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 121C. 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 (100C) 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 121C. 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 121C. 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 SO47H2O 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 121C. 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 (CoCl26H2O) 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 121C. 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 121C. 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 121C. 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 121C.

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 121C.

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 121C. Cool to 50C.

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

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 4C.

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 121C. 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 (48-50C) 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 35C 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 121C.

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 m 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 121C. 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 121C. 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 121C. 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 chloride6H2O 5 g
Agar 20 g

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

Enrichment. Bacto EY tellurite enrichment.

Complete medium. Aseptically add 5 ml prewarmed (45-50C) 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 20-25C 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 121C, 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 45-50C. 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 121C. Cool to 50C. 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 121C. Cool to 45-50C. 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 121C. 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 121C 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 48C. 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 121C.

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 121C. 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 121C.

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 121C. 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 121C. Final pH, 7.0 0.2. Sterilize stock solutions of carbohydrates (50% w/v) separately by autoclaving or, preferably, by filtration (0.2 m 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 121C. 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 121C (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 m, using a syringe filter if # 25 ml. Store solution 5 days at 4C, 14 days at -20C, and 5 months at -70C. 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 4C. 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 50C (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 4C. 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 4C 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 121C. 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 42C 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 -20C. 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 m syringe filter. Freeze unused portions in 5 ml amounts at -70C 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 121C. 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 m) 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 121C, 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 50C 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 121C.

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 121C.

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 m 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 SO47H2O (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 -20C up to 4 weeks.

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

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 -70C. 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 80C (10 min in 50C water bath). Add solution B (Irgasan) and mix well. Cool to 50-55C. 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 g streptomycin, and 50 g gentamicin per ml)

Mix on magnetic stirrer, filter through 0.20 m membrane, and dispense into sterile 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5C. 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 118C 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 121C.

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 121C. 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 121C. Cool to 50-55C. 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 121C 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 121C. Final pH, 7.2 0.2C. 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 121C.

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 121C. 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
Na2HPO47H2O 10 g
Raffinose 4 g
Distilled water 1 liter

Dissolve ingredients and sterilize by autoclaving for 15 min at 121C. 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-AsparagineH2O 150.0 mg
Choline chloride 1.0 mg
L-Aspartic acid 13.3 mg
Folic acid 1.0 mg
L-Cystine2HCl 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 HClH2O 42.0 mg
Thiamine HCl 1.0 mg
L-Isoleucine 52.0 mg
Glucose 1000.0 mg
L-Leucine 52.0 mg
CaCl22H2O 265.0 mg
L-Lysine HCl 72.5 mg
KCl 400.0 mg
L-Methionine 15.0 mg
Mg SO47H2O 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
NaH2PO4H2O 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
CaCl22H2O 265 mg
MgSO47H2O 200 mg
NaH2PO4H2O 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 121C. 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 121C. Final pH, 6.9 0.2.

M50. EC-MUG Medium
Prepare as for EC broth (M49), but add 50 mg 4-methylumbelliferyl--D-glucuronide (MUG) per liter before autoclaving (15 min, 121C). 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 4C 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 121C 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 30C. 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 55C. Adjust pH to 7.0 0.2. Dispense 4 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 121C. 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 121C. Cool to 45-50C. 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 121C. Cool to 45-50C. 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 g
Distilled water 100 ml

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

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-Cysteine2HCl 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 HClH2O 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
CaCl22H2O 44.10 mg
D-Phenylalanine 4.96 mg
CuSO45H2O 0.0025 mg
L-Proline 11.50 mg
FeSO47H2O 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 SO47H2O 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
NaH2PO4H2O 290.0 mg
Hypoxanthine 4.08 mg
ZnSO47H2O 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 121C 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 48C. 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 121C. 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 121C. 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 4C. 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 121C.

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 121C. 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 121C. 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 118C. Final pH, 7.2 0.2.

M67. Irgasan-Ticarcillin-Chlorate (ITC) Broth
Tryptone 10 g
Yeast extract 1 g
MgCl26H2O 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 121C 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 118C. 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 121C. 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 121C. Cool to 50C.

Carbohydrates, 10%. Dissolve 10 g quantities of carbohydrates in 100 ml distilled water. Sterilize by filtration through 0.22 m 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 121C. 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
NaNH4HPO4H2O 1.5 g
KH2PO4 (monobasic) 1 g
Mg SO4 7H2O 0.2 g
Sodium citrate2H2O 3 g
Distilled water 1 liter

Dispense into screw-cap tubes as desired. Autoclave 15 min at 121C. 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-AsparagineH2O 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 m membrane and dispense into 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5C.

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 121C 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 50-60C 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 121C. If not used within 8 h, deaerate by heating at 50-70C 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 121C. 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--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 121C. 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 121C. 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 121C. 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 121C. 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 121C. 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 121C for 15 min. Cool to 48-50C 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 121C. 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 121C. Cool to 50C.

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 35C 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.

M85. Long-term Preservation Medium
Yeast extract, 0.3% 3 g
Peptone 10 g
NaCl 30 g
Agar 3 g
Distilled water 1 liter

Heat to dissolve ingredients. Dispense 4 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121C. Cool and tighten caps for storage. No pH adjustment is necessary.

M86. Lysine Arginine Iron Agar (LAIA)
Peptone 5 g
Yeast extract 3 g
Glucose 1 g
L-Lysine 10 g
L-Arginine 10 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.04 g
Bromcresol purple 0.02 g
Agar 15 g
Adjust pH to 6.8.

Heat to boiling and dispense 5 ml into each 13 x 100 mm screw-cap culture tube. Autoclave at 121C for 12 min. Cool tubes in slanted position. (This medium may also be prepared by supplementing Difco lysine iron agar (LIA) with 10 g L-arginine per liter.)

M87. Lysine Decarboxylase Broth (Falkow)
(for Salmonella)
Gelysate or peptone 5 g
Yeast extract 3 g
Glucose 1 g
L-Lysine 5 g
Bromcresol purple 0.02 g
Distilled water 1 liter

Heat until dissolved. Dispense 5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave loosely capped tubes 15 min at 121C. Screw the caps on tightly for storage and after inoculation. Final pH, 6.8 0.2. For halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M88. Lysine Decarboxylase (LDC) Medium
(for Gram-negative nonfermentative bacteria)
L-Lysine HCl 0.5 g
Dextrose 0.5 g
KH2PO4 0.5 g
Distilled water 100 ml

Dissolve ingredients. Adjust pH to 4.6 0.2. Autoclave 15 min at 121C. Aseptically dispense 1 ml portions to sterile 13 x 100 mm tubes.

M89. Lysine Iron Agar (Edwards and Fife)
Gelysate or peptone 5 g
Yeast extract 3 g
Glucose 1 g
L-Lysine hydrochloride 10 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate (anhydrous) 0.04 g
Bromcresol purple 0.02 g
Agar 15 g
Distilled water 1 liter

Heat to dissolve ingredients. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 12 min at 121C. Let solidify in slanted position to form 4 cm butts and 2.5 cm slants. Final pH, 6.7 0.2.

M90. Lysozyme Broth
Base. Prepare nutrient broth as recommended. Dispense 99 ml portions to 170 ml bottles. Autoclave 15 min at 121C. Cool to room temperature before use.
Lysozyme solution. Dissolve 0.1 g lysozyme in 65 ml sterile 0.0l N HCl. Heat to boiling for 20 min. Dilute to 100 ml with sterile 0.0l N HCl. Alternatively, dissolve 0.1 g lysozyme in 100 ml distilled water. Sterilize by filtration through 0.45 m membrane. Test for sterility before use. Add 1 ml lysozyme solution to 99 ml nutrient broth. Mix and dispense 2.5 ml portions to sterile 13 x 100 mm tubes.

M91. MacConkey Agar
Proteose peptone or polypeptone 3 g
Peptone or gelysate17 g
Lactose 10 g
Bile salts No. 3 or bile salts mixture 1.5 g
NaCl 5 g
Neutral red 0.03 g
Crystal violet 0.001 g
Agar 13.5 g
Distilled water 1 liter

Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121C, cool to 45-50C, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed. DO NOT USE WET PLATES. Final pH, 7.1 0.2.

M92. Malonate Broth
Yeast extract 1 g
(NH4)2SO4 2 g
K2HPO4 0.6 g
KH2PO4 0.4 g
NaCl 2 g
Sodium malonate 3 g
Glucose 0.25 g
Bromthymol blue 0.025 g
Distilled water 1 liter

Dissolve by heating, if necessary. Dispense 3 ml portions into 13 x 100 mm test tubes. Autoclave 15 min at 121C. Final pH, 6.7 0.2.

