Reference: Merker, R. I. and Solomon, H. M. 1998. Media and Reagents. Appendix 3. In Food and Drug
Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). R.L. Merker (Ed.). AOAC
International, Gaithersburg, MD.

The media and reagents listed here were recommended by the BAM-8 authors. Formulations were reviewed for BAM-8 and approved by . Changes to this section in the current revision were implemented by Robert I. Merker.

MEDIA

M1. A-1 Medium
Tryptone 20 g
Lactose 5 g
NaCl 5 g
*Triton X-100 (Rohm & Haas) 1 ml
Salicin 0.5 g
Distilled water 1 liter

Dissolve ingredients in 1 liter distilled water. Adjust pH to 6.9 ± 0.1. Dispense 10 ml portions of single strength broth into 18 x 150 mm tubes containing inverted fermentation vials. For double strength broth, use 22 x 175 mm tubes containing inverted fermentation vials. Medium may be cloudy before sterilization. Autoclave 10 min at 121°C. Store in dark up to 7 days. (Commercially available A-1 medium is unacceptable.)
*Triton X-100 may be purchased from Fisher Scientific Company, Fairlawn, NJ 07410.

M2. Acetamide Medium
Stock basal medium
KH₂PO₄ 0.5 M 14 ml
K₂HPO₄ 0.5 M 6 ml
Agar 0.5 g
Distilled water 400 ml

Heat with agitation to dissolve agar. Add 1 ml PR-CV (500X concentrate).

Stock acetamide, 1%
Acetamide 1 g
Distilled water 100 ml

Store over CHCl₃ in screw-cap container. Stable indefinitely at room temperature.

PR-CV (500X concentrate)
Phenol red 2 g
Crystal violet 0.2 g
Distilled water 200 ml

Add 5 N NaOH until ingredients are dissolved.

Final medium. Add 0.8 ml basal medium to 13 x 100 mm tube. Add 0.2 ml acetamide solution. Steam (100°C) 10 min. Cool.

M3. Acetate Agar
Sodium acetate 2 g
NaCl 5 g
Mg SO₄ (anhydrous) 0.2 g
Ammonium phosphate1 g
K₂HPO₄ 1 g
Bromthymol blue 0.08 g
Agar 20 g
Distilled water 1 liter

Add all ingredients except Mg SO₄ to 1 liter distilled water. Heat to boiling with stirring. Add Mg SO₄ and adjust pH. Dispense 8 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121°C. Incline tubes to obtain 5 cm slant. Final pH, 6.7. NOTE: This medium contains a slightly smaller concentration of acetate than the formula recommended in Ewing (1986).

M4. Acid Broth
Proteose peptone 5 g
Yeast extract 5 g
Dextrose 5 g
K₂HPO4 4 g
Distilled water l liter

Dissolve ingredients and dispense 12-15 ml portions into 20 x 150 mm tubes. Autoclave 15 min at 121°C. Final pH, 5.0.

M5. AE Sporulation Medium, Modified (for C. perfringens)
(AE base is available commercially)
Polypeptone 10.0 g
Yeast extract 10.0 g
Na₂HPO₄ 4.36 g
KH₂PO₄ 0.25 g
Ammonium acetate1.5 g
Mg SO₄·7H₂O 0.2 g
Distilled water 1 liter

Dissolve ingredients and adjust to pH 7.5 ± 0.1, using 2 M sodium carbonate. Dispense 15 ml into 20 x 150-mm screw-cap tubes and sterilize by autoclaving for 15 min at 121°C. After sterilization, add 0.6 ml of sterilized 10% raffinose and 0.2 ml each of filter-sterilized 0.66 M sodium carbonate and 0.32% cobalt chloride (CoCl₂·6H₂O) dropwise to each tube. Check pH of one or two tubes; it should be 7.8 ± 0.1. Just before use, steam medium for 10 min; after cooling, add 0.2 ml of filter-sterilized 1.5% sodium ascorbate (prepared daily) to each tube.

M6. Agar Medium P
Beef extract 3 g
K₂HPO₄ 0.25 g
Peptone 5 g
Polysorbate 80 1 g
Tryptone 1.7 g
Bromcresol purple 0.06 g
Soytone 0.3 g
Agar 15 g
Dextrose 5.25 g
Distilled water l liter
NaCl 0.5 g

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Final pH, 7.8 ± 0.2.

M7. AKI Medium
Peptone 15 g
Yeast extract 4 g
NaCl 5 g
Distilled water 970 ml
NaHCO₃, 10% aqueous, filter-sterilized 30 ml

On day of use, dissolve peptone, yeast extract, and NaCl in distilled water. Autoclave 15 min at 121°C. Cool. Add 30 ml freshly prepared, filter-sterilized NaHCO₃, and mix. Dispense aseptically into screw-capped tubes (use 15 ml for 16 x 125 mm tubes). Final pH, 7.4 ± 0.2.

M8. Alkaline Peptone Agar
Peptone 10 g
NaCl 20 g
Agar 15 g
Distilled water 1 liter

Boil to dissolve ingredients. Adjust pH so that value after sterilization is 8.5 ± 0.2. Autoclave 15 min at 121°C. Solidify agar in tubes as slants.

M9. Alkaline Peptone Salt Broth (APS)
Peptone 10 g
NaCl 30 g
Distilled water 1 liter

Dissolve ingredients. Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense 10 ml into tubes. Autoclave 10 min at 121°C.

M10. Alkaline Peptone Water
Peptone 10 g
NaCl 10 g
Distilled water l liter

Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense into screw-cap tubes. Autoclave 10 min at 121°C.

M11. Anaerobe Agar
Base
Trypticase (tryptic) soy agar 40 g
Agar 5 g
Yeast extract 5 g
L-Cysteine (dissolved in 5 ml 1 N NaOH) 0.4 g
Distilled water l liter

Heat with agitation to dissolve agar. Adjust pH to 7.5 ± 0.2. Autoclave 15 min at 121°C. Cool to 50°C.

Hemin solution. Suspend 1 g hemin in 100 ml distilled water. Autoclave 15 min at 121°C. Refrigerate at 4°C.

Vitamin K₁ solution. Dissolve 1 g vitamin K₁ (Sigma Chemical Co., St. Louis, MO) in 100 ml 95% ethanol. Solution may require 2-3 days with intermittent shaking to dissolve. Refrigerate at 4°C.

Final medium. To 1 liter base add 0.5 ml hemin solution and 1 ml Vitamin K₁ solution. Mix and pour 20 ml portions into 15 x 100 mm petri dishes. Medium must be reduced before inoculation by 24 h anaerobic incubation in anaerobic glove box or GasPak jar.

M12. Anaerobic Egg Yolk Agar
Agar base
Yeast extract 5 g
Tryptone 5 g
Proteose peptone 20 g
NaCl 5 g
Agar 20 g
Distilled water 1 liter

Autoclave 15 min at 121°C. Adjust pH to 7.0 ± 0.2.

2 Fresh Eggs

Treatment of eggs. Wash 2 fresh eggs with stiff brush and drain. Soak eggs in 70% ethanol for 1 h. Crack eggs aseptically. Retain yolks. Drain contents of yolk sacs into sterile stoppered graduate and discard sacs. Add yolk to equal volume of sterile 0.85% saline. Invert graduate several times to mix. Egg yolk emulsion (50%) is available commercially.

Preparation of medium. To 1 liter melted medium (48-50°C) add 80 ml yolk-saline mixture (available from Difco as Bacto Egg Yolk Enrichment 50%), and mix. Pour plates immediately. After solidification dry 2-3 days at ambient temperature or at 35°C for 24 h. Check plates for contamination before use. After drying, plates may be stored for a short period in refrigerator.

M13. Andrade's Carbohydrate Broth and Indicator

Base
Beef extract 3 g
Peptone or gelysate10 g
NaCl 10 g
Distilled water 1 liter

Adjust pH to 7.2 ± 0.2. Autoclave 15 min at 121°C.

Andrade's indicator
Acid fuchsin 0.2 g
Distilled water 10 ml
1 N NaOH 16 ml

Allow to decolorize before use. Add 1-2 ml NaOH if necessary. Add 10 ml indicator to 1 liter base.

Carbohydrate stock solution. Prepare dextrose, lactose, sucrose, and mannitol in 10% solutions. Prepare dulcitol, salicin, and other carbohydrates in 5% solutions. Sterilize by filtration through 0.20 µm membrane. Dilute sugar solutions 1:10 in base with Andrade's indicator to give recommended concentration. Mix gently.

M14. Antibiotic Medium No. 1 (Agar Medium A)
Gelatone or gelysate 6 g
Tryptone or trypticase 4 g
Yeast extract 3 g
Beef extract 1.5 g
Dextrose 1 g
Agar 15 g
Distilled water 1 liter

Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Penassay Seed Agar or BBL Seed Agar.

M15. Antibiotic Medium No. 4 (Agar Medium B)
Tryptone or trypticase 6 g
Yeast extract 3 g
Beef extract 1.5 g
Dextrose 1 g
Agar 15 g
Distilled water 1 liter

Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Yeast Beef Agar or BBL Yeast Beef Agar.

M16. Arginine-Glucose Slant (AGS)
Peptone 5 g
Yeast extract 3 g
Tryptone 10 g
NaCl 20 g
Glucose 1 g
L-Arginine (hydrochloride) 5 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.3 g
Bromocresol purple 0.02 g
Agar 13.5 g
Distilled water 1 liter

Suspend ingredients in distilled water and boil to dissolve. Dispense into tubes (for 13 x 100 mm tubes use 5 ml). Autoclave 10-12 min at 121°C. After sterilization, solidify as slants. Final pH, 6.8-7.0.

M17. Baird-Parker Medium, pH 7.0

Basal medium
Tryptone 10 g
Beef extract 5 g
Yeast extract 1 g
Sodium pyruvate10 g
Glycine 12 g
Lithium chloride·6H₂O 5 g
Agar 20 g

Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2. If desired for immediate use, maintain melted medium at 48-50°C before adding enrichment. Otherwise, store solidified medium at 4 ± 1°C up to 1 month. Melt medium before use.

Enrichment. Bacto EY tellurite enrichment.

Complete medium. Aseptically add 5 ml prewarmed (45-50°C) Bacto EY tellurite enrichment to 95 ml melted base. Mix well (avoiding bubbles) and pour 15-18 ml portions into sterile 15 x 100 mm petri dishes. The medium must be densely opaque. Dry plates before use. Store prepared plates at 20-25°C for up to 5 days. See AOAC Official Methods, 15th Edition (1990), p. 429, for more information.

M18. Bile Esculin Agar
Beef extract 3 g
Peptone 5 g
Esculin 1 g
Oxgall 40 g
Ferric citrate 0.5 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve. Dispense into tubes, autoclave 15 min at 121°C, and slant until solidified. Final pH, 6.6 ± 0.2.

