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Appendix G
Media Glossary
February 2006
Glossary of agars, broths and other reagents
Section P
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(PDF Version - 50 K)| Basal Medium | |
|---|---|
| Peptone | 23.0 g |
| Starch | 1.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Yeast | 3.0 g |
| Agar | 13.0 g |
| Mannitol | 10.0 g |
| Ferric ammonium citrate | 0.5 g |
| Esculin | 0.8 g |
| Dextrose | 0.5 g |
| Lithium chloride (LiCl) | 15.0 g |
| Phenol red | 0.08 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
| Supplements (per 500 mL basal media) | |
| Polymyxin-B-sulphate | 5.0 mg |
| Ceftazidime | 10.0 mg |
| Acriflavine | 2.5 mg |
| Distilled water | 5.0 mL |
Complete Medium:
Suspend the basal medium ingredients in 1.0 L of distilled water and adjust pH if necessary. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min. Cool to 50oC. Aseptically add supplement solution to 500 mL of cooled agar base (double the amount for 1.0 L). Mix thoroughly and dispense. Store under refrigeration in the dark (acriflavine can photo-oxidize to form compounds inhibitory to Listeria spp.)
Supplement solution : Suspend supplement components in 5.0 mL of distilled water. Filter sterilize.
| Basal Medium | |
|---|---|
| Peptone | 23.0 g |
| Yeast extract | 5.0 g |
| Lithium chloride (LiCl) | 10.0 g |
| Esculin | 0.8 g |
| Ammonium iron(III) citrate | 0.5 g |
| D(-) mannitol | 5.0 g |
| Phenol red | 0.08 g |
| Soybean lecithin | 1.0 g |
| Tween 80™ | 2.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
| Supplements (per 500 mL basal medium) | |
| Polymyxin-B-sulphate | 5.0 mg |
| Ceftazidime | 10.0 mg |
| Acriflavine | 2.5 mg |
| Distilled water | 5.0 mL |
Complete Medium:
Suspend the basal medium ingredients in 1.0 L of distilled water and adjust pH if necessary. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min. Cool to 50oC. Aseptically add supplement solution to 500 mL of cooled agar base (double the amount for 1.0 L). Mix thoroughly and dispense. Store under refrigeration in the dark (acriflavine can photo-oxidize to form compounds inhibitory to Listeria spp.)
Supplement solution : Suspend supplement components in 5.0 mL of distilled water. Filter sterilize.
| Basal Medium (Brucella broth base) | |
|---|---|
| Pancreatic digest of casein | 10.0 g |
| Peptic digest of animal tissue | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Yeast extract | 2.0 g |
| Glucose | 1.0 g |
| Sodium pyruvate (optional) | 0.25 g |
| Sodium bisulphite (NaHSO3) | 0.1 g |
| Distilled water | 950 mL |
| pH 7.0 ± 0.2 | |
| Supplements | |
| Lysed horse blood | 50.0 mL |
| Supplement A | |
| Vancomycin | 0.01 g |
| Trimethoprim lactate | 0.01 g |
| Distilled water | 5.0 mL |
| Supplement B | |
| Sodium cefoperazone | 0.032 g |
| Cycloheximide (optional) | 0.1g |
| Distilled water | 5.0 mL |
Complete Medium:
Suspend basal medium ingredients in 950 mL of distilled water . Mix thoroughly. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min. Cool to 25oC. Aseptically add the lysed horse blood and 5.0 mL of supplement A and mix thoroughly. Aseptically dispense 100 mL into 250 mL flasks. Aseptically add 0.5 mL of supplement B to 100 mL of media .
Supplement A : Suspend supplement components in 5.0 mL of distilled water, mix thoroughly and filter sterilize.
Supplement B : Suspend supplement components in 5.0 mL of distilled water, mix thoroughly and filter sterilize.
Note: If the samples have been stressed, particularly by freezing, incubate the 100 mL of inoculated media for 3-4 h before adding 0.5 mL of Supplement B.p
| Basal Medium | |
|---|---|
| Penicillin G | 500,000 IU |
| Streptomycin | 500,000 µg |
| Distilled water | 100 mL |
Complete Medium:
Dissolve antibiotics in water and filter sterilize. Store refrigerated.