M93. Malt Extract Agar
(Cosmetics-General Microbiology)
Malt extract 30 g
Agar 20 g
Distilled water 1 liter

Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color.Autoclave 15 min at 121C. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 0.2.

For cosmetics, cool medium to 47-50C after autoclaving. Dispense 4 ml stock filter-sterilized chlortetracycline HCl solution (1 g/100 ml) per liter of medium to yield final concentration of 40 ppm chlorotetracycline HCl. Mix thoroughly and dispense 20 ml portions into 15 x 100 mm petri dishes.

M94. Malt Extract Broth (Difco)
Malt extract base 6.0 g
Maltose, technical 1.8 g
Dextrose 6.0 g
Yeast extract 1.2 g
Final pH, 4.7 0.2.

M95. Mannitol-Egg Yolk-Polymyxin (MYP) Agar
Base

Beef extract 1 g
Peptone 10 g
Mannitol 10 g
NaCl 10 g
Phenol red (1% solution in 95% ethanol) 2.5 ml
Agar 15 g
Distilled water 900 ml

Heat with agitation to dissolve agar. Adjust pH so that value after sterilization is 7.2 0.2. Dispense 225 ml portions to 500 ml Erlenmeyer flasks. Autoclave 15 min at 121C. Cool to 50C. MYP agar is commercially available from Difco.

Polymyxin B solution, 0.1%. Dissolve 500,000 units polymyxin B sulfate in 50 ml distilled water. Filter-sterilized and store in the dark at 4C until needed.

Egg yolk emulsion, 50% (see M51). Also available from commercial suppliers.Final medium. To 225 ml melted base add 2.5 ml polymyxin B solution and 12.5 ml egg yolk emulsion. Mix and dispense 18 ml portions to sterile 15 x 100 mm petri dishes. Dry plates at room temperature for 24 h before use.

M96. Mannitol Maltose Agar
Phytone 5 g
Polypeptone 5 g
Beef Extract 5 g
D-Mannitol 10 g
Maltose 10 g
NaCl 20 g
Agar 13 g
1000X Dye stock solution* 1 ml
Distilled water 1 liter

Suspend ingredients and boil to dissolve. Adjust to pH 7.8 0.2 Autoclave 15 min at 121C.

*1000X Dye stock solution. See formulation under modified cellobiose-polymyxin B-colistin (mCPC) agar (M98).

M97. Mannitol Salt Agar
Beef extract 1 g
Polypeptone 10 g
NaCl 75 g
Mannitol 10 g
Phenol red 0.025 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve agar and boil 1 min. Dispense 20 ml portions into 15 x 100 mm petri dishes.Autoclave 15 min at 121C. Final pH, 7.4 0.2.

M98. Modified Cellobiose-Polymyxin B-Colistin (mCPC) Agar
Solution 1

Peptone 10 g
Beef extract 5 g
NaCl 20 g
1000X Dye stock solution* 1 ml
Agar 15 g
Distilled water 900 ml

Adjust to pH 7.6. Boil to dissolve agar. Cool to 48-55C.

*1000X Dye stock solution
 Bromthymol blue 4.0 g
Cresol red 4.0 g
Ethanol, 95%100 ml

For consistent medium color, use dye solution rather than repeatedly weighing out dry dyes. Dissolve dyes in ethanol for 4% (w/v) stock solution. Using 1 ml of this solution per liter of mCPC agar gives 40 mg bromthymol blue and 40 mg cresol red per liter.

Solution 2
Cellobiose 10 g
Colistin 400,000 units
Polymyxin B 100,000 units
Distilled water 100 ml

Dissolve cellobiose in distilled water by heating gently. Cool. Add antibiotics. Add Solution 2 to cooled Solution1, mix, and dispense into petri dishes. Final color, dark green to green-brown.

NOTE: This medium, like TCBS, is very inhibitory and does not require autoclaving. Medium may be stored 2weeks at refrigeration temperatures.

M99. Motility-Indole-Ornithine (MIO) Medium
Yeast extract 3 g
Peptone 10 g
Tryptone 10 g
L-Ornithine HCl 5 g
Dextrose 1 g
Agar 2 g
Bromcresol purple 0.02 g
Distilled water 1 liter

Dispense 4 ml portions into 13 x 100 mm tubes. Autoclave 15 min at 121C. Final pH, 6.5 0.2.

M100. Motility Medium (for B. cereus)
Trypticase 10 g
Yeast extract 2.5 g
Dextrose 5 g
Na2HPO4 2.5 g
Agar 3 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Dispense 100 ml portions to 170 ml bottles. Autoclave 15 min at 121C. Final pH, 7.4 0.2. Cool to 50C. Aseptically dispense 2 ml portions to sterile 13 x 100 mm tubes. Store at room temperature 2 days before use.

M101. Motility Nitrate Medium (for Cosmetics)
(for Gram-negative nonfermentative bacteria)
Tryptose 10 g
Heart infusion agar (Difco) 8 g
Potassium (or sodium) nitrate1 g
Glucose 0.5 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121C. Available as prepared medium from BBL and Scott. NOTE: Make sure nitrates are nitrite-free.

M102. Motility-Nitrate Medium, Buffered (for C. perfringens)
Beef extract 3 g
Peptone (Difco) 5 g
KNO3 1 g
Na2HPO3 2.5 g
Agar 3 g
Galactose 5 g
Glycerin (reagent grade) 5 ml
Distilled water 1 liter

Dissolve all ingredients except agar. Adjust pH to 7.3 0.1. Add agar and heat to dissolve. Dispense 11 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121C. If not used within 4 h, heat 10 min in boiling water or flowing steam. Chill in cold water.

M103. Motility Test Medium (Semisolid)
Beef extract 3 g
Peptone or gelysate10 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 screw-cap tubes.Autoclave 15 min at 121C. Final pH, 7.4 0.2.

For Salmonella: Dispense 20 ml portions into 20 x 150 mm screw-cap tubes, replacing caps loosely. Autoclave 15min at 121C. Cool to 45C after autoclaving. Tighten caps, and refrigerate at 5-8C. To use, remelt in boiling water or flowing steam, and cool to 45C. Aseptically dispense 20 ml portions into sterile 15 x 100 mm petri plates. Cover plates and let solidify. Use same day as prepared. Final pH, 7.4 0.2.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M104. MR-VP Broth
Medium 1

Buffered peptone-water powder (Difco or BBL) 7 g
Glucose 5 g
K2HP04 5 g
Distilled water 1 liter

Medium 2
Pancreatic digest of casein 3.5 g
Peptic digest of animal tissue 3.5 g
Dextrose 5.0 g
Potassium phosphate 5.0 g
Distilled water 1 liter

Dissolve ingredients in water with gentle heat if necessary. Dispense 10 ml into 16 x 150 mm test tubes andautoclave 15 min at 118-121C. Final pH, 6.9 0.2.

Medium 3
Peptone 5.0 g
Glucose 5.0 g
Phosphate buffer 5.0 g
Distilled water 1 liter

Dissolve ingredients in water. Dispense 10 ml into 16 x 150 mm test tubes and autoclave 15 min at 121C. Final pH, 7.5 0.2.

For Salmonella: Dispense 10 ml into 16 x 150 mm test tubes and autoclave 12-15 min at 121C.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M105. Mucate Broth
Peptone 10 g
Mucic acid 10 g
Bromthymol blue0.024 g
Distilled water 1 liter

Dissolve peptone. Dissolve mucic acid by slowly adding 5 N NaOH and stirring. Dispense 5 ml portions into 13 x100 mm screw-cap tubes. Autoclave 10 min at 121C. Final pH, 7.4 0.1.

M106. Mucate Control Broth
Peptone 10 g
Bromthymol blue 0.024 g
Distilled water 1 liter

Dissolve ingredients. Dispense 5 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 10 min at 121C. Final pH, 7.4 0.1.

M107. Mueller-Hinton Agar
Beef, infusion from 300 g
Acidicase peptone (BBL) or casamino acids (Difco) 17.5 g
Starch 1.5 g
Agar 17 g
Distilled water 1 liter

Heat to boiling for 1 min. Autoclave 15 min at 116C. Final pH, 7.3 0.2.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M108. Nitrate Broth
Beef extract 3 g
Peptone 5 g KNO3 (nitrite-free) 1 g
Distilled water 1 liter

Dissolve ingredients. Dispense 5 ml portions into 16 x 125 mm tubes. Autoclave 15 min at 121C. Final pH, 7.0 0.2.