M19. Bismuth Sulfite Agar (Wilson and Blair)
Polypeptone (or peptone)10 g
Beef extract 5 g
Dextrose 5 g
Na₂HPO₄ (anhydrous) 4 g
FeSO₄ (anhydrous) 0.3 g
Bismuth sulfite (indicator) 8 g
Brilliant green 0.025 g
Agar 20 g
Distilled water 1 liter

Mix thoroughly and heat with agitation. Boil about 1 min to obtain uniform suspension. (Precipitate will not dissolve.) Cool to 45-50°C. Suspend precipitate by gentle agitation, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let plates dry about 2 h with lids partially removed; then close plates. Final pH, 7.7 ± 0.2 DO NOT AUTOCLAVE. Prepare plates on day before streaking and store in dark. Selectivity decreases in 48 h.

M20. Blood Agar
Tryptone 15 g
Phytone or soytone 5 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C. Add 5 ml defibrinated sheep red blood cells to 100 ml melted agar. Mix and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Final pH of base, 7.3 ± 0.2. Tryptic soy agar, tryptic soy agar blood base, or trypticase soy agar [soybean-casein digest agar (M152)] may be used as the basal medium. Commercially available sheep blood agar plates are satisfactory. Blood agar base is available from Difco, BBL, and Oxoid. For Vibrio hollisae, add NaCl to a final concentration of 1%.

M20a. Blood Agar Base
(commercially available blood agar base may be used)
Infusion from beef heart 500 g
Tryptose 10 g
NaCl 5 g
Agar 15 g
Sheep blood, sterile, defibrinated 50 ml
Distilled water 1 liter

Suspend ingredients except blood to dissolve. Autoclave 15 min at 121°C. Cool to 45-50°C. Add 50 ml blood (room temperature), and mix. Dispense into sterile petri dishes. Final pH, 6.8 ± 0.2.

M21. Blood Agar Base (Infusion Agar)
Heart muscle, infusion from 375 g
Thiotone 10 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Heat gently to dissolve. Autoclave 20 min at 121°C. Final pH, 7.3 ± 0.2. Commercially available dehydrated heart infusion agars may be used.

M22. Blood Agar Base #2 (Difco)
Proteose peptone15 g
Liver digest 2.5 g
Yeast extract 5 g
NaCl 5 g
Agar 12 g
Distilled Water 1 liter

Autoclave at 121°C for 15 min. For blood agar, reduce water to 950 ml. Add 50 ml defibrinated (whole or lysed) horse blood and FBP (4 ml to agar + blood) after autoclaving and cooling to 48°C. Final pH, 7.4 ± 0.2.

M23. Brain Heart Infusion (BHI) Agar (0.7%)
(for staphylococcal enterotoxin)

Prepare a suitable quantity of brain heart infusion broth (M24). Adjust pH to 5.3 with 1 N HCl. Add agar to give 0.7% concentration. Dissolve by minimal boiling. Dispense 25 ml portions into 25 x 200 mm test tubes. Autoclave 10 min at 121°C.

M24. Brain Heart Infusion (BHI) Broth and Agar
(Acceptable formulations are available from Difco, BBL, and OXOID)
Formulations used by selected manufacturers are represented below; these media are normally available as pre-mixed dry powder.

Medium 1
Calf brain, infusion from 200 g
Beef heart, infusion from 250 g
Proteose peptone (Difco) or polypeptone (Bioquest) 10 g
NaCl 5 g
Na₂HPO₄ * 2.5 g
Dextrose 2.0 g
Distilled water 1 liter
*Difco does not specify waters of hydration.

Medium 2
Brain heart-infusion 6.0 g
Peptic digest of animal tissue 6.0 g
NaCl 5.0 g
Dextrose 3.0 g
Pancreatic digest of gelatin 14.5 g
Na₂HPO₄ *2.5 g
Distilled water 1 liter
*BBL does not specify waters of hydration.

Dissolve ingredients of Medium 1 in distilled water with gentle heat. Suspend ingredients of Medium 2 in distilled water and boil for 1 min to completely dissolve. For both Medium 1 and Medium 2, dispense broth into bottles or tubes for storage. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Commercially available BHI is acceptable.

To prepare brain heart infusion agar, add 15 g agar to 1 liter BHI broth. Heat to dissolve agar before dispensing into bottles or flasks. Autoclave 15 min at 121°C.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M25. Brilliant Green Lactose Bile Broth

Peptone 10 g
Lactose 10 g
Oxgall 20 g
Brilliant green 0.0133 g
Distilled water 1 liter

Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1% aqueous brilliant green in distilled water. Add distilled water to make 1 liter. Dispense into fermentation tubes, making certain that fluid level covers inverted vials. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1.

M26. Bromcresol Purple Broth
Base
Peptone 10 g
Beef extract 3 g
NaCl 5 g
Bromcresol purple 0.04 g
Distilled water 1 liter

Dispense 2.5 ml portions of base solution into 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. Autoclave 10 min at 121°C. Final pH, 7.0 ± 0.2. Sterilize stock solutions of carbohydrates (50% w/v) separately by autoclaving or, preferably, by filtration (0.2 µm pore size). Add 0.278 ± 0.002 ml stock carbohydrate solution to 2.5 ml basal medium to give 5% w/v final carbohydrate concentration.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M27. Bromcresol Purple Dextrose Broth (BCP)
Dextrose 10 g
Beef extract 3 g
Peptone 5 g
Bromcresol purple (1.6% in ethanol) 2 ml
Distilled water 1 liter

Dissolve ingredients in distilled water. Dispense 12-15 ml into tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ±  0.2.

M28-M30 have been replaced in the 8th Edition Revision. M28a, M29a, and M30a-d are new to the 8th Edition Revision A.

M28a. Campylobacter enrichment broth (Bolton formula)
(Oxoid AM-7526 (manufactured by Med-Ox Chemicals Ltd.)or Malthus Diagnostics Lab)Enrichment Broth Base
Meat Peptone 10 g
Lactalbumin Hydrolysate 5 g
Yeast Extract 5 g
NaCl 5 g
Haemin 0.01 g
Sodium Pyruvate 0.5 g
a-Ketoglutamic Acid 1 g
Sodium Metabisulphite 0.5 g
Sodium Carbonate 0.6 g
Distilled Water 1000 ml
Final pH, 7.4 ± 0.2.

THOROUGHLY MIX 27.61 G IN 1L WATER AND LET SOAK 10 MIN. SWIRL AGAIN AND autoclave 15 min at 121°C (in screw-capped bottles if possible). Before use, add 50 ml lysed horse blood and 2 rehydrated [5 ml per vial 50:50 sterile filtered H 0-Ethanol solution] vials of Campylobacter enrichment broth (Bolton formula)2 supplement (Oxoid NDX131 or Malthus Diagnostics X-131). If supplement is not available add 4 ml each of antibiotic concentrates (formulas below, solutions made separately).

Store powdered media in a tightly fastened container in a cool, dry area to reduce oxygen infusion and peroxide formation, which can inhibit recovery of microaerophiles. Use prepared broth within 1 month of preparation (preferably less than 2 weeks).

Campylobacter Enrichment Broth Supplements (Prepare each solution separately)

1) Sodium cefoperazone Weigh 0.5 g into 100 ml distilled water in volumetric flask and dissolve. Only prepare amount required and filter-sterilize, 0.22 µm, using a syringe filter if # 25 ml. Store solution 5 days at 4°C, 14 days at -20°C, and 5 months at -70°C. Freeze in sterile plastic tubes or bottles. Powder may be purchased from Sigma or obtained free from Pfizer by writing Roering Division, Pfizer, 235 E. 42nd St., New York, NY 10017. Request 2-4 g for in vitro use. Final concentration is 20 mg/l.

2) i. Trimethoprim lactate (Sigma Cat. No. T0667). Dissolve 0.66 g in 100 ml distilled water, and filter. May be stored 1 year at 4°C. Add 4 ml/liter to yield a final concentration of 20 mg/liter Trimethoprim. or

ii. Trimethoprim (Sigma Cat. No. T7883)[a lower cost alternative]. To prepare Trimethoprim (TMP)-HCl: add 0.5 g TMP to 30 ml 0.05N HCl at 50°C (stir until dissolved using hot plate with magnetic stirrer). Adjust volume to 100 ml with distilled water. Add 4 ml/liter to yield a final concentration of 20 mg/liter Trimethoprim.

3) Vancomycin (Sigma). Dissolve 0.5 g in 100 ml distilled water and filter. Store up to 2 months at 4°C. Because of short shelf life, prepare smaller amounts. Add 4 ml/liter for final concentration of 20 mg/liter.

4) Cycloheximide Dissolve 1.25 g in 20-30 ml ethanol in a 100 ml volumetric flask and bring to line with water. Filter-sterilize. Store at 4°C up to 1 year. Add 4 ml for final concentration of 50 mg/L.

M29a. Abeyta-Hunt-Bark Agar
Heart infusion agar (Difco) 40 g
Yeast extract 2 g
Distilled water 950 ml

Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Cool and add sodium cefoperazone (6.4 ml if using broth preparation or 4 ml of the agar preparation[below]), 4 ml rifampicin, 4 ml amphotericin B, and 4 ml FBP.After pouring plates, dry plates overnight on bench.If plates must be used the same day, place them in 42°C incubator for several hours. Do not dry in a hood with lids open. Even very brief surface drying will inhibit campylobacter growth.

1) Sodium cefoperazone Prepare as described for broth for final concentration of 32 mg/liter, adding 6.4 ml to agar. OR dissolve 0.8 g in 100 ml water in a 100 ml * volumetric, filter and add 4 ml to agar.

2) Rifampicin Dissolve 0.25 g slowly into 60-80 ml alcohol in a 100 volumetric, swirling repeatedly. When powder is dissolved completely, bring to the line * with distilled water. Store up to 1 year at -20°C. Final concentration is 10 mg/liter.

3) Amphotericin B Dissolve 0.05 g in water in a 100 ml volumetric flask and bring to the line. * Filter sterilize and store at -20 C for 1 year. Final concentration is 2 mg/L.o

4) FBP Dissolve 6.25 g Sodium pyruvate in 10-20 ml distilled water. Pour into a 100 ml volumetric. Add 6.25 g Ferrous sulfate and 6.25 g Sodium metabisulfite. Bring to the line with distilled water and filter sterilize. Use 4 ml/liter agar. FBP is light sensitive and absorbs oxygen rapidly. Only prepare the amount needed. 10-25 ml amounts can be filtered with a 0.22 µm syringe filter. Freeze unused portions in 5 ml amounts at -70°C as soon as possible after preparation. It can be stored at -70° for 3 mos or -20° for 1 mo.

M30a. Modified Campylobacter Blood-Free Selective Agar Base (CCDA)
CCDA agar base (OXOID) 45.5 g
Yeast extract 2 g
Distilled water 1 liter

Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Cool and add of sodium cefoperazone (6.4 ml of strength used for broth or 4 ml of the solution for A-H agar), 4 ml rifampicin, and 4 ml amphotericin B. See A-H directions for precautions when drying plates.

