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Appendix G
Media Glossary
January 2006
Glossary of agars, broths and other reagents
Section L
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(PDF Version - 27 K)| Basal Medium | |
|---|---|
| Beef extract | 3.0 g |
| Peptone | 5.0 g |
| Lactose | 5.0 g |
| Distilled water | 1.0 L |
| pH 6.9 ± 0.2 | |
Complete Medium:
Dissolve ingredients in 1.0 L of distilled water. Dispense and autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptose | 15.0 g |
| Yeast extract | 10.0 g |
| Lactose | 10.0 g |
| Phenol red (1% solution in 95% ethanol) | 5.0 ml |
| Gelatin* | 120 g |
| Distilled water | 1.0 L |
| pH 7.5 ± 0.2 | |
| * Problems associated with the gelatin used sometimes occur. Quality control is important to ensure that the gelatin used is satisfactory. | |
Complete Medium:
Heat tryptose, yeast extract and lactose in 400 mL water to dissolve. Suspend gelatin in 600 mL water and heat at 50-60oC with swirling to dissolve. Mix the 2 solutions and adjust the pH to 7.5 " 0.2. Add phenol red and mix. Dispense into screw-cap tubes and autoclave for 10 min at 121oC . If not used within 8 h, deaerate by heating at 50-70oC for 2-3 h or heat for 10 min in a boiling waterbath or flowing steam.
| Basal Medium | |
|---|---|
| Tryptose, tryptone or trypticase | 20.0 g |
| Lactose | 5.0 g |
| Dipotassium phosphate (K2HPO4) | 2.75 g |
| Monopotassium phosphate (KH2PO4) | 2.75 g |
| Sodium chloride (NaCl) | 5.0 g |
| Sodium lauryl sulphate | 0.1 g |
| Distilled Water | 1.0 L |
| pH 6.8 ± 0.2 | |
Complete Medium:
Dissolve ingredients in 1.0 L of distilled water (in 500 mL if double strength is required), dispense in 10.0 mL volumes in tubes containing gas vials. Sterilize at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 10.0 g |
| Yeast extract | 5.0 g |
| Sodium Chloride (NaCl) | 10.0 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.5 ± 0.2 | |
Complete Medium:
Suspend components in water and heat to boiling to dissolve completely. Autoclave for 15 min at 121oC, cool to 45-50oC and dispense into Petri dishes.
| Basal Medium | |
|---|---|
| Peptone | 10.0 g |
| Lactose | 10.0 g |
| Dipotassium phosphate (K2HPO4) | 2.0 g |
| Agar | 15.0 g |
| Eosin Y | 0.4 g |
| Methylene blue | 0.065 g |
| Distilled water | 1.0 L |
| pH 7.1 ± 0.2 | |
Complete Medium:
Add all ingredients to distilled water. Heat to boiling to obtain complete solution. Mix well, dispense as required (usually in 100-200 mL volumes), and sterilize at 121oC for 15 min. Avoid overheating, as the agar is likely to become soft and unsuitable for streaking. When the agar is to be used as a streaking medium, melt it in flowing steam or boiling water. Gently swirl the contents of the flask to distribute the flocculant precipitate, and pour a thick layer into Petri plates. Continue intermittent swirling while pouring plates. Dry surface completely . A dry surface promotes growth of distinct individual colonies which are easier to differentiate. Plates can be stored at 2 to 7oC in the dark for 1 month. Incubate some uninoculated plates as part of quality control procedures.
In Levine's EMB agar there is no sucrose but more lactose. This gives good differentiation of Escherichia coli from Enterobacter aerogenes. Coliform colonies usually show a dark center and have a greenish metallic sheen.
| Basal Medium | |
|---|---|
| Proteose peptone | 5.0 g |
| Tryptone | 5.0 g |
| Beef extract | 5.0 g |
| Yeast extract | 5.0 g |
| Sodium chloride ( NaCl) | 20.0 g |
| Monopotassium phosphate ( KH2PO4) | 1.35 g |
| Dibasic sodium phosphate (Na2HPO4) | 12.0 g |
| Esculin | 1.0 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
| Supplement UVM 1 (commercially available) For 500 mL Broth | |
| Nalidixic Acid | 10.0 mg |
| Acriflavine | 6.0 mg |
| Sterile distilled water | 2.0 mL |
| Supplement UVM 2 ( commercially available) For 500 mL Broth | |
| Nalidixic Acid | 10.0 mg |
| Acriflavine | 12.5 mg |
| Sterile distilled water | 2.0 mL |
Complete Medium:
Suspend ingredients in 1.0 L of distilled water. Autoclave at 121oC for 15 min. and cool to 50oC .