M109. Nitrate Broth, Enriched (CDC)
Infusion broth 25 g
KNO3 (nitrite-free) 2 g
Distilled water 1 liter

Dispense 4 ml portions into 13 x 100 mm tubes with inverted Durham tubes. Autoclave 15 min at 121C. Final pH, 7.3 0.2.

M110. Nitrate Reduction Medium and Reagents
Culture medium. Prepare nitrate broth (M108) from nutrient broth (M114) containing 1.0 g/L potassium nitrate.

Reagent A. Dissolve 0.5 g alpha-naphthylamine (a carcinogen) in 100 ml 5 Nacetic acid by gently heating. Prepare 5 N acetic acid by adding distilled water to 28.7 ml glacial acetic acid (17.4 N)to give final volume of 100 ml.

Reagent B. Dissolve 0.8 g sulfanilic acid in 100 ml 5 N acetic acid by gently heating.

Reagent C. Dissolve 1 g alpha-naphthol in 200 ml acetic acid.

Zinc powder.Cadmium reagent. Place zinc rods in 20% solution of cadmium sulfate for several hours. Draw off precipitated cadmium and add to 1 N HCl.

M111. Nonfat Dry Milk (Reconstituted)
Nonfat dry milk100 g
Distilled water 1 liter

For Salmonella: Suspend 100 g dehydrated nonfat dry milk in 1 liter distilled water. Swirl until dissolved.Autoclave 15 min at 121C.

For monkey kidney cell culture. Dispense 500 ml into 1 liter Erlenmeyer flasks.

M112. Nutrient Agar
Beef extract 3 g
Peptone 5 g
Agar 15 g
Distilled water 1 liter

Heat to boiling to dissolve ingredients. Dispense into tubes or flasks. Autoclave 15 min at 121C. Final pH, 6.8 0.2. If used as base for blood agar, add 8 g NaCl to prevent hemolysis of blood cells.

M113. Nutrient Agar (for B. cereus)
For slants, prepare nutrient agar and dispense 6.5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave 15 min at 121C. Slant tubes until medium solidifies. For plates, dispense 100-500 ml portions into bottles or flasks andautoclave. Cool to 50C and dispense 18-20 ml into sterile 15 x 100 mm petri dishes. Dry plates for 24-48 h at room temperature before use.

M114. Nutrient Broth
Beef extract 3 g
Peptone 5 g
Distilled water 1 liter

Heat to dissolve. Dispense 10 m1 portions into tubes or 225 ml portions into 500 ml Erlenmeyer flasks. Autoclave15 min at 121C. Final pH, 6.8 0.2.

M115. Nutrient Gelatin (CDC)
(for Gram-negative nonfermentative bacteria)

Infusion broth 25 g
Gelatin 120 g
Distilled water 1 liter

Heat with agitation to dissolve. Cool to 55C and adjust pH to 7.4 0.2. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121C.

M116. OF Glucose Medium, Semisolid
Tryptone (trypticase) 2 g
NaCl 5 g
Dipotassium phosphate 0.3 g
Bromthymol blue dye 0.03 g
Agar 2 g
Glucose 10 g
Distilled water 1 liter

Boil ingredients to dissolve. For halophilic Vibrio spp., add 15 g NaCl (2% NaCl, final concentration) to medium.Dispense 5 ml into 13 x 100 mm tubes and autoclave 15 min at 121C. Final pH, 6.8 0.2.

To use OF medium with sugars other than glucose, prepare medium without glucose in 900 ml water, sterilize as above, and cool to 45-50C. Add 100 ml of 10% solution of filter-sterilized sugar to basal medium. Aseptically dispense 5 ml into sterile 13 x 100 mm tubes.

For Campylobacter, prepare half the tubes with glucose and half without. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M117. Oxidative-Fermentative (OF) Test Medium
Base

Peptone 2 g
NaCl 5 g
K2HPO4 0.3 g
Bromthymol blue 0.03 g
Agar 2.5 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Dispense 3 ml portions into 13 x 100 mm tubes. Autoclave 15 min at 121C.Cool to 50C; pH, 7.1.

Carbohydrate stock solution. Dissolve 10 g carbohydrate in 90 ml distilled water. Sterilize by filtration through 0.22 m membrane. Add 0.3 ml stock solution to 2.7 ml base in tube. Mix gently and cool at room temperature.Inoculate tubes in duplicate. Layer one tube with sterile mineral oil. Incubate 48 h at 35C.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M118. Oxford Medium
Columbia blood agar base39.0 g
Esculin 1.0 g
Ferric ammonium citrate 0.5 g
Lithium chloride15.0 g
Cycloheximide 0.4 g
Colistin sulfate 0.02 g
Acriflavin 0.005 g
Cefotetan 0.002 g
Fosfomycin 0.010 g
Distilled water 1 liter

Add the 55.5 g of the first 4 components (basal medium) to 1 L distilled water. Bring gently to boil to dissolve completely. Sterilize by autoclaving at 121C for 15 min. Cool to 50C and aseptically add supplement, mix, and pour into sterile petri dishes. To prepare supplement, dissolve cycloheximide, colistin sulfate, acriflavin, cefotetan,and fosfomycin in 10 ml of 1:1 mixture of ethanol and distilled water. Filter-sterilize supplement before use. Oxford basal medium and supplement mixture are available commercially.

M118a. PALCAM Listeria Selective Agar
NOTE
: Equivalent basal medium powders and lyophilized supplement mixtures are available commercially; follow the manufacturer’s instructions for preparing the medium. If prepared medium is not available, a representative formulation and preparation instructions are listed below.

Basal medium
Peptone 11.5 g
Starch 0.5 g
NaCl 2.5 g
Columbia agar 6.5 g
Mannitol 5 g
Ferric ammonium citrate 0.25 g
Esculin (aesculin) 0.4 g
Dextrose (glucose) 0.25 g
Lithium chloride 7.5 g
Phenol red 0.04 g
TOTAL Dry Ingredients 34.44
Distilled Water 500 ml

Selective agents
Polymyxin B sulfate10 mg
Acriflavin 5 mg
Ceftazidine 20 mg

To make 500 ml of medium, weigh 34.4 g basal medium powder (all ingredients except the three selective agents) and suspend in 500 ml distilled water. Sterilize by autoclaving at 121C for 15 min. Dissolve the selective agent supplement mixture in sterile distilled water at a rate of 17.5 mg/ml and filter sterilize. Add 1 ml selective agent supplement solution to 500 ml sterile basal medium which has been cooled to 50C. Mix gently and pour plates.Final pH, 7.2 0.1.

M119. Penicillin-Streptomycin Solution
(antibiotic concentrate)

Penicillin G 500,000 IU
Streptomycin 500,000 g
Distilled water 100 ml
Dissolve antibiotics in water and sterilize by filtration. Store at 5C.

For V. cholerae, use commercially available penicillin G-streptomycin sulfate solution. Add 5 ml solution per 500 ml medium. Store at -20C.

M120. Peptone Sorbitol Bile Broth
Na2HPO4 8.23 g
NaH2PO4H2O 1.2 g
Bile salts No. 3 1.5 g
NaCl 5 g
Sorbitol 10 g
Peptone 5 g
Distilled water 1 liter

Dispense 100 ml into Wheaton bottles. Autoclave 15 min at 121C. Final pH, 7.6 0.2.

M121. Phenol Red Carbohydrate Broth
Trypticase or proteone peptone No. 3 10 g
NaCl 5 g
Beef extract (optional) 1 g
Phenol red (7.2 ml of 0.25% phenol red solution) 0.018 g
Distilled water 1 liter
Carbohydrate*

*Dissolve either 5 g dulcitol, 10 g lactose, or 10 g sucrose (as specified in the Salmonella test) in this basal broth.
 Dispense 2.5 ml portions into 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. Autoclave10 min at 118C. Final pH, 7.4 0.2. Alternatively, dissolve ingredients, omitting carbohydrate, in 800 ml distilled water with heat and occasional agitation. Dispense 2.0 ml portions into 13 x 100 mm test tubes containing inverted fermentation tubes. Autoclave 15 min at 118C and let cool. Dissolve carbohydrate in 200 ml distilled water and sterilize by passing solution through bacteria-retaining filter. Aseptically add 0.5 ml sterile filtrate to each tube of sterilized broth after cooling to less than 45C. Shake gently to mix. Final pH, 7.4 0.2.