M30b. Freezing medium
Bolton broth base (9.5 ml), 1 ml fetal bovine serum (filtered, 0.22 µm) and 1 ml glycerol (10%). Mix well before use.

M30c. Semi-Solid Medium, modified, for Culture Storage
Campylobacter Enrichment Broth (Bolton)(Oxoid AM-7526) 27.6 g
Agar 1.8 g
Sodium Citrate 0.1 g
Distilled water 1000 ml

Mix ingredients, pH to 7.4 ± 0.2, boil and dispense 10 ml per 16 X 125 screw-cap tube. Autoclave 121 C, 15 min.o Keep tubes tightly capped during storage.

M30d. Semisolid Medium, modified, for Biochemical Identification
Campylobacter Enrichment Broth (Bolton)(Oxoid AM-7526) 27.6 g
Agar 1.8 g
Distilled water 1000 ml
Neutral red solution (see below)10 ml
Biochemicals (see below)

Mix ingredients, except the neutral red solution, boil and separate batch into 4 portions of 250 ml. Add 2.5 ml neutral red to each of 3 portions and omit neutral red from the 4th portion. Add one biochemical (sects. b-e below) to one of each 250 ml portion.Final pH of each portion, 7.4 ± 0.2. Dispense 10 ml per 16 X 125 mm screw-cap tube. Autoclave 121°C, 15 min.

a. Neutral red solution, 0.2% Dissolve 0.2 g neutral red in 10 ml EtOH in a 100 ml volumetric and bring to line with d. water.

b. Potassium nitrate, 1% Add 10 g/liter or 2.5 g/250 ml semi-solid mixture without neutral red.

c. Glycine, 1% Add 10 g/liter or 2.5 g/250 ml semi-solid mixture with neutral red.

d. NaCl, 3.5% Add 30 g/liter or 7.5 g/250 ml semi-solid mixture with neutral red.

e. Cysteine-HCl, 0.02% Add 0.2 g/liter or 0.05 g/250 ml semi-solid mixture with neutral red.

M31. Cary-Blair Transport Medium
Sodium thioglycollate 1.5 g
Na₂HPO₄ 1.1 g
NaCl 5 g
Agar 5 g
CaCl₂ (1% solution) 9 ml
Distilled water 991 ml

Heat with agitation to dissolve dry ingredients. Cool to 50°C and add CaCl₂ .Adjust pH to 8.4. Dispense 7 ml portions into 9 ml screw-cap tubes and immediately steam exactly 15 min. Cool, and tighten caps.

M32. Casamino Acids-Yeast Extract (CYE) Broth
Casamino acids 30 g
Yeast extract 4 g
K₂HPO₄ 0.5 g
Distilled water 1 liter

Dissolve ingredients. Adjust pH to 7.4 ± 0.2. Dispense 10 ml into 50 ml flasks. Autoclave 15 min at 121°C.

M33. Not used in 8th Edition, Bacteriological Analytical Manual

M34. Casamino Acids-Yeast Extract-Salts (CA-YE) Broth (Gorbach)
Casamino acids 20 g
Yeast extract 6 g
NaCl 2.5 g
K₂HPO₄ 8.71 g
Trace salts solution (below), optional 1 ml
Distilled water 1 liter

Adjust pH so that value after autoclaving is 8.5 ± 0.2. Autoclave 15 min at 121°C.

Trace salts solution (optional)
Mg SO₄ 50 g
MnCl₂ 5 g
FeCl₂ 5 g
Distilled water 1 liter

Suspend ingredients. Add enough 0.1 N H₂SO₄ to dissolve. Sterilize by filtration through 0.45 µm membrane. Add 1 ml to 1 liter base. Dispense 10 ml portions into 50 ml flasks.

M35. Cefsulodin-Irgasan Novobiocin (CIN) Agar or Yersinia Selective Agar (YSA)

A. Basal medium
Special peptone 20 g
Yeast extract 2 g
Mannitol 20 g
Pyruvic acid (Na salt) 2 g
NaCl 1 g
Mg SO₄·7H₂O (10 mg/ml) 1 ml
Agar 12 g
Distilled water 756 ml

B. Irgasan (Ciba-Geigy) solution
Irgasan 0.40% in 95% ethanol 1 ml
May be stored at -20°C up to 4 weeks.

C. Desoxycholate solution
Sodium desoxycholate0.5 g Distilled water 200 ml
Bring to boil with stirring; cool to 50-55°C.

D. Sodium hydroxide, 5 N 1 ml

E. Neutral red, 3 mg/ml 10 ml

F. Crystal violet, 0.1 mg/ml 10 ml

G. Cefsulodin (Abbott Labs) 1.5 mg/ml 10 ml

May be stored at -70°C. Thaw to room temperature just before use.

H. Novobiocin, 0.25 mg/ml 10 ml

I. Strontium chloride, 10%; filter-sterilized 10 ml

Preparation: Add ingredients for solution A (basal medium) to water and bring to boil with stirring. Cool to about 80°C (10 min in 50°C water bath). Add solution B (Irgasan) and mix well. Cool to 50-55°C. Add solution C (desoxycholate); solution should remain clear. Add solutions D through H. Slowly add solution I with stirring. Adjust pH to 7.4 with 5 N NaOH. Dispense 15-20 ml into each petri dish. Commercially prepared dehydrated Yersinia selective agar (Difco) with supplements may be substituted. Follow manufacturer's instructions for preparation.

M36. Cell Growth medium
Mix the following sterile solutions aseptically:

Minimal essential medium with Hanks' salts (MEMH) 1 liter
L-15 medium (Leibovitz) 1 liter
(containing 100 units penicillin G, 100 µg streptomycin, and 50 µg gentamicin per ml)

Mix on magnetic stirrer, filter through 0.20 µm membrane, and dispense into sterile 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5°C. Just before use add:
Fetal bovine serum 200 ml
NaHCO₃ (7.5%) 50 ml

M37. Cetrimide Agar
Pancreatic digest of gelatin 20.0 g
Magnesium chloride1.4 g
Potassium sulfate10.0 g
Agar 13.6 g
Cetrimide (cetyl trimethyl ammonium bromide) 0.3 g

Suspend 45.3 g of ingredients or commercial powder (Pseudosel™, BBL; Becton-Dickinson) in 1 liter of purified water. Add 10 ml glycerol. Mix well. Heat to boiling, agitating frequently; maintain boiling for 1 min to dissolve powder. Autoclave at 118°C for 15 min. Final pH, 7.2 ± 0.2. Bacto cetrimide agar base plus glycerol (Difco) is a similar medium.

M38. Chopped Liver Broth
Fresh beef liver 500 g
Peptone 10 g
K₂HPO₄ 1 g
Soluble starch 1 g
Distilled water 1 liter

Grind liver into the water. Heat to boiling and simmer 1 h. Cool, adjust pH to 7.0, and boil 10 min. Filter through cheesecloth and press out excess liquid. Add other ingredients and adjust pH to 7.0. Add water to make 1 liter. Filter through coarse filter paper. Store broth and meat separately in freezer. To 18 x 150 or 20 x 150 mm test tubes, add chopped liver to depth of 1.2-2.5 cm and 10-12 ml of broth. Autoclave 15 min at 121°C.

M39. Christensen Citrate Agar
Sodium citrate 3 g
Glucose 0.2 g
Yeast extract 0.5 g
Cysteine monohydrochloride 0.1 g
Ferric ammonium citrate 0.4 g
KH₂PO₄ 1 g
NaCl 5 g
Sodium thiosulfate 0.08 g
Phenol red 0.012 g
Agar 15 g
Distilled water 1 liter

Suspend ingredients, mix thoroughly and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave for 15 min at 121°C. Final pH, 6.9 ± 0.2. Before media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt. The Difco formulation does not include ferric ammonium citrate and sodium thiosulfate.

M40. Christensen's Urea Agar
Base
Peptone 1 g
NaCl 5 g
Dextrose 1 g
KH₂PO₄ 2 g
Phenol red (6 ml of 1:500 solution)0.012 g Agar 15 g Distilled water 900 ml

Urea concentrate
Urea 20 g
Distilled water 100 ml

Dissolve all ingredients except urea in 900 ml water (basal medium). For halophilic Vibrio spp., add extra 15 g NaCl (final NaCl concentration, 2%). Autoclave 15 min at 121°C. Cool to 50-55°C. Dissolve urea in 100 ml water.Filter-sterilize; add aseptically to cooled basal medium. Mix. Final pH, 6.8 ± 0.1. Dispense to sterile tubes or petri dishes. Slant tubes for 2 cm butt and 3 cm slant.

M41. Congo Red BHI Agarose (CRBHO) Medium
*Brain heart infusion (M20) 37 g
MgCl₂ 1 g
Agarose 12 g
Congo Red dye (375 mg/100 ml of distilled water) 20 ml

Dissolve, autoclave at 121°C for 15 min and pour 20 ml per plate.

*From S. Bhaduri, USDA, Philadelphia, PA. ASM Abstracts, 1989, No. P26. U.S. Patent application ser. no. 07/493,662.

M42. Cooked Meat Medium
Beef heart 454 g
Proteose peptone 20 g
Dextrose 2 g
NaCl 5 g
Distilled water 1 liter

Follow directions as in M38, or suspend 12.5 g commercial dehydrated cooked medium in 100 ml cold distilled water. Mix and let stand 15 min to wet particles thoroughly. Or distribute 1.25 g into 20 x 150 mm test tubes, add 10 ml cold distilled water, and mix thoroughly to wet all particles. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2°C. Steam the sterilized medium and cool, without agitation, just before use.

M43. Cooked Meat Medium (Modified)
(a) Cooked meat medium (commercially available in dehydrated form)
Beef heart 454 g
Proteose peptone 20 g
Dextrose 2 g
NaCl 5 g

(b) Diluent (not available commercially)
Tryptone 10 g
Sodium thioglycollate 1 g
Soluble starch 1 g
Dextrose 2 g
Neutral red (1% aqueous) 5 ml
Distilled water 1 liter

Adjust pH to 6.8 ± 0.2. Add 1 g dehydrated (a) and 15 ml (b) to 20 x 150 mm tubes. Let meat particles rehydrate. Autoclave 15 min at 121°C.