For V. cholerae use commercially available penicillin G-streptomycin sulfate solution. Add 500 mL solution per 500 mL medium. Store at -20oC.
| Basal Medium | |
|---|---|
| Peptone | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Mix thoroughly and dispense. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Peptone | 10.0 g |
| Sodium chloride (NaCl) | 30.0 g |
| Potassium nitrate (KNO3) | 1.0 g |
| Distilled water | 1.0 L |
| pH 7.6 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Mix thoroughly and dispense. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Peptone | 1.0 g |
| Distilled water | 1. 0 L |
| pH 7.0 ± 0.2 | |
Complete Medium:
Suspend 1.0 g of peptone in 1.0 L of distilled water. Mix thoroughly, and dispense into dilution bottles or tubes in amounts that will allow for volume lost due to autoclaving. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Peptone | 1.0 g |
| Tween 80™ | 10.0 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of warm distilled water. Mix thoroughly until dissolved, and dispense into dilution bottles or tubes in amounts that will allow for volume lost due to autoclaving. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| ABTS | 10 mg |
| Citric acid (0.05 M) | 10 mL |
| Hydrogen peroxide, 30% (H2O2) | 30 µL |
Complete Medium:
Mix basal ingredients together. Prepare 5 min before use. Sufficient for one 96-well microwell plate.
| Solution A | |
|---|---|
| 4-Chloro-1-naphthol | 60.0 mg |
| Methanol (cold) | 20 mL |
| Solution B | |
| Hydrogen peroxide, 30% (H2O2) | 60 µL |
| Tris-buffered saline (TBS) | 100 mL |
Complete Medium:
Solution A : Dissolve 4-chloro-1-naphthol in cold methanol. Store at -20oC .
Solution B: Mix the 2 ingredients together at room temperature.
Complete Solution: Mix ice cold Solution A with room temperature Solution B immediately before use.
| Basal Mediumm | |
|---|---|
| Trypticase or proteose Peptone no. 3 | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Beef extract (optional) | 1.0 g |
| Phenol red (or 7.2 mL of 0.25% solution of phenol red) | 0.018 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
| Supplement | |
| Carbohydrate required for test | 5.0 g |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Dissolve 5.0 g of the required carbohydrate in this basal broth. Mix thoroughly and dispense into test tubes containing inverted fermentation tubes. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Beef extract | 1.0 g |
| Enzymatic digest of animal tissues | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Phenol red | 0.018 g |
| D-sorbitol | 5.0 g |
| 4-methylumbelliferyl- β -D-glucuronide (MUG) (or MUG supplement ) | 0.05 g |
| Agar | 20.0 g |
| Distilled water | 1.0 L |
| pH 6.8 - 6.9 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min. Cool and dispense (thick plates).
| Stock Solution | |
|---|---|
| Potassium phosphate monobasic (KH2PO4) | 34.0 g |
| Distilled water | 500 mL |
| pH to 7.2 | |
Complete Medium:
Adjust pH to 7.2 with 1N NaOH and bring volume to 1.0 L with distilled water. Sterilize at 121oC for 15 min and store in refrigerator.
Dilution blanks:
Take 1.25 mL of stock solution and bring volume to 1.0 L with distilled water. Dispense required volume and sterilize at 121oC for 15 min.
| Stock Solution 1 | |
|---|---|
| Sodium phosphate dibasic anhydrous reagent grade (Na2HPO4) | 28.4 g |
| Sodium chloride reagent grade (NaCl) | 85.0 g |
| Distilled water | 1.0 L |
| pH is approximately 8.2 | |
| Stock Solution 2 | |
| Sodium phosphate monobasic monohydrate reagent grade (NaH2PO4.H2O) | 27.6 g |
| Sodium chloride reagent grade (NaCl) | 85.0 g |
| Distilled water | 1.0 L |
| pH is approximately 5.6 | |
Complete Medium:
Make 1 to 9 dilutions of aliquots of each stock solution to obtain a final volume of 500 mL of diluted solution 1, and 100 mL of diluted solution 2. By means of a pH meter, titer the diluted solution 1 to a pH of 7.3 - 7.4 by adding approximately 65.0 mL of diluted solution 2. The resulting solution will be 0.02 M phosphate saline buffer.
