Microbiology A Laboratory Manual 10th

Edition Cappuccino Solutions Manual

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 Appendix 2
Biochemical Test Reagents

Barritt’s reagent, for detection of Kovac’s reagent, for detection of indole

acetylmethylcarbinol p-Dimethylaminobenzaldehyde 5.0 g
Solution A Amyl alcohol 75.0 ml
Alpha-naphthol 5.0 g Hydrochloric acid (concentrated) 25.0 ml
Ethanol, absolute 95.0 ml Note: Dissolve the p-dimethylaminobenzaldehyde
Note: Dissolve the alpha-naphthol in the ethanol in the amyl alcohol. Add the hydrochloric acid.
with constant stirring.
Solution B McFarland Barium Sulfate Standards,
Potassium hydroxide 40.0 g for STAPH-IDENT procedure
Creatine 0.3 g Prepare 1% aqueous barium chloride and 1% sulfu-
Distilled water 100.0 ml ric acid solutions. Using the table below, add the
Note: Dissolve the potassium hydroxide in 75 ml amounts of barium chloride and sulfuric acid to
of distilled water. The solution will become warm. clean 15- × 150-mm screw-cap test tubes. Label the
Allow to cool to room temperature. Add the crea- tubes 1 through 10.
tine and stir to dissolve. Add the remaining water.
Store in a refrigerator.
Preparation of MacFarland Standards
Biotin-histidine solution, for Ames test
l-histidine HCl 0.5 mM Corresponding
Biotin 0.5 mM approximate
Distilled water 10.0 ml Barium Sulfuric density of
chloride acid bacteria
Tube 1% (ml) 1% (ml) (million/ml)
Buffered glycerol (pH 7.2),
for immunofluorescence 1 0.1 9.9 300
2 0.2 9.8 600
Glycerin 90.0 ml
Phosphate buffered saline 10.0 ml 3 0.3 9.7 900
4 0.4 9.6 1200
5 0.5 9.5 1500
Diphenylamine reagent, for detection of
6 0.6 9.4 1800
nitrates
7 0.7 9.3 2100
Dissolve 0.7 g diphenylamine in a mixture of 60 8 0.8 9.2 2400
ml concentrated sulfuric acid and 28.8 ml of dis- 9 0.9 9.1 2700
tilled water. Cool and slowly add 11.3 ml of con- 10 1.0 9.0 3000
centrated hydrochloric acid. Allow to stand for 12
hours. Sedimentation indicates that the reagent is
saturated. Methyl cellulose, for microscopic
observation of protozoa
Ferric chloride reagent
Methyl cellulose 10.0 g
Ferric chloride 10.0 g Distilled water
Distilled water 90.0 ml
100.0 ml

Gram’s iodine, for detection of starch Methyl red solution, for detection of acid
As in Gram stain Methyl red 0.1 g
95% ethyl alcohol 300.0 ml
Hydrogen peroxide, 3%, for detection Distilled water 200.0 ml
of catalase activity Note: Dissolve the methyl red in the 95% ethyl
Note: Refrigerate when not in use. ­alcohol. Dilute to 500 ml with distilled water.
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 Nessler’s reagent, for detection of Sodium barbital buffer, for
ammonia immunofluorescence
Potassium iodide 50.0 g Sodium barbital 6.98 g
Distilled water (ammonia-free) 35.0 ml Sodium chloride 6.0 g
Add saturated aqueous solution 1 N hydrochloric acid 27.0 ml
of mercuric chloride until a Distilled water, q.s. to 1000 ml
slight precipitate persists.
Potassium hydroxide
Toluidine blue solution, 0.1%, for
(50% aqueous) 400.0 ml detection of DNase activity
Note: Dilute to 1000 ml with ammonia-free distilled 1% toluidine blue solution 0.1 ml
water. Let stand for 1 week, decant supernatant liq- Distilled water 99.9 ml
uid, and store in a tightly capped amber bottle. Tris-acetate buffer 10∙
Nitrate test solution, for detection of Tris base 48.4 g
Glacial acetic acid 11 g
nitrites
EDTA (0.5 M) 20 ml
Solution A, Sulfanilic acid Distilled water 1000 ml
Sulfanilic acid 8.0 g Note: Add ingredients to 1-liter volumetric flask and
Acetic acid, 5 N: 1 part glacial then add water to volume.
acetic acid to 2.5 parts
distilled water 1000.0 ml Tris-acetate buffer 1∙
Solution B, Alpha-naphthylamine Tris-acetate buffer 10×
Alpha-naphthylamine 5.0 g (see previous entry) 100 ml
Acetic acid, 5 N 1000.0 ml Distilled water 900 ml
Note: Buffer can be stored indefinitely at room
Oxidase Reagent temperature.
Tetramethyl-p-phenylene diamine Tris-borate buffer 5∙
dihydrochloride 1.0 g
Tris base 54 g
Distilled water 100 ml
Boric acid 27.5 g
EDTA (0.5 M, pH 8.0) 20 ml
Orthonitrophenyl-ß-D-galactoside (ONPG),
Distilled water 1000 ml
for enzyme induction
0.1 M sodium phosphate Tris-borate buffer 1∙ working solution
buffer (pH 7.0) 50.0 ml Tris-borate buffer 5×
ONPG (8 × 10–4 M) 12.5 mg (see previous entry) 200 ml
Distilled water 800 ml
p-Aminodimethylaniline oxalate, for
detection of oxidase activity Trommsdorf’s reagent, for detection of
p-Aminodimethylaniline oxalate 0.5 g nitrite
Distilled water 50.0 ml Slowly add 100 ml of 20% aqueous zinc chloride so-
Note: To dissolve fully, gently warm the solution. lution to a mixture of 4 g of starch in water. Heat
until the starch is dissolved as much as possible
Phosphate-buffered saline, 1% (pH 7.2–7.4), and the solution is almost clear. Dilute with water
for immunofluorescence and add 2 g of potassium iodide. Dilute to 1000 ml,
Solution A filter, and store in an amber bottle.
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 So-
lution 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.
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