M44. Decarboxylase Basal Medium (Arginine, Lysine, Ornithine)
Base
Peptone or gelysate 5 g
Yeast extract 3 g
Glucose 1 g
Bromcresol purple 0.02 g
Distilled water 1 liter

For arginine broth, add 5 g L-arginine to 1 liter base; for lysine (Falkow) broth, add 5 g L-lysine to 1 liter base; for ornithine, add 5 g L-ornithine to 1 liter base. Adjust pH so that value after sterilization is 6.5 ± 0.2. Dispense 5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave loosely capped tubes 10 min at 121°C. Screw the caps on tightly for storage and after inoculation. For control, use unsupplemented base.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M45. Duncan-Strong (DS) Sporulation Medium, Modified(for C. perfringens)
Proteose peptone 15 g
Yeast extract 4 g
Sodium thioglycollate 1 g
Na₂HPO₄·7H₂O 10 g
Raffinose 4 g
Distilled water 1 liter

Dissolve ingredients and sterilize by autoclaving for 15 min at 121°C. Adjust to pH 7.8 ± 0.1, using filter-sterilized 0.66 M sodium carbonate.

M46. Eagle's Minimal Essential Medium (MEME) (with Earle's salts and nonessential amino acids)
L-Alanine 8.9 mg
L-Valine 46.0 mg
L-Arginine HCl 126.0 mg
D-Calcium pantothenate 1.0 mg
L-Asparagine·H₂O 150.0 mg
Choline chloride 1.0 mg
L-Aspartic acid 13.3 mg
Folic acid 1.0 mg
L-Cystine·2HCl 31.29 mg
Isoinositol 2.0 mg
L-Glutamic acid 14.7 mg
Nicotinamide 1.0 mg
L-Glutamine 292.0 mg
Pyridoxal HCl 1.0 mg
L-Glycine 7.5 mg
Riboflavin 0.1 mg
L-Histidine HCl·H₂O 42.0 mg
Thiamine HCl 1.0 mg
L-Isoleucine 52.0 mg
Glucose 1000.0 mg
L-Leucine 52.0 mg
CaCl₂·2H₂O 265.0 mg
L-Lysine HCl 72.5 mg
KCl 400.0 mg
L-Methionine 15.0 mg
Mg SO₄·7H₂O 200.0 mg
D-Phenylalanine 32.0 mg
NaCl 6800.0 mg
L-Proline 11.5 mg
NaHCO₃ 2200.0 mg
L-Serine 10.5 mg
NaH₂PO₄·H₂O 140.0 mg
L-Threonine 48.0 mg
Phenol red 10.0 mg
L-Tryptophan 10.0 mg
Distilled water 1 liter
L-Tyrosine (disodium salt) 52.1 mg

Dissolve ingredients in distilled water. Sterilize by filtration. Final pH, 7.2 ± 0.2.

M47. Earle's Balanced Salts (Phenol Red-Free)
NaCl 6.8 g
KCl 400 mg
CaCl₂·2H₂O 265 mg
MgSO₄·7H₂O 200 mg
NaH₂PO₄·H₂O 140 mg
Glucose 1.0 g
NaHCO₃ 2.2 g
Distilled water 1 liter

Dissolve ingredients in the water. Sterilize by filtration. Final pH, 7.2 ± 0.2.

M48. EB Motility Medium
Beef extract 3 g
Peptone or gelysate 10 g
NaCl 5 g
Agar 4 g
Distilled water 1 liter

Heat with agitation and boil 1-2 min to dissolve agar. Dispense 8 ml portions into 16 x 150 mm screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

M49. EC Broth
Trypticase or tryptose 20 g
Bile salts No. 31.5 g
Lactose 5 g
K₂HPO₄ 4 g
KH₂PO₄ 1.5 g
NaCl 5 g
Distilled water 1 liter

Distribute 8 ml portions to 16 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.9 ± 0.2.

M50. EC-MUG Medium
Prepare as for EC broth (M49), but add 50 mg 4-methylumbelliferyl-ß-D-glucuronide (MUG) per liter before autoclaving (15 min, 121°C). EC-MUG medium is available commercially.

M51. Egg Yolk Emulsion, 50%
Wash fresh eggs with a stiff brush and drain. Soak eggs 1 h in 70% ethanol. Drain ethanol. Crack eggs aseptically and discard whites. Remove egg yolks with sterile syringe or wide-mouth pipet. Place yolks in sterile container and mix aseptically with equal volume of sterile 0.85% saline. Store at 4°C until use. Egg yolk emulsion (50%) is available commercially.

M52. Enrichment Broth, pH 7.3 ± 0.1
TSBYE supplemented with:
Monopotassium phosphate (anhydrous) 1.35 g/liter
Disodium phosphate (anhydrous) 9.6 g/liter
Acriflavin HCl 10 mg/liter
Nalidixic acid (sodium salt) 40 mg/liter
Cycloheximide 50 mg/liter
Pyruvic acid (sodium salt) (Sigma),10% (w/v) aqueous solution 11.1 ml/liter

Sterilize enrichment broth without the 3 selective agents by autoclaving at 121°C for 15 min. Then add 2.5 ml 10% (w/v) filter-sterilized sodium pyruvate. Add the 3 selective ingredients aseptically to 225 ml enrichment broth plus the 25 g food sample after 4 h incubation at 30°C. Prepare acriflavin and nalidixic supplements as 0.5% (w/v) stock solutions in distilled water. Prepare cycloheximide supplement as 1.0% (w/v) stock solution in 40% (v/v) solution of ethanol in water. Filter-sterilize the 3 selective ingredients. Add stock solutions: 0.455 ml acriflavin, 1.8 ml nalidixic, and 1.15 ml cycloheximide to 225 ml enrichment broth plus the 25 g food sample.

M53. Esculin Agar, Modified (CDC)
Infusion agar 40 g
Esculin 1 g
Ferric citrate 0.5 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Cool to 55°C. Adjust pH to 7.0 ± 0.2. Dispense 4 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 121°C. Slant tubes.

M54. Gelatin Agar (GA)
Peptone 4 g
Yeast extract 1 g
Gelatin 15 g
Agar 15 g
Distilled water 1 liter

Suspend ingredients with constant stirring to prevent scorching gelatin, and boil to dissolve gelatin and agar. Adjust to pH 7.2 ± 0.2. Autoclave 15 min at 121°C. Cool to 45-50°C. Pour plates.

M55. Gelatin Salt Agar (GS)
Prepare gelatin agar (M54), but add 30 g NaCl/liter. Suspend ingredients and boil to dissolve gelatin and agar. Adjust to pH 7.2 ± 0.2. Autoclave 15 min at 121°C. Cool to 45-50°C. Pour plates. If necessary, to inhibit spreading by Vibrio spp. such as V. alginolyticus, use 25-30 g agar/liter.

M56. Gelatinase Solution, 5% replaced by M56a. Papain Solution, 5%

M56a. Papain Solution, 5%
Papain 5 g
Distilled water 95 ml

Add papain to sterile, distilled water and swirl to dissolved completely. Dispense 100 ml portion into bottles.

M57. Gentamicin Sulfate Solution
Gentamicin sulfate 500,000 µg
Distilled water 100 ml

Sterilize by filtration through 0.20 µm membrane. Store at -20°C.

M58. Ham's F-10 Medium
(commercial preparation is preferred)
L-Alanine 8.91 mg
Sodium pyruvate 110 mg
L-Arginine HCl 211.00 mg
Thymidine 0.727 mg
L-Asparagine-H₂O 15.00 mg
Biotin 0.024 mg
L-Aspartic acid 35.12 mg
Choline chloride 0.698 mg
L-Cysteine·2HCl 146.20 mg
Folic acid 1.320 mg
L-Glutamine 14.70 mg
Isoinositol 0.541 mg
L-Glutamic acid 21.00 mg
Niacinamide 0.615 mg
Glycine 7.51 mg
D-Calcium pantothenate 0.715 mg
L-Histidine HCl·H₂O 21.00 mg
Pyridoxine HCl 0.206 mg
L-Isoleucine 2.60 mg
Riboflavin 0.376 mg
L-Leucine 13.10 mg
Thiamine HCl 1.010 mg
L-Lysine HCl 29.30 mg
Vitamin B₁₂ 1.360 mg
L-Methionine 4.48 mg
CaCl₂·2H₂O 44.10 mg
D-Phenylalanine 4.96 mg
CuSO₄·5H₂O 0.0025 mg
L-Proline 11.50 mg
FeSO₄·7H₂O 0.83 mg
L-Serine 10.50 mg
KCl 285.00 mg
L-Threonine 3.57 mg
KH₂PO₄ 83.00 mg
L-Tryptophan 0.60 mg
Mg SO₄·7H₂O 152.80 mg
L-Tyrosine 1.81 mg
NaCl 7400.00 mg
L-Valine 3.50 mg
NaHCO₃ 1200.00 mg
Glucose 1100.0 mg
NaH₂PO₄·H₂O 290.0 mg
Hypoxanthine 4.08 mg
ZnSO₄·7H₂O 0.028 mg
Lipoic acid 0.2 mg
Distilled water 1 liter
Phenol red 1.2 mg

Dissolve ingredients in water. Sterilize by filtration. Final pH, 7.0 ± 0.2. Check sterility before use.

M59. Heart infusion agar (HIA) (Difco)
Beef heart infusion (Difco) 500 g
Tryptose 10 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Or

Blood Agar Base No. 2 (Difco)1 liter
Proteose peptone 15 g
Liver digest 2.5 g
Yeast extract 5 g
NaCl 5 g
Agar 12 g

Autoclave either base at 121°C for 15 min. If preparing blood agar, reduce water to 950 ml. Add 50 ml defibrinated (whole or lysed) horse blood and FBP (4 ml to agar + blood) after autoclaving and cooling to 48°C. Final pH, 7.4 ± 0.2.

M60. Heart Infusion (HI) Broth and Agar (HIA) (for Vibrio)
Beef heart, infusion from 500 g 1 liter
Tryptose 10 g
NaCl 5 g

Dissolve ingredients and dispense into tubes. For halophilic Vibrio spp. add an extra 15 g NaCl/liter. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. For heart infusion agar, add 15 g agar/L and boil to dissolve before dispensing and sterilizing.Commercially available from Difco.

M61. Hektoen Enteric (HE) Agar
Peptone 12 g
Sodium thiosulfate 5 g
Yeast extract 3 g
Ferric ammonium citrate 1.5 g
Bile salts No. 39 g
Bromthymol blue 0.065 g
Lactose 12 g
Acid fuchsin 0.1 g
Sucrose 12 g
Agar 14.0 g
Salicin 2 g
Distilled water 1 liter
NaCl 5 g

Heat to boiling with frequent agitation to dissolve. Boil no longer than 1 min. Do not overheat. Cool in water bath. Pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let dry 2 h with lids partially removed. Final pH, 7.5 ± 0.2. Do not store more than 1 day.

M62. HC (Hemorrhagic colitis E. coli strains) Agar
Tryptone 20.0 g
Bile salts No. 3 1.12 g
NaCl 5.0 g
Sorbitol 20.0 g
MUG reagent 0.1 g
Bromcresol purple 0.015 g
Agar 15.0 g
Distilled water (deionized) 1 liter

Dissolve ingredients in distilled water by heating with stirring. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2. NOTE: The MUG reagent is not essential for the enzyme-labeled monoclonal antibody procedure.

