stored indefinitely at room temperature.

Tris-borate buffer 5 Tris base Boric acid EDTA (0.5 M, pH 8.0) Distilled water

54 g 27.5 g 20 ml 1000 ml

Tris-borate buffer 1 working solution Tris-borate buffer 5 (see previous entry) Distilled water

200 ml 800 ml

Trommsdorf’s reagent, for detection of nitrite Slowly add 100 ml of 20% aqueous zinc chloride solution to a mixture of 4 g of starch in water. Heat until the starch is dissolved as much as possible and the solution is almost clear. Dilute with water and add 2 g of potassium iodide. Dilute to 1000 ml, filter, and store in an amber bottle.

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