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.
| Basal Medium | |
|---|---|
| Sodium phosphate monobasic monohydrate reagent grade (NaH2PO4.H2O) | 0.55 g |
| Sodium phosphate dibasic anhydrous reagent grade (Na2HPO4) | 2.85 g |
| Sodium chloride (NaCl) | 8.7 g |
| Distilled Water | 1.0 L |
| pH 7.4 | |
Complete Medium:
Dissolve in 500 mL distilled water. Adjust pH to 7.4. Add additional distilled water to one liter.
| Basal Medium | |
|---|---|
| Tryptone | 5.0 g |
| Glucose | 1.0 g |
| Yeast extract | 2.5 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.0 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling to dissolve all ingredients. Dispense into appropriate glassware and autoclave at 121oC for 15 min. For shelf stable milk products, addition of 1.0 mL of resterilised product, to the tempered media before pouring the plates, may aid in recovery of microorganisms.
| Basal Medium | |
|---|---|
| Plate count agar (see above) | 1.0 L |
| Chloramphenicol | 100.0 mg |
| pH 7.0 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients of plate count agar in 1.0 L of distilled water. Heat to boiling to dissolve all ingredients, and add 100 mg of chloramphenicol. Mix thoroughly. Dispense into appropriate glassware and autoclave at 121oC for 15 min.
| Basal Medium (Available from Oxoid under the name 'Bacillus cereus Selective Agar'.) | |
|---|---|
| Peptone | 1.0 g |
| D-mannitol | 10.0 g |
| Magnesium sulphate hepta hydrate (MgSO4.7H2O) | 0.1 g |
| Sodium chloride (NaCl) | 2.0 g |
| Sodium phosphate dibasic (Na2HPO4) | 2.5 g |
| Potassium phosphate monobasic (KH2PO4) | 0.25 g |
| Bromothymol Blue | 0.12 g |
| Sodium pyruvate (C3H3O3Na) | 10.0 g |
| Agar | 18.0 g |
| Distilled water | 940 mL |
| pH 7.4 ± 0.2 | |
| Supplement | |
| Polymyxin B solution (10,000 units/mL) | 10.0 mL |
| Egg yolk emulsion 50% | 50.0 mL |
Complete Medium:
Suspend basal medium ingredients in 940 mL of distilled water. Heat to boiling to dissolve completely. Adjust to pH 7.4. Autoclave at 121oC for 15 min. Cool to 50-55oC. Add aseptically 10.0 mL of the pre-warmed (50-55oC) Polymyxin B solution and 50.0 mL of the pre-warmed (50-55oC) egg yolk emulsion to the tempered (50-55oC) basal medium suspension. Mix thoroughly but gently and dispense into sterile Petri dishes. (If commercial egg yolk emulsion is used, add the egg yolk to a final concentration of 2.5%.).
Polymyxin B solution: Filter-sterilize the solution
Egg yolk emulsion (50%) (commercially available as an enrichment and usually contains about 50% egg yolk): Soak fresh clean eggs in 70% alcohol for 15 min. Separate egg yolks aseptically and blend with an equal amount of physiological saline for about 5 min on a magnetic stirrer at low speed (do not heat).
| Basal Medium | |
|---|---|
| Potatoes | 200.0 g |
| Dextrose | 20.0 g |
| Agar | 20.0 g |
| Distilled water | 1.0 L |
| pH 5.6 ± 0.2 | |
Complete Medium:
Boil 200 g sliced, unpeeled potatoes in 1.0 L of distilled water for 30 min. Filter through cheesecloth saving the effluent. Add other ingredients to the potato infusion and boil to dissolve completely.
Or suspend 39 g of commercially available dehydrated media and 5.0 g of agar in 1.0 L of distilled water. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min and dispense into sterile Petri dishes. Medium should not be remelted more than once.
| Basal Medium | |
|---|---|
| PDA (see above) | 1.0 L |
| Chloramphenicol | 100.0 mg |
| pH 5.6 ± 0.2 | |
Complete Medium:
Prepare 1.0 L of PDA, and add 100.0 mg of chloramphenicol. Mix thoroughly, and autoclave at 121oC for 15 min. Dispense into sterile Petri dishes.
| Basal Medium | |
|---|---|
| PDA (see above) | 1.0 L |
| Sodium chloride (NaCl) | 75.0 g |
| pH 5.6 ± 0.2 | |
Complete Medium:
Prepare 1.0 L of PDA, and add 75.0 g of sodium chloride. Mix thoroughly, and autoclave at 121oC for 15 min. Dispense into sterile Petri dishes.
| Basal Medium | |
|---|---|
| PDA (see above) | 1.0 L |
| Supplement | |
| 10% Tartaric acid | 16.0 mL |
| final pH 3.5 ± 0.1 | |
Complete Medium:
Prepare 1.0 L of PDA and autoclave at 121oC for 15 min. Cool to 45-50oC. Aseptically add 16.0 mL of sterile 10% tartaric acid and mix thoroughly. Dispense into sterile Petri plates. Do not reheat the acidified medium as this will hydrolyse the agar.