For DNA probe: If colonies are not to be isolated by growth and metabolic characteristics, MUG reagent may be omitted. Plates may be kept 3-4 weeks if wrapped and stored at 4°C. Colonies do not attach well on filters if plates are too dry. If HC agar is to be utilized fully, see FDA BAM Chapter 24, ref. 105. MUG reagent may be purchased from HACH Company, P.O. Box 389, Loveland, CO.

M63. Hugh-Leifson Glucose Broth (HLGB)
Peptone 2 g
Yeast extract 0.5 g
NaCl 30 g
Dextrose 10 g
Bromcresol purple 0.015 g
Agar 3 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Adjust pH to 7.4 ± 0.2. Autoclave 15 min at 121°C.

M64. Indole Medium
L-Tryptophan 1 g
NaCl 1 g
K₂HPO₄ 3.13 g
KH₂PO₄ 0.27 g
Distilled water 200 ml

Dissolve ingredients. Dispense 1 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

M65. Indole Medium (CDC)
Tryptone or trypticase 20 g
Distilled water 1 liter

Adjust pH to 7.3 ± 0.2. Dispense 4 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

M66. Indole Nitrite Medium (Tryptic Nitrate)
Trypticase 20 g
Na₂HPO₄ 2 g
Dextrose 1 g
KNO₃ 1 g
Agar 1 g
Distilled water 1 liter

Heat with agitation to dissolve ingredients. Mix and dispense 11 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 118°C. Final pH, 7.2 ± 0.2.

M67. Irgasan-Ticarcillin-Chlorate (ITC) Broth
Tryptone 10 g
Yeast extract 1 g
MgCl₂·6H₂O 60 g
NaCl 5 g
Potassium chlorate 1 g
Malachite green, 0.2% 5 ml
Distilled water 1 liter

Adjust to pH 7.6 ± 0.2 and autoclave at 121°C for 15 min. Add the following:
Ticarcillin (1 mg/ml) 1 ml
Irgasan DP300 (1 mg/ml) 1 ml

M68. Iron Milk Medium (Modified)
Fresh whole milk1 liter
Ferrous sulfate.7H₂O 1 g
Distilled water50 ml

Dissolve ferrous sulfate in 50 ml distilled water. Add slowly to 1 liter milk and mix with magnetic stirrer. Dispense 11 ml medium into 16 x 150 mm culture tubes. Autoclave 12 min at 118°C. Prepare fresh medium before use.

M69. King's B Medium
Proteose peptone No. 320 g
Glycerol, C.P.10 ml
K₂HPO₄ 1.5 g
Mg SO₄ 1.5 g
Agar 15 g
Distilled water 1 liter

Add all ingredients except Mg SO₄ . Heat with agitation to dissolve agar. Adjust pH to 7.2 ± 0.2. Add Mg SO₄ slowly and mix. Dispense 4 ml portions to 13 x 100 tubes. Autoclave 15 min at 121°C. Incline tubes to give half butt and half slant.

M70. King's O/F Basal Medium
Base
Trypticase (or casitone) 2 g
Phenol red, 1.5% solution 2 ml
Agar 3 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Adjust pH to 7.3 ± 0.2. Dispense 100 ml portions to flasks. Autoclave 15 min at 121°C. Cool to 50°C.

Carbohydrates, 10%. Dissolve 10 g quantities of carbohydrates in 100 ml distilled water. Sterilize by filtration through 0.22 µm membrane. Add 10 ml concentrate to 90 ml melted base and mix. Aseptically dispense 3 ml portions to sterile 13 x 100 mm tubes.

M71. Kligler Iron Agar
Polypeptone peptone 20 g
Lactose 20 g
Dextrose 1 g
NaCl 5 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.5 g
Agar 15 g
Phenol red 0.025 g
Distilled water 1 liter

Heat with agitation to dissolve. Dispense into 13 x 100 mm screw-cap tubes and autoclave 15 min at 121°C. Cool and slant to form deep butts. Final pH, 7.4 ± 0.2.Commercially available from Difco, BBL, and Oxoid.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M72. Koser's Citrate Broth
NaNH₄HPO·4H₂O 1.5 g
KH₂PO₄ (monobasic) 1 g
Mg SO₄ ·7H₂O 0.2 g
Sodium citrate·2H₂O 3 g
Distilled water 1 liter

Dispense into screw-cap tubes as desired. Autoclave 15 min at 121°C. Final pH, 6.7 ± 0.2. This formulation is listed in Official Methods of Analysis of the AOAC, and Standard Methods for the Examination of Wastewater of the APHA. It differs from the composition of commercially available dehydrated medium. The latter is satisfactory.

M73. L-15 Medium (Modified) Leibovitz
D-Galactose 90 mg
L-Tyrosine 300 mg
Phenol red, Na10 mg
DL-Valine 200 mg
Sodium pyruvate 550 mg
D-Calcium pantothenate 1 mg
DL-a-Alanine 450 mg
Choline chloride 1 mg
L-Arginine (free base) 500 mg
Folic acid 1 mg
L-Asparagine·H₂O 250 mg
i-Inositol 2 mg
L-Cysteine (free base) 120 mg
Nicotinamide 1 mg
L-Glutamine 300 mg
Pyridoxine HCl 1 mg
Glycine 200 mg
Riboflavin-5-phosphate, Na 0.1 mg
L-Histidine (free base) 250 mg
Thiamin monophosphate.2H₂O 1.0 mg
L-Isoleucine 250 mg
CaCl₂ (anhydrous) 140 mg
L-Leucine HC1 125 mg
KC1 400 mg
DL-Methionine 150 mg
KH₂PO₄ 60 mg
L-Phenylalanine 250 mg
MgCl₂ (anhydrous) 93.68 mg
L-Serine 200 mg
NaCl 8000 mg
DL-Threonine 600 mg
Na₂HPO₄ (anhydrous) 190 mg
L-Tryptophan 20 mg
Distilled water 1 liter

Filter through 0.20 µm membrane and dispense into 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5°C.

M74. Lactose Broth
Beef extract 3 g
Peptone 5 g
Lactose 5 g
Distilled water 1 liter

Dispense 225 ml portions into 500 ml Erlenmeyer flasks. After autoclaving 15 min at 121°C and just before use, aseptically adjust volume to 225 ml. Final pH, 6.9 ± 0.2.

M75. Lactose-Gelatin Medium (for C. perfringens)
Tryptose 15 g
Yeast extract 10 g
Lactose 10 g
Phenol red (1% solution in 95% ethanol) 5.0 ml
Gelatin 120 g
Distilled water 1 liter

Heat to dissolve tryptose, yeast extract, and lactose in 400 ml water. Suspend gelatin in 600 ml water and heat at 50-60°C with agitation to dissolve. Mix 2 solutions. Adjust pH to 7.5 ± 0.2. Add phenol red and mix. Dispense 10 ml portions into 16 x 150 mm screw-cap tubes. Autoclave 10 min at 121°C. If not used within 8 h, deaerate by heating at 50-70°C for 2-3 h before use.

M76. Lauryl Tryptose (LST) Broth
Tryptose or trypticase 20 g
Lactose 5 g
K₂HPO₄ 2.75 g
KH₂PO₄ 2.75 g
NaCl 5 g
Sodium lauryl sulfate 0.1 g
Distilled water 1 liter

Dispense 10 ml portions into 20 x 150 mm tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

M77. Lauryl Tryptose MUG (LST-MUG) Broth
Tryptose or trypticase 20 g
Lactose 5 g
K₂HPO₄ 2.75 g
KH₂PO₄ 2.75 g
24NaCl 5 g
Sodium lauryl sulfate 0.1 g
4-Methylumbelliferyl-ß-D-glucuronide (MUG)* 50 mg
Distilled water 1 liter

Prepare lauryl tryptose broth and add MUG. Dissolve with gentle heat if necessary. Dispense 10 ml portions into 20 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.
*MUG may be obtained from several sources, e.g., HACH Co., Loveland, CO.

M78. Letheen Agar (Modified)
Letheen agar (Difco or BBL) 32 g
Trypticase peptone 5 g
Thiotone peptone10 g
Yeast extract 2 g
NaCl 5 g
Sodium bisulfite 0.1 g
Agar 5 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Aseptically dispense 20 ml into 15 x 100 mm petri dishes. Final pH, 7.2 ± 0.2.

M79. Letheen Broth (Modified)
Letheen broth 26.7 g
Trypticase peptone 5 g
Thiotone peptone10 g
Yeast extract 2 g
Sodium bisulfite 0.1 g
Distilled water 1 liter

Dispense 90 ml into screw-cap bottle. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

M80. Levine's Eosin-Methylene Blue (L-EMB) Agar
Peptone 10 g
Lactose 10 g
K₂HPO₄ 2 g
Agar 15 g
Eosin Y 0.4 g
Methylene blue 0.065 g
Distilled water 1 liter

Boil to dissolve peptone, phosphate, and agar in 1 liter of water. Add water to make original volume. Dispense in 100 or 200 ml portions and autoclave 15 min at not over 121°C. Final pH, 7.1 ± 0.2. Before use, melt, and to each 100 ml portion add (a) 5 ml sterile 20% lactose solution; (b) 2 ml aqueous 2% eosin Y solution; and (c) 4.3 ml 0.15% aqueous methylene blue solution. When using complete dehydrated product, boil to dissolve all ingredients in 1 liter water. Dispense in 100 or 200 ml portions and autoclave 15 min at 121°C. Final pH, 7.1 ± 0.2.

M81. Lithium Chloride-Phenylethanol-Moxalactam (LPM) Medium
Phenylethanol agar (Difco) 35.5 g
Glycine anhydride (NOTE: not glycine)10 g
Lithium chloride 5 g
Moxalactam stock solution, 1% in phosphate buffer, pH 6.0 2 ml
Distilled water 1 liter

Sterilize medium (without moxalactam) at 121°C for 15 min. Cool to 48-50°C and add filter-sterilized moxalactam solution.

Moxalactam stock solution consists of 1 g moxalactam salt (ammonium or sodium) in 100 ml 0.1 M potassium phosphate buffer, pH 6.0. Store filter-sterilized stock solution frozen in 2 ml aliquots.

Moxalactam (Eli Lilly Co.) is retailed by Sigma Chemical Co. The LPM medium is most effective in the Henry illumination system when poured thin, i.e., 12-15 ml per standard petri dish. To avoid drying of thin agar, refrigerate and use plates rapidly. LPM basal medium is commercially available as a powder.

M82. LPM Plus Esculin and Ferric Iron
Esculin 1.0 g
Ferric ammonium citrate 0.5 g

Add these components to those for LPM (M81). Sterilize medium, temper, and add filter-sterilized moxalactam, as described for LPM medium.