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

M122. Phenol Red Glucose Broth
Proteose peptone No. 3 10 g
NaCl 5 g
Beef extract (optional) 1 g
Dextrose 5 g
Phenol red (7.2 ml of 0.25% solution) 0.018 g
Distilled water 1 liter

Dispense 2.5 ml portions into 13 x 100 mm tubes. Autoclave 10 min at 118C. Final pH, 7.4 0.2.

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

M123. Phenylalanine Deaminase Agar
Yeast extract 3 g
L-Phenylalanine or1 g
DL-Phenylalanine 2 g
Na2HPO4 1 g
NaCl 5 g
Agar 12 g
Distilled water 1 liter

Heat gently to dissolve agar. Tube and autoclave 10 min at 121C. Incline tubes to obtain long slant. Final pH, 7.3 0.2.

M124. Plate Count Agar (Standard Methods)
Tryptone 5 g
Yeast extract 2.5 g
Dextrose 1 g
Agar 15 g
Distilled water 1 liter

Heat to dissolve ingredients. Dispense into suitable tubes or flasks. Autoclave 15 min at 121C. Final pH, 7.0 0.2.

For viable yeasts and molds, dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes.

M125. PMP Broth
Na2HPO4 7.9 g
NaH2PO4 1.1 g
Peptone 2.5 g
D-Mannitol 2.5 g
Distilled water 1 liter

Adjust pH to 7.6. Autoclave at 121C for 15 min.

M126. Potassium Cyanide (KCN) Broth
Potassium cyanide 0.5 g
Proteose peptone No. 3 or polypeptone 3 g
NaCl 5 g
KH2PO4  0.225 g
Na2HPO4 5.64 g
Distilled water 1 liter

Dissolve above ingredients (except potassium cyanide) and autoclave 15 min at 121C. Cool and refrigerate at5-8C. Final pH, 7.6 0.2. Prepare KCN stock solution by dissolving 0.5 g KCN in 100 ml sterile distilled water cooled to 5-8C. Using bulb pipetter, add 15 ml cold KCN stock solution to 1 liter cold, sterile base. DO NOT PIPETTE BY MOUTH. Mix and aseptically dispense 1.0-1.5 ml portions to 13 x 100 mm sterile tubes. Using aseptic technique, stopper tubes with No. 2 corks impregnated with paraffin. Prepare corks by boiling in paraffin about 5min. Place corks in tubes so that paraffin does not flow into broth but forms a seal between rim of tubes and cork.Store tubes at 5-8C no longer than 2 weeks before use.

M127. Potato Dextrose Agar
Potato infusion 200 g
Dextrose 20 g
Agar 20 g
Distilled water 1 liter

To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min. Filter through cheese cloth, saving effluent, which is potato infusion (or use commercial dehydrated form). Mix in other ingredients and boil to dissolve. Autoclave 15 min at 121C. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes.Final pH, 5.6 0.2. Medium should not be re-melted more than once. Commercially-prepared medium powder is available, but extra agar may be required to obtain a final concentration of 20 g/L. To BBL or Difco dehydrated medium, add 5 g of agar.

For potato dextrose salt agar, prepare potato dextrose agar, as above, and add 75 g NaCl per liter.

For cosmetics, cool medium to 47-50C after autoclaving. Add 40 ppm (final concentration) chlortetracycline. Mix thoroughly and dispense 20 ml portions into 15 x 100 mm petri dishes. Dispense 4 ml of stock filter-sterilized chlortetracycline HCl (1 g/100 ml) per liter of medium.

M128. Pseudomonas Agar F (for fluorescein production)
Use Difco product, if available.
Proteose peptone No. 3 (Difco) 20.0 g
Tryptone (Difco) 10.0 g
Dipotassium phosphate 1.5 g
Magnesium sulfate 0.73 g
Glycerol (Difco) 10.0 g
Agar 15.0 g
Distilled water 1 liter

Add medium powder and glycerol to water; mix. Heat to boiling to dissolve ingredients. Autoclave at 121C for 15min. Final pH, 7.0.

M129. Pseudomonas Agar P (for pyocyanine production)
Peptone (Difco) 20.0 g
Potassium sulfate 10.0 g
Magnesium chloride 1.4 g
Glycerol (Difco) 10.0 g
Agar 15.0 g
Distilled or deionized water 1 liter

Prepare as described for Pseudomonas Agar F (M128, above).

M130. Purple Carbohydrate Broth
Proteose peptone No. 3 10 g
Beef extract 1 g
NaCl 5 g
Bromcresol purple 0.02 g
Distilled water 1 liter

Prepare as for phenol red carbohydrate broth (M121). Final pH, 6.8 0.2.

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

M130a. Purple Carbohydrate Fermentation Broth Base
Purple broth base (Becton-Dickinson) 15 g
Distilled water 900 ml

Add purple broth base to distilled water. Dispense 9 ml into 16 x 125 mm tubes containing Durham tubes. Autoclave at 121C for 15 min. Prepare carbohydrates, except esculin, as sterile 5% solutions. Filter-sterilize or autoclave, as appropriate. Add 1 ml carbohydrate solution to 9 ml broth base to yield 0.5% carbohydrate in broth.Add esculin directly into base broth to make a 0.5% solution and autoclave 15 min at 115C. At room temperature a 5% solution of esculin is a gel that cannot be pipetted.

M131. Pyrazinamidase Agar*
Tryptic soy agar (M152) 30 g
Yeast extract 3 g
Pyrazine-carboxamide 1 g
0.2 M Tris-maleate, pH 6.0 1 liter

Heat to boiling; dispense 5 ml in 16 x 125 mm tubes. Autoclave at 121C for 15 min. Cool slanted.

*See FDA BAM Chapter 8, Yersinia, ref. 26.

M132. Rappaport-Vassiliadis Medium Broth base
Tryptone 5 g
NaCl 8 g
KH2PO4 1.6 g
Distilled water 1 liter

Magnesium chloride solution
MgCl26H2O 400 g
Distilled water 1 liter

Malachite green oxalate solution
Malachite green oxalate 0.4 g
Distilled water 100 ml

To prepare the complete medium, combine 1000 ml broth base, 100 ml magnesium chloride solution, and 10 ml malachite green oxalate solution (total volume of complete medium is 1110 ml). Broth base must be prepared on same day that components are combined to make complete medium. Magnesium chloride solution may be stored in dark bottle at room temperature up to 1 year. To prepare solution, dissolve entire contents of MgCl26H2O from newly opened container according to formula, because this salt is very hygroscopic. Malachite green oxalate solution may be stored in dark bottle at room temperature up to 6 months. Merck analytically pure malachite green oxalate is recommended because other brands may not be equally effective. Dispense 10 ml volumes of complete medium into 16 x 150 mm test tubes. Autoclave 15 min at 115C. Final pH, 5.5 0.2. Store in refrigerator and use within 1 month.

This medium must be made from its individual ingredients. Use of commercially available dehydrated media is not recommended. Users of this medium should be aware that there are formulations and incubation temperatures, other than those recommended in this manual, for this medium.

M133. Sabouraud's Dextrose Broth and Agar
Polypeptone or neopeptone 10 g
Dextrose 40 g
Distilled water 1 liter

Dissolve completely and dispense 40 ml portions into screw-cap bottles. Final pH, 5.8. Autoclave 15 min at 118-121C. Do not exceed 121C.

For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel strength desired. Final pH, 5.6 0.2. Dispense into tubes for slants and bottles or flasks for pouring plates. Autoclave 15 min at 118-121C.

M134. Selenite Cystine Broth
Medium 1 (modification of Leifson's formulation for selenite broth)

Tryptone or polypeptone 5 g
Lactose 4 g
Sodium acid selenite (NaHSeO3) 4 g
Na2HPO4 10 g
L-Cystine 0.01 g
Distilled water 1 liter

Heat to boiling to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Final pH, 7.0 0.2. The medium is not sterile. Use same day as prepared.

Medium 2 (North-Bartram modification)
Polypeptone 5 g
Lactose 4 g
Sodium acid selenite (NaHSeO3) 4 g
Na2HPO4 5.5 g
KH2PO4 4.5 g
L-Cystine 0.01 g
Distilled water 1 liter

Heat with agitation to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Use same day as prepared.

