Barritt S Reagent, for Detection of Acetylmethylcarbinol

Full file at

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 0.5 mM

Biotin 0.5 mM

Distilled water 10.0 ml

Buffered glycerol (pH 7.2), for immunofluorescence

Glycerin 90.0 ml

Phosphate buffered saline 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 10.0 g

Distilled water 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.

Preparation of MacFarland Standards

Corresponding
approximate
BariumSulfuric density of
chlorideacidbacteria
Tube1% (ml)1% (ml)(million/ml)

10.19.9300

20.29.8600

30.39.7900

40.49.61200

50.59.51500

60.69.41800

70.79.32100

80.89.22400

90.99.12700

101.09.03000

Methyl cellulose, for microscopic observation of protozoa

Methyl cellulose 10.0 g

Distilled water 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 8.0 g

Acetic acid, 5 N: 1 part glacial
acetic acid to 2.5 parts
distilled water 1000.0 ml

Solution B, Alpha-naphthylamine

Alpha-naphthylamine 5.0 g

Acetic acid, 5 N 1000.0 ml

Orthonitrophenyl-ß-D-galactoside (ONPG), for enzyme induction

0.1 M sodium phosphate
buffer (pH 7.0) 50.0 ml

ONPG (8  10–4 M) 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.

Phosphate-buffered saline, 1% (pH 7.2–7.4), for immunofluorescence

Solution A

Disodium phosphate 1.4 g

Distilled water 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 6.98 g

Sodium chloride 6.0 g

1 N hydrochloric acid 27.0 ml

Distilled water, q.s. to 1000 ml

Toluidine blue solution, 0.1%, for detection of DNase activity

1% toluidine blue solution 0.1 ml

Distilled water 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 54 g

Boric acid 27.5 g

EDTA (0.5 M, pH 8.0) 20 ml

Distilled water 1000 ml

Tris-borate buffer 1 working solution

Tris-borate buffer 5

(see previous entry) 200 ml

Distilled water 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.