M83. Liver-Veal Agar
Liver, infusion from 50 g
Casein, isoelectric 2 g
Veal, infusion from 500 g
NaCl 5 g
Proteose peptone 20 g
Sodium nitrate 2 g
Neopeptone 1.3 g
Gelatin 20 g
Tryptone 1.3 g
Agar 15 g
Dextrose 5 g
Distilled water 1 liter
Starch, soluble 10 g

Heat with agitation to dissolve. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2.

M84. Liver-Veal-Egg Yolk Agar
Fresh eggs, yolks only 2 or 3
Liver veal agar 48.5 g
Distilled water 500 ml

Heat with agitation to dissolve. Autoclave 15 min at 121°C. Cool to 50°C.

Egg yolk emulsion. To each 500 ml of melted liver veal agar, add 40 ml egg yolk-saline suspension (see M51 for instructions). Mix thoroughly and pour into sterile 15 x 100 mm petri dishes. Dry plates at room temperature for 2 days or at 35°C for 24 h. Check plates for sterility and store sterile plates in refrigerator. In certain instances the medium may be used without addition of egg yolk emulsion.

Rl2a. Chlorine Solution, 200 ppm,
Containing Q.1% Sodium Dodecyl Sulfate
Commercial bleach (5.25% sodium hypochlorite) 8 ml
Distilled water containing 1 g sodium dodecyl sulfate 992 ml

Dissolve 1 g sodium dodecyl sulfate in 992 ml distilled water. Add 8 ml commercial bleach and mix well. Make immediately before use.

R13. 0.05 M Citric Acid (PH 4.0)
Citric acid (monohydrate)10.5 g
Double distilled water to make l liter

Dissolve citric acid in 900 ml distilled water. Adjust pH to 4.0 with 6 M NaOH and dilute to 1 liter. Store in refrigerator.

Rl4. Clark's Flagellar Stain
Solution A
Basic fuchsin, special 1.2 g
95% ethanol 100 ml

Mix and let stand overnight at room temperature.

Solution B
Tannic acid 3.0 g
NaCl 1.5 g
Distilled water 200 ml

Mix solutions A and B. Adjust pH to 5.0 with 1 N NaOH or 1 N HCl, if necessary. Refrigerate 2-3 days before use. Stain is stable l month at 4°C or may be stored frozen indefinitely (50 ml portions). To use, thaw stain, remix, and store at 4°C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4°C), stain a known flagellated organism on 3 or more cleaned slides for various times (e.g., 5, 10, 15 min). Mark best staining time on all containers.

IMPORTANT: Stain will not work unless slides are clean. To clean slides, soak them 4 days at room temperature in cleaning solution (either acid dichromate or 3% concentrated HCl in 95% ethanol). Rinse 10 times in fresh tap water and twice in distilled water. Air-dry at room temperature. Store in covered container.

Staining Procedure

To prepare suspension, pick small amount of growth from 18-24 h plate (equivalent to 1 mm colony). Do not pick up agar. Suspend gently in 3 ml distilled water. (Flagella can be knocked off.) Suspension should be faintly opalescent.

To prepare slide, pass cleaned slide through blue part of burner flame several times to remove residual dirt. Cool slide, flamed side up, on paper towel. Mark wax line across slide to give area 2.5 x 4.5 cm. Place large loopful of suspension in center of slide adjacent to wax line. Tilt slide, letting drop run down center of slide to end. If drop does not run evenly, slide is dirty. Discard it. Air-dry slide on level surface.

R15. Coating Solution for V. vulnificus EIA
Phosphate-buffered saline 100 ml
Triton X-100 (a polyoxyethylene ether)20 µl

Mix Triton X-100 with PBS, pH 7.4.

R16. Crystal Violet Stain (for Bacteria)

1. Crystal violet in dilute alcohol
Crystal violet (90% dye content) 2 g
Ethanol (95%) 20 ml
Distilled water 80 ml

2. Ammonium oxalate crystal violet (Hucker's (See R32)

Either solution is generally considered suitable as a simple stain to observe morphology.

R17. 50X Denhardt's Solution
Ficoll (Av. MW 400,000) 2 g
Polyvinyl pyrrolidone (Av. MW 360,000) 2 g
Bovine serum albumin 2 g

Add distilled water to make 200 ml. Store at -20°C in 10 ml aliquots.

R18. Disinfectants
(for preparation of canned foods for microbiological analysis)

2. Sodium hypochlorite solution
*Sodium hypochlorite 5.0-5.25 g
Distilled water 100 ml

*Laundry bleach, which is 5.25% sodium hypochlorite (NaOCl), may be used.

R19. Dulbecco's Phosphate-Buffered Saline (DPBS)
NaCl 8.0 g
KCl 200 mg
Na₂HPO₄ 1.15 g
KH₂PO₄ 200 mg
CaCl₂ 100 mg
MgCl₂·6H₂O 100 mg
Distilled water 1 liter

Dissolve ingredients in water. Sterilize by filtration. Final pH, 7.2.

R20. 0.5 M EDTA
Na₂EDTA 186.12 g
Dissolve in 800-900 ml dH₂O . Adjust pH to 8.0 with 10 N NaOH. Add distilled water to make 1 liter.

R21. EIA (V. vulnificus) Wash Solution
NaCl 87.65 g
Tween 205.0 ml

Dissolve ingredients in 10 liters of deionized water.

R22. ELISA Buffer (for Cholera Toxin Test)
Bovine serum albumin 1.0 g
NaCl 8.0 g
KH₂PO₄ 0.2 g
Na₂HPO₄2.9 g
KCl 0.2 g
Distilled water 1 liter
Tween 20 0.5 ml

Adjust pH to 7.4 and add Tween 20. Store frozen. Thaw before use.

R23. Ethanol Solution, 70%
Ethanol, 95% 700 ml
Distilled wateradd to final volume of 950 ml

R24. Evans Blue Dye Solution
(commercially available)

Evans Blue dye 2 g
NaCl 0.5 g
Distilled water to make 100 ml (final volume)

CAUTION: Evans Blue dye is a suspected carcinogen.

R25. Ferric Chloride. 10%
FeCl₃ 10 g
Distilled water 90 ml

R26. Formalinized Phosphate-Buffered Saline for V. vulnificus flaqellar seroloqy)
No longer used in Bacteriological Analytical Manual

R27. Formalinized Physiological Saline Solution
Formaldehyde solution (36-38%) 6 ml
NaCl 8.5 ml
Distilled water 1 liter

Dissolve 8.5 g NaCl in 1 liter distilled water. Autoclave 15 min at 121°C. Cool to room temperature. Add 6 ml formaldehyde solution. Do not autoclave after addition of formaldehyde.

R28. Gel Diffusion Agar. 1.2%
NaCl 8.5 g
Sodium barbital 8.0 g
Merthiolate (crystalline) 0.1 g
Noble special agar (Difco) 12.0 g
Distilled water 1 liter

Dissolve NaCl, sodium barbital, and merthiolate in 900 ml distilled water. Adjust pH to 7.4 with 1 N HCl and/or 1 N NaOH. Bring volume to l liter. Add Noble agar. Melt agar mixture in Arnold steamer. Filter in steamer, while hot, through 2 layers of analytical grade filter paper (e.g., No. 588, Schleicher and Schuell or equivalent). Dispense in small (15-25 ml) portions into 4 oz prescription bottles. Do not remelt more than twice.

R29. Gel-Phosphate Buffer
Gelatin 2 g
Na₂HPO₄ 4 g
Distilled water 1 liter

Use gentle heat to dissolve ingredients. Sterilize 20 min at 121°C. Final pH, 6.2.

R30. Giemsa Stain
Giemsa stain (Matheson Coleman & Bell, Norwood, OH 45212) 1 g
Glycerol 66 ml
Methanol (absolute) 66 ml
Distilled stain in glycerol by heating 1.5-2.0 h at 55-60°C. Add methanol. Store stain in tightly stoppered bottle at 22°C for at least 2 weeks. Dilute stock solution with distilled water (1+9) before use.

R31. Glycerin-Salt Solution (Buffered)
Glycerin (reagent grade) 100 ml
K₂HPO₄ (anhydrous) 12.4 g
KH₂PO₄ (anhydrous) 4 g
NaCl 4.2 g
Distilled water 900 ml

Distilled NaCl and bring volume to 900 ml with water. Add glycerin and phosphates. Adjust pH to 7.2. Autoclave 15 min at 121°C. For double strength (20%) glycerin solution, use 200 ml glycerin and 800 ml distilled water.

R32. Gram Stain
(commercial staining solutions are satisfactory)

Hucker's crystal violet
Solution A
Crystal violet (90% dye content) 2 g
Ethanol, 95% 20 ml
Solution B
Ammonium oxalate 0.8 g
Distilled water 80 ml
Mix solutions A and B. Store 24 h and filter through coarse filter paper.

Gram's iodine
Iodine 1 g
Potassium iodide (KI) 2 g
Distilled water 300 ml
Place KI in mortar, add iodine, and grind with pestle for 5-10 s. Add 1 ml water and grind; then add 5 ml of water and grind, then 10 ml and grind. Pour this solution into reagent bottle. Rinse mortar and pestle with amount of water needed to bring total volume to 300 ml.

Hucker's counterstain (stock solution)
Safranin O (certified) 2.5
Ethanol, 95% 100 ml
Working solution: Add 10 ml stock solution to 90 ml distilled water.

Staining Procedure

(Gram stain)Fix air-dried films of food sample in moderate heat. Stain films 1 min with crystal violet-ammonium oxalate solution. Wash briefly in tap water and drain. Apply Gram's iodine for 1 min. Wash in tap water and drain. Decolorize with 95% ethanol until blue color is no longer released (about 30 s). Alternatively, flood slides with ethanol, pour off immediately, and reflood with ethanol for 10 s. Wash briefly with water, drain, and apply Hucker's counterstain (safranin solution) for 10-30 s. Wash briefly with water, drain, blot or air-dry, and examine.

R32a. Endospore Stain (Schaeffer-Fulton)Solution AMalachite green10 g Distilled water 100 mlFilter to remove undissolved dye.Solution BSafranin O0.25 g Distilled water20 ml

R33. Hippurate Solution, 1%

Dissolve 0.1 g sodium hippurate in 10 ml distilled water. Filter-sterilize and store refrigerated or in 0.4 ml aliquots at -20°C. Commercial preparations are also available.

R34. Horseradish Peroxidase
(color development solution)

Solution A (horseradish)
HRP color development reagent 60 mg
Ice cold methanol 20 ml

Mix to dissolve. Protect from light and prepare fresh.

Solution B
Ice cold hydrogen peroxide, 30% 60 µl
Tris-buffered saline 100 ml

Prepare fresh before use. Mix ice cold solution A with room temperature solution B. Use immediately.