M135. Sheep Blood Agar
Blood agar base (Oxoid No. 2) 95 ml
Sterile sheep blood, defibrinated 5 ml

Rehydrate and sterilize base as recommended by manufacturer. Agar and blood should both be at 45-46C before blood is added and plates are poured. Commercial pre-poured sheep blood agar plates may be used.

M136. Shigella Broth
Base

Tryptone 20 g
K2HPO4 2 g
KH2PO4 2 g
NaCl 5 g
Glucose 1 g
Tween 80 1.5 ml
Distilled water 1 liter

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

Novobiocin solution. Weigh 50 mg novobiocin into 1 liter distilled water. Sterilize by filtration through 0.45 m membrane. Add 2.5 ml concentrate to 225 ml base.

M137. SIM Motility Medium
Pancreatic digest of casein (casitone) 20.0 g
Peptic digest of animal tissue (beef extract) 6.1 g
Ferrous ammonium sulfate 0.2 g
Sodium thiosulfate 0.2 g
Agar 3.5 g

Rehydrate and add 6 ml medium per 16 x 125 mm screw-cap tube. Sterilize according to manufacturer's instructions. Final pH, 7.3 0.2. Medium is available from BBL. DO NOT use Difco SIM.

M138. Simmons Citrate Agar
Sodium citrate*2 g
NaCl 5 g
K2HPO4 1 g
NH4H2PO4 1 g
MgSO4 0.2 g
Bromthymol blue 0.08 g
Agar 15 g
Distilled water 1 liter

*Difco does not specify waters of hydration.Heat gently with occasional agitation. Boil 1-2 min until agar dissolves. Fill 13 x 100 or 16 x 150 mm screw-cap tubes 1/3 full. Autoclave 15 min at 121C. Before medium solidifies, incline tubes to obtain 4-5 cm slants and 2-3 cm butts. Final pH, 6.8 0.2.

M139. Sorbitol-MacConkey Agar
Peptone or gelysate 17.0 g
Protease peptone No. 3 or polypeptone 3.0 g
Sorbitol 10.0 g
Bile salts, purified 1.5 g
NaCl 5.0 g
Agar 13.5 g
Neutral red 0.03 g
Crystal violet 0.001 g
Distilled water 1 liter

Dissolve ingredients in distilled water by heating with stirring. Autoclave 15 min at 121C. Final pH, 7.1 0.2.

M140. Sporulation Broth (for C. perfringens)
Polypeptone 15 g
Yeast extract 3 g
Starch, soluble 3 g
MgSO4 (anhydrous) 0.1 g
Sodium thioglycollate 1 g
Na2HPO4 11 g
Distilled water 1 liter

Adjust pH to 7.8 0.1. Dispense 15 ml portions into 20 x 150 mm screw-cap tubes. Autoclave 15 min at 121C.

M141. Spray's Fermentation Medium (for C. perfringens)
Tryptone 10 g
Neopeptone 10 g
Agar 2 g
Sodium thioglycollate 0.25 g

Dissolve all ingredients except agar and adjust pH to 7.4 0.2. Add agar and heat with agitation to dissolve. Dispense 9 ml portions into 16 x 125 mm tubes. Autoclave 15 min at 121C. Before use, heat in boiling water or flowing steam for 10 min. Add 1 ml of sterile 10% carbohydrate solution to 9 ml base.

M142. Staphylococcus Agar No. 110
Gelatin 30 g
Trypticase peptone 10 g
Yeast extract 2.5 g
Lactose 2 g
Mannitol 10 g
NaCl 75 g
K2HPO4 5 g
Agar 15 g
Distilled water 1 liter

M143. Starch Agar
Nutrient agar 23 g
Potato starch 10 g
Distilled water 1 liter

Heat to dissolve agar in 500 ml water. Dissolve starch in 250 ml water. Combine and dilute to 1 liter. Autoclave 15 min at 121C. NOTE: add 3 g agar to Difco's dehydrated starch agar.

M144. T1N1 Medium
Trypticase 10 g
NaCl 10 g
Agar (if solid medium is preferred) 20 g
Distilled water 1 liter

Heat if necessary to dissolve ingredients. Dispense into 16 x 125 mm screw-cap tubes (if tubed medium is required). Autoclave 15 min at 121C. Slant tubes until cool or let medium cool to 50C and pour into 15 x 100 mm petri dishes. Final pH, 7.1 0.2.

M145. Tetrathionate BrothTetrathionate broth base
Polypeptone 5 g
Bile salts 1 g
Calcium carbonate 10 g
Sodium thiosulfate·5H2O 30 g
Distilled water 1 liter

Suspend ingredients in 1 liter distilled water, mix, and heat to boiling. DO NOT AUTOCLAVE. (Precipitate will not dissolve completely.) Cool to less than 45C. Store at 5-8C. Final pH, 8.4 0.2.

Iodine-Potassium Iodide (I -KI) solution
Potassium iodide 5 g
Iodine, resublimed 6 g
Distilled water, sterile 20 ml

Dissolve potassium iodide in 5 ml sterile distilled water. Add iodine and stir to dissolve. Dilute to 20 ml.

Brilliant green solution
Brilliant green dye, sterile 0.1 g
Distilled water, sterile 100 m1

On day of use, add 20 ml I -KI solution and 10 ml brilliant green solution to 1 liter base. Resuspend precipitate by gentle agitation and aseptically dispense 10 ml portions into 20 x 150 or 16 x 150 mm sterile test tubes. Do not heat medium after addition of I -KI and dye solutions.2

M146. Thioglycollate Medium (Fluid) (FTG)
L-Cystine 0.5 g
Agar (granulated) 0.75 g
NaCl 2.5 g
Dextrose 5 g
Yeast extract 5 g
Tryptone 15 g
Sodium thioglycollate or thioglycollic acid 0.5 g
Resazurin, sodium solution (1:1000), fresh 1 ml
Distilled water 1 liter

Mix L-cystine, NaCl, dextrose, yeast extract, and tryptone with 1 liter water. Heat in Arnold steamer or water bath until ingredients are dissolved. Dissolve sodium thioglycollate or thioglycollic acid in solution and adjust pH so that value after sterilization is 7.1 0.2. Add sodium resazurin solution, mix, and autoclave 20 min at 121C. If commercial media are used, dispense 10 ml portions to 16 x 150 mm tubes and autoclave 15 min at 121C.

M147. Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar
Yeast extract 5 g
NaCl 10 g
Peptone 10 g
Ferric citrate 1 g
Sucrose 20 g
Bromthymol blue O.04 g
Sodium thiosulfate·5H2O 10 g
Thymol blue 0.04 g
Sodium citrate·2H2O 10 g
Agar 15 g
Sodium cholate 3 g
Distilled water 1 liter
Oxgall 5 g

Prepare in flask at least 3 times larger than required volume of medium. Add ingredients to warm distilled water and heat to dissolve. Bring just to boil, and immediately remove from heat. DO NOT AUTOCLAVE. Cool to 50C and pour into petri dishes. Dry the plates overnight or at 37-45C before use.

M148. Toluidine Blue-DNA Agar
Deoxyribonucleic acid (DNA) 0.3 g
Agar 10 g
CaCl2 (anhydrous) 1.1 mg
NaCl 10 g
Toluidine blue O 0.083 g
Tris (hydroxymethyl) aminomethane 6.1 g
Distilled water 1 liter

Dissolve Tris (hydroxymethyl) aminomethane in 1 liter distilled water. Adjust pH to 9.0. Add the remaining ingredients except toluidine blue O and heat to boiling to dissolve. Dissolve toluidine blue O in medium. Dispense to rubber-stoppered flasks. Sterilization is not necessary if used immediately. The sterile medium is stable at room temperature for 4 months and is satisfactory after several melting cycles.

M149. Triple Sugar Iron Agar (TSI)
Medium 1Medium 2
Polypeptone 20 gBeef extract 3 g
NaCl 5 g Yeast extract 3 g
Lactose 10 g Peptone 15 g
Sucrose 10 gProteose peptone 5 g
Glucose 1 g Glucose 1 g
Fe(NH4)2(SO4)26H2O 0.2 g Lactose 10 g
Na2S2O3 0.2 g Sucrose 10 g
Phenol red 0.025 g FeSO4 0.2 g
Agar 13 g NaCl 5 g
Distilled water 1 liter Na2S2O3 0.3 g
Phenol red 0.024 g
Agar 12 g
Distilled water 1 liter

These two media are interchangeable for general use.Suspend ingredients of Medium 1 in distilled water, 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 Medium 1 for 15 min at 118C. Prepare Medium 2 in the same manner as Medium 1, except autoclave 15 min at 121C. Before the media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt. Final pH, 7.3 0.2 for Medium 1 and 7.4 0.2 for Medium 2.