Aseptically add filter sterilized UVM 1 or UVM 2 supplement to 500 mL of cooled basal media (double the amount for 1.0 L). Mix well and dispense. Store media under refrigeration (2-7oC) in the dark (acriflavine can photo-oxidize to form compounds inhibitory to Listeria spp.) for up to 1 month.
| Basal Medium | |
|---|---|
| Glycine anhydride | 10.0 g |
| Lithium chloride | 5.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Pancreatic digest of casein | 5.0 g |
| Peptic digest of animal tissue | 5.0 g |
| Beef extract | 3.0 g |
| Phenylethyl alcohol | 2.5 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
| Supplement: Moxalactam solution | |
| Moxalactam (ammonium or sodium salt) | 1.0 g |
| Potassium phosphate buffer, 0.1 M, pH 6.0 | 100 mL |
Complete Medium:
Sterilize the basal medium at 121oC for 15 min. Cool to 45-50oC and add 2.0 mL of moxalactam solution. Mix thouroughly. Pour 12.0-15.0 mL into Petri dishes and store at 4oC. The basal medium cannot be made in advance and reheated.
Supplement: Filter sterilize. Store the solution frozen in 2.0 mL aliquots
| scope="colgroup">Basal Medium | |
|---|---|
| Beef liver, infusion from | 50.0 g |
| Veal, infusion from | 500 g |
| Gelatin | 20.0 g |
| Proteose peptone | 20.0 g |
| Enzymatic digest of protein | 1.3 g |
| Pancreatic digest of casein | 1.3 g |
| Dextrose | 5.0 g |
| Soluble starch | 10.0 g |
| Casein | 2.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Sodium nitrate | 2.0 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Add components to distilled water. Mix thoroughly, gently heat and bring to a boil. Dispense, making sure that some liver and veal particles are distributed to each aliquot. Sterilize at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Liver veal agar base - see LVA above. | |
Complete Medium:
Egg-yolk emulsion*
Soak clean, uncracked eggs in 80% ethanol for 10 min, remove them with a spoon and flame off the alcohol. Carefully crack the eggs, discard the white, break the yolk sac, withdraw a minimum of 10.0 mL of yolk with a wide-mouth pipette avoiding the chalaza (yolk ligament), and transfer to a sterile bottle. Add an equal volume of saline (0.85% NaCl) and mix.
Complete Medium:
Add 80 mL of egg-yolk emulsion per L of agar base. Mix gently and pour plates.
*Egg-yolk emulsions are also commercially available, but these are generally inferior to freshly prepared emulsions.
| Basal Medium | |
|---|---|
| Yeast extract, 0.3 % | 3.0 g |
| Peptone | 10.0 g |
| Sodium Chloride (NaCl) | 30.0 g |
| Agar | 3.0 g |
| Distilled water | 1.0 L |
Complete Medium:
Add ingredients to distilled water and heat to dissolve. Dispense 4.0 mL into 13x100 mm screw-cap tubes. Autoclave 12 min at 121oC. Cool and tighten caps for storage. No pH adjustment is necessary.
| Basal Medium | |
|---|---|
| Peptone | 5.0 g |
| Yeast extract | 3.0 g |
| Dextrose | 1.0 g |
| L-Lysine | 10.0 g |
| Ferric ammonium citrate | 0.5 g |
| Sodium thiosulfate (Na2S2O3) | 0.04 g |
| Bromcresol purple | 0.02 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 6.7 ± 0.2 | |
Complete Medium:
Suspend ingredients in distilled water and heat to boiling with frequent swirling. Dispense appropriate volume of medium into tubes, and autoclave at 121oC for 15 min. Cool in slanted position to form deep butts.
Lysostaphin Solution
Complete Medium:
Dissolve lysostaphin in phosphate saline buffer (0.02 M; pH 7.3-7.4) to obtain a concentration of 50.0 µg lysostaphin per mL.