R35. Hybridization Mixture
(6X SSC, 5X Denhardt's, 1 mM EDTA, pH 8.0)

20X SSC 15.0 ml
50X Denhardt's solution 5.0 ml
0.5 M EDTA 0.1 ml
Distilled water 28.9 ml

For hybridization with 10X Denhardt's, add 10 ml 50X Denhardt's, and decrease water volume to 23.9 ml.

R36. 1 N Hydrochloric Acid
HCl (concentrated) 89 ml
Distilled water to make 1 liter

R37. 10X Kinase Buffer
2.0 M Tris, pH 7.6 2.5 ml
1.0 M MgCl₂ 1.0 ml
0.5 M Dithiothreitol 1.0 ml
10 mM Spermidine 1.0 ml
0.5 M EDTA, pH 8.0 20 µl
Distilled water 4.5 ml
Store at 4°C

R38. Kovacs' Reagent
p-Dimethylaminobenzaldehyde 5 g
Amyl alcohol (normal only) 75 ml
HCl (concentrated) 25 ml

Dissolve p-dimethylaminobenzaldehyde in normal amyl alcohol. Slowly add HCl. Store at 4°C. To test for indole, add 0.2-0.3 ml reagent to 5 ml of 24 h bacteria culture in tryptone broth. Dark red color in surface layer is positive test for indole.

R39. 0.1 N Lithium Hydroxide
Lithium hydroxide (anhydrous) 2.395 g
Distilled water 1 liter

R40. Lugol's Iodine Solution
Potassium iodide (KI)10 g
Iodine 5 g
Distilled water 100 ml

Dissolve KI in about 20-30 ml of distilled water. Add iodine and heat gently with constant mixing until iodine is dissolved. Dilute to 100 ml with distilled water. Store in amber glass-stoppered bottle in the dark.

R41. May-Grunwald Stain
May-Grunwald stain (Matheson Coleman & Bell, Norwood, OH 45212) 2.5 g
Methanol (absolute) 1 liter

Weigh stain into 50 ml methanol, dissolve by grinding, and dilute to 1 liter with methanol. Stir 16 h at 37°C. Hold stain 1 month at 22°C (room temperature). Filter before use.

R42. McFarland Nephelometer
Make suspensions of barium sulfate as follows:
1. Prepare 1.0% solution of CP (chemically pure) sulfuric acid.
2. Prepare 1.0% solution of CP barium chloride.
3. Prepare 10 standards as follows:

4. Seal about 3 ml of each standard suspension of barium sulfate precipitate in small test tube. Select test tubes carefully for uniformity of wall thickness, diameter, and color. (These preparations are available commercially from several laboratory supply firms.)

R43. Mercuric Chloride Solution, 0.1%
No longer used. See R12a, Chlorine Solution, 200 ppm.

R44. Methyl Red Indicator
Methyl red 0.10 g
Ethanol, 95% 300 ml
Distilled water to make 500 ml

Dissolve methyl red in 300 ml ethanol. Bring volume to 500 ml with distilled water.

R45. Methylene Blue Stain (Loeffler's)
Solution A
Methylene blue (90% dye content) 0.3 g
Ethanol (95%) 30 ml

Solution B
Diluted potassium hydroxide (0.01%) 100 ml

Mix solutions A and B.

R46. Mineral Oil
Autoclave 30 min at 121°C. Use screw-cap containers, about 1/2 full with 20-50 ml.

R47. Ninhydrin Reagent (commercially available)
Dissolve 3.5 g ninhydrin in 100 ml of 1:1 mixture of acetone and butanol. Store refrigerated.

R48. Nitrite Detection Reagents

To prepare 5 N acetic acid, add 28.75 ml glacial acetic acid to 71.25 ml distilled water. Store reagents in glass-stoppered brown bottles. To perform test, add 0.1-0.5 ml each of reagent A and either reagent B or reagent C (as specified in method) to culture grown in liquid or semisolid medium. Development of red-violet color with reagents A and B or orange color with reagents A and C indicates that nitrate has been reduced to nitrite. Since color produced with reagents A and B may fade or disappear within a few minutes, record reaction as soon as color appears. If no color develops, test for presence of nitrate by adding small amount of zinc dust. If color develops, nitrate has not been reduced.

Nitrate reduction test for enteropathogenic E. coli. To 3 ml of 18-24 h culture in indole-nitrite medium, add 2 drops each of reagents A and B.Red-violet color indicates that nitrate has been reduced to nitrite. Check negative tests by adding small amount of zinc dust; if red-violet color does not appear, nitrate has been reduced.

CAUTION: The alpha-naphthylamine reagent recommended in previous editions of the Bacteriological Analytical Manual should not be used because of the possible hazard to laboratory personnel. Any supplies of alpha-naphthylamine on hand should be inventoried and secured by supervisory personnel because of the strict rules governing the use of carcinogenic substances in laboratories of the U.S. Department of Health and Human Services.

R49. North Aniline (Oil)-Methylene Blue Stain
Aniline oil 3 ml
Ethanol (95%) 10 ml
HCl (concentrated) 1.5 ml
Methylene blue (saturated solution) 30 ml
Distilled water to make 100 ml

Mix aniline oil with ethanol. Slowly add HCl with constant agitation. Add saturated methylene blue solution, dilute to 100 ml with water, and filter.

R50. Novobiocin Solution (100 mg/ml)
Novobiocin (sodium salt)100 mg
Deionized water l.0 ml

Dissolve novobiocin. Filter-sterilize, using 0.2 µm filter and syringe. May be stored for several months in dark bottle at 4°C.

R51. O/129 Disks
(2,4-Diamino-6,7-diisopropyl pteridine)

2,4-Diamino-6,7-diisopropyl pteridine (O/129) or phosphate salt
Distilled water, sterilized
Filter paper disk blanks, 5-6 mm or 1/4 inch, sterilized
Micropipettor, calibrated to deliver 10 µl

Mark disks to differentiate 10 µg from 150 µg disks. Sterilize filter paper disks in glass petri plates. Dissolve O/129 or O/129-PO₄ (the phosphate salt dissolves more easily) in sterile water. For 150 g disks, place 10 µl of 15 mg/ml O/129 or 20.8 mg/ml O/129-PO₄ on each disk. For 10 µg disks, make l:l5 dilutions of the O/129 solutions and place 10 µl of 1.O mg/ml O/129 or 1.4 mg/ml O/129-PO₄ on each disk. Dry disks and store desiccated and protected from light in refrigerator. O/129 disks are commercially available.

R52. O157 Monoclonal Antibody Solution
Dispense 10 µl ascitic fluid into 1 ml 1% gelatin-Tris-buffered saline (TBS). Add 3 µl horseradish peroxidase-protein A conjugate, and stir mixture at 4°C for 1 h. Then dilute to 10 ml with 1% gelatin-TBS. Sufficient for 1 hydrophobic grid membrane filter.

R53. ONPG Test

Monosodium phosphate solution. 1.0 M, pH 7
NaH₂PO₄·H₂0 6.9 g
Distilled water 45 ml
NaOH solution, 30% (w/v) 3 ml

Dissolve NaH₂PO₄·H₂O in distilled water. Add 30% NaOH solution and adjust to pH 7. Bring volume to 50 ml with distilled water and store in refrigerator (about 4°C).

0.0133 M o-Nitrophenyl-beta-D-galactoside (ONPG)
ONPG 80 mg
Distilled water, 37°C 15 ml
Monosodium phosphate solution, 1.0 M, pH 7 5 ml

Dissolve ONPG in distilled water at 37°C. Add 1.0 M NaH₂PO₄solution. Solution should be colorless. Store in refrigerator (about 4°C). Before use, warm appropriate portion (sufficient for number of tests) of ONPG solution to 37°C.

Procedure
Inoculate cultures to be tested onto triple sugar iron agar slants and incubate for 18 h at 37°C (or other appropriate temperature, if required). Nutrient (or other) agar slants containing 1.0% lactose may also be used. In sterile 13 x 100 mm tube containing 0.25 ml of physiological saline, emulsify large loopful of culture growth to form heavy suspension. Add 1 drop of toluene to each tube and shake well to liberate enzyme. Let tubes stand 5 min at 37°C. Add 0.25 ml buffered 0.0133 M ONPG solution to each suspension to be tested. Incubate tubes at 35-37°C. Examine tubes at intervals up to 24 h. Yellow color is positive result.ONPG disks are available commercially. Emulsify growth from 18 h lactose-containing medium in 0.2 ml sterile physiological saline in sterile 13 x 100 mm tube. Add disk and agitate gently. Incubate and read tubes as above.

R54. Oxidase Reagent
N,N,N',N'-Tetramethyl-p-phenylenediamine·2HCl 1 g
Distilled water 100 ml

This is the preferred reagent. Use freshly prepared. However, reagent can be used up to 7 days if stored in a dark glass bottle under refrigeration.Apply freshly prepared solution directly to young culture (24 h) on either agar plate or slant. Oxidase-positive colonies develop a pink color and progressively turn dark purple. If cultures are to be preserved, complete the transfer from plates to which reagent has been added within 3 min, since reagent is toxic to organisms.

Optional method: Transfer small amount of culture to reagent-impregnated filter paper. Dark purple color within 10 s indicates positive test. Use platinum wire or sterile wooden stick (toothpicks or applicator sticks), since wire containing iron (e.g., nichrome wire in ordinary inoculating needles and loops) gives false-positive reactions.

Alternatively, the test may be performed with a 1% solution of N,N-dimethyl-p-phenylenediamine hydrochloride. Apply solution directly to culture plate or slant.

R55. Penicillinase (ß-Lactamase)

Commercially available from Difco Laboratories, Box 1058A, Detroit, MI 48232; BBL, Division of Bioquest, P.O. Box 234, Cockeysville, MD 21030; ICN Nutritional Biochemicals, 26201 Miles Road, Cleveland, OH 44128; SchwartzMann, Orangeburg, NY 10962; and Calbiochem, 10933 N. Torrey Pines Road, La Jolla, CA 93037. Obtain Penicillin G reference standard from U.S.P. reference standards, 12601 Twin Brook Parkway, Rockville, MD 20852.

R56. Peptone Diluent, 0.1%
Peptone 1 g
Distilled water 1 liter

Autoclave 15 min at 121°C. Final pH, 7.0 + 0.2.

R57. Peroxidase Substrate for Membrane ELISA
Solution A
4-Chloro-1-naphthol 60 mg
Methanol (cold)20 ml

Dissolve 4-chloro-1-naphthol in cold methanol. Store at -20°C.

Solution B
Hydrogen peroxide, 30% 60 µl
Tris-buffered saline (TBS) 100 ml

Add 30% H₂O₂ to TBS at room temperature. Immediately before use, mix ice cold Solution A with room temperature ySolution B.

R58. Peroxidase Substrate Solution (ABTS)
(2,2"-Azino-di-[3-ethyl benzthiazoline sulfonate (6)]
(ABTS) substrate)

ABTS 10 mg
Citric acid (0.05 M) 10 ml
Hydrogen peroxide, 30% 30 µl

Prepare 5 min before use. One recipe is sufficient for one 96-well microwell plate.