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

M150. Trypticase Novobiocin (TN) Broth
Trypticase soy broth 30 g
Bile salts No. 3 1.5 g
Dipotassium phosphate 1.5 g
Novobiocin 20 mg
Distilled water 1 liter

Dissolve all ingredients except novobiocin by heating and stirring; autoclave at 121C for 15 min. Prepare stock solution of novobiocin by adding 20 mg monosodium novobiocin per ml of distilled water. Filter-sterilize. Make fresh stock each time of use, or store frozen at -10C in the dark (compound is light-sensitive) for not more than 1 month (half-life is several months at 4C). Add 1 ml stock solution per liter of medium.

M151. Trypticase-Peptone-Glucose-Yeast Extract Broth (TPGY)
Trypticase 50 g
Peptone 5 g
Yeast extract 20 g
Dextrose 4 g
Sodium thioglycollate 1 g
Distilled waterl liter

Dissolve solid ingredients and dispense 15 ml in 20 x 150 mm tubes. Autoclave tubes 10 min at 121°C. Final pH, 7.0 0.2. Refrigerate at 5°C.

M151a. Trypticase-Peptone-Glucose-Yeast Extract Broth with Trypsin (TPGYT)Base
Trypticase 50 g
Peptone 5 g
Yeast extract 20 g
Dextrose 4 g
Sodium thioglycollate 1 g
Distilled water 1 liter

Dissolve solid ingredients of base and dispense 15 ml in 20 x 150 mm tubes or 100 ml in 170 ml prescription bottles. Autoclave tubes 10 min at 121C and bottles 15 min at 121C. Final pH, 7.0 0.2. Refrigerate at 5C. Add trypsin immediately before use.

Trypsin solution
Trypsin (1:250) 1.5 g
Distilled water 100 ml

Stir trypsin in water to suspend. Let particles settle and filter-sterilize supernatant through 0.45 m membrane. Before use, steam or boil base for 10 min to expel dissolved oxygen. Add 1 ml trypsin to each 15 ml of broth or 6.7 ml trypsin to 100 ml of broth.

M152. Trypticase (Tryptic) Soy Agar
Trypticase peptone 15 g
Phytone peptone 5 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Boil 1 min. Dispense into suitable tubes or flasks. Autoclave 15 min at 121C. Final pH, 7.3 0.2. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M153. Trypticase Soy Agar with 0.6% Yeast Extract (TSAYE)
Trypticase soy agar 40 g
Yeast extract 6 g
Distilled water 1 liter

Weigh ingredients, add water, mix, and autoclave 15 min at 121C. Final pH, 7.3 0.2. After autoclaving, swirl to disperse molten agar.

M154. Trypticase (Tryptic) Soy Broth
Trypticase peptone 17 g
Phytone peptone 3 g
NaCl 5 g
K2HPO4 2.5 g
Glucose 2.5 g
Distilled water 1 liter

Heat with gentle agitation to dissolve. Dispense 225 ml into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121C. Final pH, 7.3 0.2. For trypticase soy broth without dextrose, prepare as above, but omit 2.5 g dextrose. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

M154a. Trypticase (Tryptic) Soy Broth with 10% NaCl and 1% Sodium Pyruvate
Trypticase or tryptose (pancreatic digest of casein) 17 g
Phytone (papaic digest of soya meal)3 g
NaCl 100 g
K2HPO4 2.5 g
Dextrose 2.5 g
Sodium pyruvate 10 g
Distilled water 1 liter

Dehydrated trypticase or tryptic soy broth is satisfactory with 95 g NaCl and 10 g sodium pyruvate added per liter. Adjust to pH 7.3. Heat gently if necessary. Dispense 10 ml into 16 x 150 mm tubes. Autoclave 15 min at 121C. Final pH, 7.3 0.2. Store up to 1 month at 4 1C.

M155. Trypticase (Tryptic) Soy Broth (TSB) with Glycerol
Trypticase peptone (tryptone) 17 g
Phytone peptone (soytone) 3 g
NaCl 15 g
K2HPO4 2.5 g
Glycerol 240 ml
Distilled water 760 ml

Suspend ingredients in distilled water and heat to dissolve. Dispense into bottles or vials. Autoclave 15 min at 121°C. Final pH, 7.3 0.2.

M156. Trypticase Soy Broth Modified (mTSB)
Trypticase soy broth 30.0 g
Bile salts No. 3 1.5 g
Dipotassium phosphate 1.5 g
Novobiocin solution (R50) 0.2 ml
Deionized water 1 liter

M157. Trypticase Soy Broth with 0.6% Yeast Extract (TSBYE)
Trypticase soy broth 30 g
Yeast extract 6 g
Distilled water 1 liter

Weigh ingredients and dissolve in water. Autoclave 15 min at 121C. Final pH, 7.3 0.2.

M158. Trypticase Soy-Polymyxin Broth
Prepare trypticase soy broth (M154) and dispense 15 ml portions into 20 x 150 mm tubes. Autoclave 15 min at 121C.

Polymyxin B solution, 0.15%. Dissolve 500,000 units polymyxin B sulfate in 33.3 ml distilled water. Filter-sterilize and store in the dark at 4C until needed. Before use, add 0.1 ml sterile 0.15% polymyxin B solution to 15 ml tryptic soy broth, and mix thoroughly.

M159. Trypticase Soy-Sheep Blood Agar

Prepare trypticase soy agar (M152). Sterilize as recommended and cool to 50C. Add 5 ml defibrinated sheep blood to 100 ml agar. Mix and dispense 20 ml portions to 15 x 100 mm petri dishes. (Commercial trypticase soy-blood agar plates are satisfactory.)

M160. Trypticase Soy-Tryptose Broth
Trypticase soy broth (commercial, dehydrated)15 g
Tryptose broth (commercial, dehydrated)13.5 g
Yeast extract 3 g
Distilled water 1 liter

Dissolve ingredients in 1 liter water. Heat gently to dissolve. Dispense 5 ml portions into 16 x 150 mm test tubes. Autoclave 15 min at 121C. Final pH, 7.2 0.2.

M161. Tryptone Broth and Tryptone Salt Broths
T1N0, T1N1, T1N3, T1N6, T1N8, T1N10

Trypticase or tryptone 10 g
NaCl 0, 10, 30, 60, 80, or 100 g
Distilled water 1 liter

Dissolve ingredients in distilled water. For T1N0, add no NaCl; for T1N1,use 10 g NaCl [1% (w/v) NaCl]. For T1N3, use 30 g NaCl/L [3% (w/v) NaCl], etc. Dispense into 16 x 125 mm screw-cap tubes. Tighten caps to maintain correct salt concentration in tube. Autoclave 15 min at 121°C. Final pH, 7.2 0.2.

M162. Tryptone Phosphate (TP) Broth
(for enteropathogenic E. coli)

Tryptone 20 g
K2HPO4 2 g
KH2PO4 2 g
NaCl 5 g
Polysorbate 80 (Tween 80) 1.5 ml
Distilled water 1 liter

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

M163. Tryptone Salt (T1N1) Agar and T1N2 Agar
Trypticase or tryptone10 g NaCl 10 g Agar 20 g Distilled water 1 literSuspend ingredients and boil to dissolve agar. For slants, dispense into tubes. Autoclave 15 min at 121 C. Solidifyo tubes as slants. Cool medium for plates to 45-50 C and pour into sterile petri dishes. For T N agar, use 20 g NaClo12 rather than the 10 g specified for T N agar.11

M164. Tryptone (Tryptophane) Broth, 1%
Tryptone or trypticase 10 g
Distilled water 1 liter

Dissolve and dispense 5 ml portions into 16 x 125 or 16 x 150 mm test tubes. Autoclave 15 min at 121C. Final pH, 6.9 0.2.

M165. Tryptone Yeast Extract Agar
Tryptone 10 g
Yeast extract 1 g
*Carbohydrate 10 g
Bromcresol purple 0.04 g
Agar 2 g
Distilled water 1 liter

Dissolve agar with heat and gentle agitation. Adjust pH to 7.0 0.2. Fill 16 x 125 mm tubes 2/3 full. Autoclave 20 min at 115C. Before use, steam medium 10-15 min. Solidify by placing tubes in ice water.