| Basal Medium | |
|---|---|
| Beef extract | 3.0 g |
| Peptone | 5.0 g |
| Lactose | 7.46 g |
| Tryptose | 9.83 g |
| Potassium phosphate dibasic (K2HPO4) | 1.35 g |
| Potassium phosphate monobasic (KH2PO4) | 1.35 g |
| Sodium chloride (NaCl) | 2.46 g |
| Sodium lauryl (dodecyl) sulfate (C12H25O4SNa) | 0.05 g |
| Bromcresol purple | 0.0085 g |
| Distilled water | 1.0 L |
| pH 6.8 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients (triple strength) in 1.0 L of distilled water. Warm gently to dissolve completely and dispense 50.0 mL into 250 mL dilution bottles. Autoclave at 121oC for 12 min., with the total autoclave time not to exceed 30 min. Cool.
| Basal Medium | |
|---|---|
| Beef extract | 10.0 g |
| Peptone | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Agar | 12.0 g |
| Distilled water | 940 mL |
| pH 7.5 ± 0.2 | |
| Antibiotics supplement | |
| Polymyxin B | 5000.0 IU |
| Rifampicin | 0.01 g |
| Trimethoprim lactate | 0.01 g |
| Cycloheximide | 0.1 g |
| Acetone: distilled water (50:50) | 10 mL |
| Supplement | |
| Lysed horse blood | 50.0 mL |
Complete Medium:
Suspend basal medium ingredients in 940 mL of distilled water. Mix thoroughly. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min . Cool to 45-50oC. Aseptically add 50.0 mL of lysed horse blood and 10.0 mL of the antibiotic supplement. Mix thoroughly and dispense into sterile Petri dishes.
Antibiotic supplement: Add antibiotic components to 10.0 mL of 50:50 acetone:distilled water and filter sterilize.
| Basal Medium | |
|---|---|
| Pancreatic digest of gelatin | 16.0 g |
| Agar | 11.0 g |
| Pancreatic digest of casein | 10.0 g |
| Potassium sulfate (K2SO4) | 10.0 g |
| Magnesium chloride hexahydrate (MgCl2. 6H20) | 1.4 g |
| Glycerol | 10.0 mL |
| Distilled water | 990 mL |
| CFC selective supplement | |
| Cephaloridine | 0.05 g |
| Fucidin | 0.01 g |
| Cetrimide | 0.01 g |
| Distilled water | 10 mL |
Complete Medium:
Suspend basal medium ingredients in 990 mL of distilled water. Mix thoroughly and gently heat to boiling. Autoclave at 121oC for 15 min. Cool to 45-50oC. Aseptically add sterile CFC selective supplement. Mix thoroughly and dispense into sterile Petri dishes.
CFC selective supplement: Add selective components to distilled water and bring the volume to 10.0 mL. Mix thoroughly and filter sterilize.
| Basal Medium | |
|---|---|
| Pancreatic digest of gelatin | 16.0 g |
| Agar | 11.0 g |
| Pancreatic digest of casein | 10.0 g |
| Potassium sulfate (K2SO4) | 10.0 g |
| Magnesium chloride hexahydrate (MgCl2. 6H20) | 1.4 g |
| Glycerol | 10.0 mL |
| Distilled water | 990 mL |
| CN selective supplement (per 500 mL basal media) | |
| Cetrimide | 0.1 g |
| Sodium nalidixate | 7.5 mg |
| Distilled water | 1.0 mL |
| Ethanol | 1.0 mL |
Complete Medium:
Suspend basal medium ingredients in 990 mL of distilled water. Mix thoroughly and gently heat to boiling. Autoclave at 121oC for 15 min. Cool to 45-50oC. Aseptically add sterile CN selective supplement. Mix thoroughly and dispense into sterile Petri dishes.
| Basal Medium | |
|---|---|
| Proteose peptone | 10.0 g |
| Beef extract | 1.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Bromcresol purple | 0.02 g |
| Distilled water | 1.0 L |
| pH 6.8 ± 0.2 | |
| Supplement | |
| Cellobiose | 10.0 g |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling to dissolve completely. Add 10.0 g of the cellobiose. Mix thoroughly. Dispense 10.0 mL into tubes and autoclave at 121oC for 15 min.