R59. Phosphate-Buffered Saline (PBS), pH 7.4
NaCl 7.650 g
Na₂HPO₄, anhydrous 0.724 g
KH₂PO₄ 0.210 g
Distilled water 1 liter

Dissolve ingredients in distilled water. Adjust pH to 7.4 (with 1 N NaOH). Autoclave 15 min at 121°C. This buffer may be used for any step in the Vibrio spp. methods when PBS is needed.

R60. O.01 M Phosphate-Buffered Saline (pH 7.5)Stock solution (0.1 M)
Na₂HPO₄ (anhydrous) 12.0 g
NaH₂PO₄·H₂0 2.2 g
NaCl 85.0 g
Distilled water 1 liter

Dissolve ingredients in distilled water and bring volume to 1 liter. Dilute stock solution 1+9 in double distilled water. Mix well. Adjust pH to 7.5 with 0.1 N HCl or 0.1 N NaOH if necessary. Commercially available in dehydrated form from Difco Laboratories and BBL.

R61. 0.02 M Phosphate Saline Buffer (PH 7.3-7.4)
Prepare stock solutions of 0.2 M mono- and disodium phosphate in 8.5% salt solutions and dilute 1:10 for preparation of 0.02 M phosphate saline buffer.

Stock solution 1
Sodium phosphate dibasic anhydrous Na₂HPO₄ (anhydrous) (reagent grade) 28.4 g
NaCl (reagent grade) 85.0 g
Distilled water to make 1 liter

Stock solution 2
Sodium phosphate monobasic monohydrate NaH₂PO₄ H₂O (monohydrate) (reagent grade)4* 227.6 g
NaCl (reagent grade) 85.0 g
Distilled water to make 1 liter

To obtain 0.02 M phosphate-buffered saline (0.85%), make 1:10 dilutions of each stock solution. For example:

Stock solution 1 50 ml	Stock solution 2 10 ml
Distilled water 450 ml	Distilled water 90 ml
Approximate pH, 8.2	Approximate pH, 5.6

Using pH meter, titer diluted solution 1 to pH 7.3-7.4 by adding about 65 ml of diluted solution 2. Use resulting 0.02 M phosphate saline buffer solution in the lysostaphin susceptibility test on S. aureus.

NOTE: Do not titer 0.2 M phosphate buffer to pH 7.3-7.4 and then dilute to 0.02 M strength. This results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2.

R62. Phosphate Saline Solution (for Y-l LT Assay)
Na₂HPO₄ 1.07 g
NaH₂PO₄ 0.24 g
NaCl 8.9 g
Distilled water 1 liter

Dissolve ingredients in water. Adjust pH to 7.5.

R63. Physiological Saline Solution 0.85% (Sterile)
NaCl 8.5 g
Distilled water 1 liter

Dissolve 8.5 g NaCl in water. Autoclave 15 min at 121°C. Cool to room temperature.

R64. Polymyxin B Disks, 50 Units
Polymyxin B sulfate
Distilled water, sterilized
Filter paper disk blanks, 5-6 mm or 1/4 inch, sterilized
Micropipettor, to deliver 10 µl

Sterilize filter paper disks in glass petri plates. Dissolve polymyxin B in sterile water to yield a final concentration of 5000 units/ml. Place 10 µl of the 5000 units/ml solution on each disk. Dry the disks. Store desiccated and protected from light in refrigerator.

Example: Polymyxin B is sold by activity in USP units per mg. Check reagent bottle for activity of the lot in use. For polymyxin B with antibiotic activity of 8090 USP units per mg, dissolve 0.618 mg in 1 ml sterile water.

Calculation:

R65. Potassium Hydroxide Solution. 40%
KOH 40 g
Distilled water to make 100 ml

R66. Saline Solution. 0.5% (Sterile)
NaCl 5 g
Distilled water 1 liter

Dissolve NaCl in water. Autoclave at 121°C for 15 min.

R67. Salts-Phosphate Buffered Saline Solution (Salts-PBS)
NaCl 121 g
KCl 15.5 g
MgCl₂ 12.7 g
CaCl₂·2H₂O 10.2 g
NaH₂PO₄·H₂0 2.0 g
Na₂HPO₄·7H₂0 3.9 g
Distilled water 1 liter

Adjust pH to 7.4.

R68. Scintillation Fluid
2,5 Diphenyloxazole 5.0 g
Toluene 1 liter

R69. Slide Preserving Solution

Prepare 1% acetic acid solution (10 ml glacial acetic acid, reagent grade + 990 ml distilled water). Add 1 ml glycerin to each 100 ml of solution.

R70. Sodium Bicarbonate Solution. 10%
Sodium bicarbonate 100 g
Distilled water to make 1 liter

Sterilize by filtration.

R72. 0.2 M Sodium Chloride Solution
NaCl 11.7 g
Distilled water to make 1 liter

Dispense in suitable containers. Autoclave 15 min at 121°C.

R73. 1 N Sodium Hydroxide Solution
NaOH 40 g
Distilled water to make 1 liter

Use for adjusting pH of culture media.

R74. 10 N Sodium Hydroxide Solution
NaOH 400 g
Distilled water to make 1 liter

R75. Sonicated Calf-Thymus or Salmon-Sperm DNA
DNA 1 g
Distilled water 1 liter

Stir 3-4 h to dissolve. May be heated to 60°C. Sonicate until MW is about 300,000 - 500,000 daltons. Store frozen in 1 ml aliquots.

R76. Spicer-Edwards EN Complex Antibody Solution

Add 0.1 ml Spicer-Edwards EN Complex (Difco) to 0.07 ml horseradish peroxidase-protein A conjugate in 1 ml 1% gelatin-Tris-buffered saline (TBS), and stir at 4°C for 1 h. Dilute to 40 ml 1% gelatin-TBS. Use within a few hours. Sufficient for 4 hydrophobic grid membrane filters.

R77. Standard Saline Citrate (SSC) Solution (20%)
NaCl (reagent grade) 175.3 g
Sodium citrate 88.2 g

Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter.

6X SSC
NaCl (reagent grade) 52.6 g
Sodium citrate 26.5 g

Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter.

3X SSC
6X SSC 500 ml
Deionized water 500 ml

2X SSC
6X SSC 333 ml
Deionized water 667 ml

R78. Tergitol Anionic 7
 
 This reagent is a sodium sulfate derivative of 3,9-diethyl tridecanol-6. Its recommended use is for wetting and emulsifying when the electrolyte is below 1% in textiles, emulsion polymers, rubber lattices, leather, and pharmaceuticals. Tergitol-7 is an anionic wetting agent manufactured by Union Carbide Corp., Chemicals and Plastics, 270 Park Avenue, New York, NY 10017.

R79. Thiazine Red R Stain
(for S. aureus enterotoxin gel diffusion technique)
Dissolve Thiazine Red R (Color Index No. 14780), 0.1%, in 1.0% acetic acid. Thiazine Red R is available from Matheson Coleman & Bell, Norwood, OH 45212.

R80. 1.0 M Tris (pH 8.0)
Tris 121.14 g
Distilled water to make 1 liter

Dissolve in several hundred ml distilled water. Adjust pH to 8.0 with concentrated HCl. Bring volume to 1 liter.

R81. Tris-Buffered Saline (TBS) (PH 7.5)
Tris 2.42 g
NaCl 29.24 g
Double distilled water to make 1 liter

Dissolve ingredients. Adjust pH to 7.5 with HCl and bring volume to 1 liter.

R82. Tris-Buffered Saline (TBS), with Gelatin

1% solution
Gelatin 1 g
TBS, pH 7.5 100 ml

3% solution
Gelatin 3 g
TBS, pH 7.5 100 ml

Add gelatin to TBS at 40°C. Stir to dissolve. Cool to room temperature before use.

R83. Tris-Buffered Saline (TBS)-Tween
Tween 2050 F1TBS, pH 7.5 100 ml

Dissolve Tween 20 in TBS.

R84. Tris-Buffered Saline (TABS), 1% or 3% Gelatin, or Tween 20

Tris 2.42 g
NaCl 29.24 g
Distilled water 1 liter

Dissolve ingredients in distilled water by heating and stirring. Adjust pH to 7.5 with HC1. Autoclave 15 min at 121°C.

For 1% and 3% Gelatin-TABS, add 10 g and 30 g gelatin, respectively, to ingredients before autoclaving. Adjust final pH to 7.5 with HCl.

For Tween-TABS, add 0.5 ml Tween 20 to ingredient and adjust pH to 7.5 before autoclaving.

R85. Tris-EDTA-Triton X-100^TM(TET) Buffer
(Triton Lytic Mix)
Triton X-100^TM 1.0 ml
1.0 M Tris (R80)5.0 ml
0.5 M Na₂EDTA (R20) 12.5 ml
Distilled water to make 100 ml

R86. Triton X-100

This reagent is the registered trademark for octylphenoxy polyethoxy ethanol. Its recommended uses include wetting agent, detergent, dispersant, emulsifier in household and industrial cleaners, textile processing, wool scouring, and emulsifying agent for insecticides and herbicides. Triton X-100 is a nonionic preparation manufactured by Rohm and Haas Company, Independence Mall West, Philadelphia, PA 19105. It is also sold in small quantities by Fisher Scientific Co., 711 Forbes Avenue, Pittsburgh, PA 15219.

R87. Trypsin-EDTA Solution, lX
Trypsin (1:250)* 0.05 g
EDTA, disodium salt 0.02 g
NaCl, 0.9% 100 ml

Dissolve trypsin and EDTA in the NaCl diluent. Filter-sterilize through 0.22 µm membrane.

R88. Verocytotoxin Antiserum

Grow E. coli 0157:H7 in trypticase soy broth at 37°C for 18 h; add 0.5% formalin and keep at 37°C for 2 weeks. Inoculate formalized culture into rabbits through ear vein. Give a total of 5 inoculations (0.5, 1.0, 2.0, 4.0 and 4.0 ml), one each, at 5-day intervals. Remove blood after 6 weeks and obtain serum. Heat serum at 56°C for 30 min and adsorb antiserum 6 times with about 10¹² cells of heat-treated (121°C, 1 h) E. coli 0157:H7 (1 ml packed cells to 20 ml antiserum). Use antibody solution at 1:5000 dilution.

R89. Voges-Proskauer (VP) Test Reagents
Solution 1
alpha-Naphthol 5 g
Alcohol (absolute)100 ml

Voges-Proskauer (VP) test. Transfer 1 ml of 48 h culture to test tube and add 0.6 ml solution 1 and 0.2 ml solution. Shake after adding each solution. To intensify and speed reaction, add a few creatine crystals to mixture. Let stand at room temperature. Read results 4 h after adding reagents. Development of eosin pink color is positive test.