*Glucose and mannitol are the carbohydrates used for identification of S. aureus.

M166. Tryptose Blood Agar Base
Tryptose 10 g
Beef extract 3 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Suspend ingredients in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave 15 min at 121C. Before media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt.

M167. Tryptose Broth and Agar (for serology)
Difco Bacto tryptose 20 g
NaCl 5 g
Dextrose 1 g
Agar 15 g
Distilled water 1 liter

Weigh ingredients, add water, and mix. Autoclave 15 min at 121C. Swirl to disperse molten agar. Final pH, 7.2 0.2.For broth, omit agar from formulation.

M168. Tryptose Phosphate Broth (TPB)(for cell culture)
Tryptose 20 g
Dextrose 2 g
NaCl 5 g
Na2HPO4 2.5 g
Distilled water 1 liter

Sterilize by filtration through 0.20 m membrane. Available from Flow Laboratories, Inc., McLean, VA 22102.

M169. Tryptose-Sulfite-Cycloserine (TSC) Agar
Tryptose 15 g
Yeast extract 5 g
Soytone 5 g
Ferric ammonium citrate (NF Brown Pearls) 1 g
>Sodium metabisulfite 1 g
Agar 20 g
Distilled water 900 ml

Heat with agitation to dissolve. Adjust pH to 7.6 0.2. Dispense 250 ml portions to 500 ml flasks. Autoclave 15 min at 121C. Maintain medium at 50C before use. Dehydrated SFP agar base (Difco) is satisfactory for base.

D-cycloserine solution. Dissolve 1 g D-cycloserine (white crystalline powder) in 200 ml of distilled water. Sterilize by filtration and store at 4C until use. D-cycloserine powder is available from Sigma Chemical Co., St. Louis, MO.

Final medium. For pour plates, add 20 ml of D-cycloserine solution to 250 ml base. To prepare prepoured plates containing egg yolk, also add 20 ml of 50% egg yolk emulsion (M51). Mix well and dispense 18 ml into 15 x 100 mm petri dishes. Cover plates with a towel and let dry overnight at room temperature before use.

M170. Tyrosine Agar
Base. Prepare nutrient agar (M112). Dispense 100 ml portions into 170 ml bottles. Autoclave 15 min at 121C. Cool to 48C.
Tyrosine suspension. Suspend 0.5 g L-tyrosine in 10 ml distilled water in 20 x 150 mm culture tube. Mix thoroughly with Vortex mixer. Autoclave 15 min at 121C.
Final medium. Combine 100 ml base with sterile tyrosine suspension. Mix thoroughly by gently inverting bottle 2 or 3 times. Aseptically dispense 3.5 ml into 13 x 100 mm tubes with frequent mixing. Slant tubes and cool rapidly to prevent separation of tyrosine.

M171. Urea Broth
Urea 20 g
Yeast extract 0.1 g
Na2HPO4 9.5 g
K2HPO4 9.1 g
Phenol red 0.01 g
Distilled water 1 liter

Dissolve ingredients in distilled water. DO NOT HEAT. Sterilize by filtration through 0.45 m membrane. Aseptically dispense 1.5-3.0 ml portions to 13 x 100 mm sterile test tubes. Final pH, 6.8 0.2.

M172. Urea Broth (Rapid)
Urea 20 g
Yeast extract 0.1 g
KH2PO4 0.091 g
Na2HPO4 0.095 g
Phenol red 0.01 g
Distilled water 1 liter

Prepare as for urea broth (M171) above.

M173. Veal Infusion Agar and Broth
Veal, infusion from 500 g Proteose peptone No. 3 10 g
NaCl 5 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Dispense 7 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121C. Incline tubes to obtain 6 cm slant. Final pH, 7.3 0.2.

For veal infusion broth, prepare as above, but omit the 15 g agar. Autoclave 15 min at 121C. Final pH, 7.4 0.2.

M174. Violet Red Bile Agar (VRBA)
Yeast extract 3.0 g Peptone or gelysate 7.0 g
NaCl 5.0 g
Bile salts or bile salts No. 3 1.5 g
Lactose 10.0 g
Neutral red 0.03 g
Crystal violet 0.002 g
Agar 15.0 g
Distilled water 1.0 liter

Suspend ingredients in distilled water and let stand for a few min.Mix thoroughly and adjust to pH 7.4 0.2. Heatwith agitation and boil for 2 min. Do not sterilize. Before use, cool to 45C and use as a plating medium. After solidification, add a cover layer above the agar of approximately 3.0 to 4.0 ml to prevent surface growth and spreading of colonies.

M175. Violet Red Bile-MUG Agar (VRBA-MUG)
Add 0.1 g 4-methylumbelliferyl-$-D-glucuronide (MUG) to the ingredients for 1 liter of VRBA (M174), and continue as for preparation of VRBA.

M176. Vogel-Johnson (VJ) Agar
Trypticase 10 g
Yeast extract 5 g
Mannitol 10 g
K2HPO4 5 g
Lithium chloride 5 g
Glycine 10 g
Phenol red 0.025 g
Agar 15 g
Distilled water 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at 121C. Cool to 50C. Add 20 ml Chapman tellurite solution, 1%, commercially available from Difco Laboratories, Detroit, MI. Mix and pour into plates. Final pH, 7.2 0.2.

M177. Voges-Proskauer Medium (Modified)
Proteose peptone 7 g
NaCl 5 g
Dextrose 5 g
Distilled water 1 liter

Dissolve ingredients in water and adjust pH if necessary. Dispense 5 ml portions into 20 x 150 mm tubes. Autoclave 10 min at 121C. Final pH, 6.5 0.2.

M178. Wagatsuma Agar
Yeast extract 3 g
Peptone 10 g
NaCl 70 g
K2HPO4 5 g
Mannitol 10 g
Crystal violet 0.001 g
Agar 15 g
Distilled water 1 liter
Human or rabbit red blood cells, fresh (24 h), with anticoagulant 50 ml

Mix fresh (within 24 h of drawing) human or rabbit blood with same or larger volume of physiological saline. Centrifuge cells at about 4000 x g at 4°C for 15 min. Pour off saline and wash 2 more times. After 3rd wash, pour off saline and resuspend cells to original volume with saline. Suspend ingredients, except blood, in distilled water and boil to dissolve agar. Adjust to pH 8.0 0.2. Steam 30 min. DO NOT AUTOCLAVE. Cool to 45-50°C. Add 50 ml of washed red blood cells to the cooled medium. Mix and pour into sterile petri dishes. Dry plates thoroughly and use promptly. Medium can be made in smaller volumes (requiring less blood) when few plates are needed.

M179. Xylose Lysine Desoxycholate (XLD) Agar
Yeast extract 3 g
Ferric ammonium citrate 0.8 g
L-lysine 5 g
Sodium thiosulfate 6.8 g
Xylose 3.75 g
NaCl 5 g
Lactose 7.5 g
Agar 15 g
Sucrose 7.5 g
Phenol red 0.08 g
Sodium desoxycholate 2.5 g
Distilled water 1 liter

Heat with agitation just until medium boils. Do not overheat. Pour into plates when medium has cooled to 50C. Let dry about 2 h with covers partially removed. Then close plates. Final pH, 7.4 0.2. Do not store more than 1 day.

M180. Y-1 Adrenal Cell Growth Medium
  E. coli V. cholerae
Ham's F-10 medium 90 ml 500 ml
Fetal bovine serum 10 ml 75 ml
Penicillin-streptomycin solution 1 (M119) 1 ml 5 ml
These ingredients are commercially available. Filter-sterilize. Store at 4-5C.

M181. Yeast Extract (YE) Agar
Proteose peptone 10 g
Yeast extract 3.0 g
NaCl 5.0 g
Agar 15.0 g
Distilled water 1 liter

Adjust pH to 7.2-7.4. Autoclave at 121C for 15 min.

M182. Malt Extract Agar - (Yeasts and Molds) (MEAYM)
Malt extract, powdered 20.0 g
Glucose 20.0 g
Peptone 1.0 g
Agar 20.0 g
Distilled water 1 liter

Final pH 5.4.Mix ingredients, heat to dissolve agar and sterilize at 121°C for 15 min. Temper media to 45°C and pour platesoo under aseptic conditions. Dehydrated MA is commercially available, but since several MA formulas exist, check for the correct composition. This medium is recommended for identification of Aspergillus and Penicillium.

M183. Dichloran rose bengal chloramphenicol (DRBC) agar
Glucose 10.0 g
Bacteriological peptone 5.0 g
Potassium phosphate, monobasic 1.0 g
Magnesium sulfate heptahydrate 0.5 g
Rose bengal (5% soln., w/v) 0.5 ml
Dichloran (2,6-dichloro-4-nitroaniline) solution 1.0 ml (0.2%(w/v) in ethanol)
Chloramphenicol 0.1 g
Distilled water 1 liter
Agar 15.0 g

Final pH should be 5.6. Mix ingredients, heat to dissolve agar and sterilize by autoclaving at 121°C for 15 min. Temper to 45 1C in a water bath and pour plates.NOTES: DRBC agar is especially useful for analyzing sample containing "spreader" molds (e.g. Mucor, Rhizopus, etc.), since the added dichloran and rose bengal effectively slow down the growth of fast-growing fungi, thus readily allowing detection of other yeast and mold propagules, which have lower growth rates. Media containing rose bengal are light-sensitive; relatively short exposure to light will result in the formation of inhibitory compounds. Keep these media in a dark, cool place until used. DRBC agar should be used for spread plates only.

M184. Dichloran 18% glycerol (DG18) agar
Glucose 10.0 g
Peptone 5.0 g
Potassium phosphate monobasic 1.0 g
Magnesium sulfate heptahydrate 0.5 g
Dichloran (0.2% in ethanol, w/v) 1.0 ml
Agar 15.0 g
Chloramphenicol 0.1 g
Distilled water 800 ml

Mix above items and steam to dissolve agar, then bring volume to 1000 mL with distilled water. Add 220 g glycerol and sterilize by autoclaving at 121 C for 15 min. The final pH should be 5.6 and the final a , 0.955. This medium isow used as a general purpose mold enumeration medium and is preferred when the aof the analyzed food is 0.95 orwlower. The low water activity of this medium reduces interference by bacteria and fast-growing fungi. When both yeasts and molds must be enumerated, DRBC agar should be used.

M185. Malt Agar (MA)
Malt extract, powdered 20.0 g
Agar 20.0 g
Distilled water 1.0 liter

Mix ingredients, steam to dissolve agar and sterilize for 15 min at 121 C. Temper medium to 45 C and pour platesoo under aseptic conditions. To prepare slants dispense 5-6 ml of steamed medium (before autoclaving) into each of several 16 x 125 mm screw-cap tubes, loosely cap tubes and sterilize as above. After autoclaving lay tubes in a slanting position and let them cool. This medium is recommended as a general maintenance medium.

REAGENTS

R1. 4 M Ammonium Acetate
Ammonium acetate 308.4 g
Distilled water to make 1 liter

R2. 0.25 M Ammonium Acetate
Ammonium acetate 19.3 g
Distilled water to make 1 liter

R3. Basic Fuchsin Staining Solution

Dissolve 0.5 g basic fuchsin dye in 20 ml 95% ethanol. Dilute to 100 ml with distilled water. Filter if necessary with Whatman No. 31 filter paper to remove any undissolved dye. (TB Carbolfuchsin ZN staining solution, available from Difco Laboratories, is satisfactory.)

R4. 0.1 M Bicarbonate Buffer (pH 9.6)
Na2CO3 1.59 g
NaHCO3 2.93 g
Distilled water 1 liter

Store at room temperature for not more than 2 weeks.

R5. Bovine Serum Albumin (BSA) (1 mg/ml)
Nuclease-free bovine serum albumin 10 mg
Distilled water 10 ml

Place 0.5 ml portions into 1.5 ml plastic conical centrifuge tubes. Store frozen.

R6. 1% Bovine Serum Albumin in Cholera Toxin ELISA Buffer
Bovine serum albumin (BSA)1 g
ELISA buffer for (cholera toxin), pH 7.4 100 ml

Dissolve BSA in ELISA buffer. Aliquot and store at -20C.

R7. 1% Bovine Serum Albumin in PBS
Bovine serum albumin (BSA)1 g
Phosphate-buffered saline, pH 7.4 100 ml

Dissolve BSA in PBS buffer. Aliquot and store at -20C.

R8. Brilliant Green Dye Solution. 1%
Brilliant green dye 1 g
Distilled water (sterile) 10 ml

Dissolve 1 g dye in sterile water. Dilute to 100 ml. Before use, test all batches of dye for toxicity with known positive and negative test microorganisms.

R9. Bromcresol Purple Dye Solution. 0.2%
Bromcresol purple dye 0.2 g
Distilled water (sterile)100 ml

Dissolve 0.2 g dye in sterile water and dilute to 100 ml.

R10. Bromthymol Blue Indicator. 0.04%
Bromthymol blue 0.2 g
0.01 N NaOH 32 ml

Dissolve bromthymol blue in NaOH. Dilute to 500 ml with distilled water.

R11. Butterfield's Phosphate-Buffered Dilution Water
Stock solution

KH2PO4 34 g
Distilled water 500 ml

Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15 min at 121C. Store in refrigerator.

Dilution blanks
Take 1.25 ml of above stock solution and bring volume to 1 liter with distilled water. Dispense into bottles to 90 or 99 1 ml. Sterilize 15 min at 121C.

R12. Catalase Test
 
 Pour 1 ml 3% hydrogen peroxide over growth on slant culture. Gas bubbles indicate positive test. Alternatively, emulsify colony in l drop 3% hydrogen peroxide on glass slide. Immediate bubbling is positive catalase test. If colony is taken from blood agar plate, any carry-over of red blood cells can give false-positive reaction.

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 4C or may be stored frozen indefinitely (50 ml portions). To use, thaw stain, remix, and store at 4C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4C), 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 -20C in 10 ml aliquots.

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

1. Alcoholic solution of iodine
Potassium iodide 10 g
Iodine 10 g
Ethanol (70%) 500 ml

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 121C. 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 121C. 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 55-60C. Add methanol. Store stain in tightly stoppered bottle at 22C 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 121C. 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 -20C. 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 4C

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 4C. 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 37C. Hold stain 1 month at 22C (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:

Add (ml 1% BaCl2solution) to (ml 1% H2SO4 solution)
1 99
2 98
3 97
4 96
5 95
6 94
7 93
8 92
9 91
10 90

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 121C. 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

A. Sulfanilic acid reagent
Sulfanilic acid 1 g
5 N acetic acid 125 ml

B. N-(l-naphthyl)ethylenediamine reagent
N-(l-naphthyl)ethylenediamine dihydrochloride 0.25 g
5 N acetic acid 200 ml

C. alpha-Naphthol reagent
alpha-Naphthol 1 g
5 N acetic acid 200 ml

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.

D. Alternative test reagents. 5-Amino-2-naphthylene sulfonic acid (Cleve's acid) and N,N-dimethyl-1-naphthylamine have been recommended as substitutes for preparation of reagent B. Absolute ethanol may be substituted for acetic acid in reagent C. However, comparative evaluations should be conducted before substitution of these alternative reagents.

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 m filter and syringe. May be stored for several months in dark bottle at 4C.

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 g from 150 g 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 g 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 4C 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
NaH2PO4H20 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 4C).

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

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

Procedure
Inoculate cultures to be tested onto triple sugar iron agar slants and incubate for 18 h at 37C (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 37C. Add 0.25 ml buffered 0.0133 M ONPG solution to each suspension to be tested. Incubate tubes at 35-37C. 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-phenylenediamine2HCl 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 (-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 121C. 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 -20C.

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 121C. 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, 8.2Approximate 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 121C. 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:

5000 Units/ml
---------------- = 0.618 mg/ml
8090 Units/mg

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 121C for 15 min.

R67. Salts-Phosphate Buffered Saline Solution (Salts-PBS)
NaCl 121 g
KCl 15.5 g
MgCl2 12.7 g
CaCl22H2O 10.2 g
NaH2PO4H20 2.0 g
Na2HPO47H20 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 121C.

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 60C. 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 4C 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 40C. 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 121C.

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-100TM(TET) Buffer
(Triton Lytic Mix)
Triton X-100TM 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 m membrane.

*1 g Trypsin (1:250) will digest 250 g casein substrate under standard conditions.

R88. Verocytotoxin Antiserum

Grow E. coli 0157:H7 in trypticase soy broth at 37C for 18 h; add 0.5% formalin and keep at 37C 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 56C for 30 min and adsorb antiserum 6 times with about 1012 cells of heat-treated (121C, 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

Solution 2
Potassium hydroxide 40 g
Distilled water to make 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.