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Appendix G
Media Glossary
January 2006
Glossary of agars, broths and other reagents
Section M
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(PDF Version - 51 K)| Basal Medium | |
|---|---|
| Yeast extract | 5.0 g |
| Tryptone | 12.5 g |
| D-mannose | 2.0 g |
| Sodium citrate | 5.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Dipotassium phosphate (K2HPO4) | 5.0 g |
| Manganese chloride (MnCl2) | 0.14 g |
| Magnesium sulfate (MgS04) | 0.8 g |
| Ferrous sulfate (FeSO4) | 0.04 g |
| Tween 80TM | 0.75 g |
| Distilled water | 1.0 L |
| pH 7.0 ± 0.2 | |
Complete Medium:
Suspend ingredients in distilled water and heat to boiling 1-2 min. Dispense and autoclave at 121°C for 15 min.
| Basal Medium | |
|---|---|
| Peptone | 20.0 g |
| Lactose | 10.0 g |
| Bile salts no. 3 | 1.5 g |
| Sodium chloride (NaCl) | 5.0 g |
| Neutral red | 0.03 g |
| Crystal violet | 0.001 g |
| Agar | 13.5 g |
| Distilled water | 1.0 L |
| Final pH 7.1 ± 0.2 | |
Complete Medium:
Suspend ingredients in 1.0 L of distilled water and heat to boiling with frequent swirling. Autoclave at 121oC for 15 min and cool to 45-50oC. Dispense medium into plates and allow to dry with lids partially removed.
| Basal Medium | |
|---|---|
| Malachite green | 5.0 g |
| Distilled water | 100 mL |
Complete Medium:
Dissolve 5.0 g of malachite green in distilled water to make 100 mL of solution. Stir.
| Basal Medium | |
|---|---|
| Malt extract | 30.0 g |
| Mycological protein | 5.0 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 5.4 ± 0.2 | |
Complete Medium:
Suspend ingredients in distilled water. Boil to dissolve completely. Autoclave at 115oC for 10 min.
*If required for osmophilic organisms, add 500 g of sucrose / L before autoclaving.
| Basal Medium | |
|---|---|
| Tryptone | 5.0 g |
| Mannitol | 5.0 g |
| Yeast extract | 2.0 g |
| Sodium chloride (NaCl ) | 3.0 g |
| Potassium chloride (KCl) | 2.0 g |
| Magnesium sulfate heptahydrate (MgSO4 . 7H2O) |
0.2 g |
| Ferric chloride, hexahydrate (FeCl3 . 6H2O) | 0.1 g |
| Bromothymol blue | 0.08 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 8.0 ± 0.2 | |
| Supplements | |
| Sodium deoxycholate | 100 mg |
Complete Medium:
Add basal ingredients, except agar, to 1.0 L of water. Dissolve at room temperature and adjust to pH 8.0. Add agar and autoclave at 121oC for 15 min. Temper and add 100 mg sodium deoxycholate. Mix well and dispense into Petri dishes. This medium can be stored for 2 months at 4°C.
| Basal Medium | |
|---|---|
| PhytoneTM (papaic digest of soybean meal) | 5.0g |
| PolypeptoneTM (equal parts pancreatic digest of casein and peptic digest of animal tissue) | 5.0 g |
| Beef extract | 5.0 g |
| D-Mannitol | 10.0 g |
| Maltose | 10.0 g |
| Sodium chloride (NaCl) | 20.0 g |
| Agar | 13.0 g |
| 1000X Dye stock solution* | 1.0 mL |
| Distilled water | 1.0 L |
| pH 7.8 ± 0.2 | |
| Supplement: *1000X Dye Stock solution | |
| Bromthymol blue | 4.0 g |
| Cresol red | 4.0 g |
| Ethanol, 95% | 100 mL |
Complete Medium:
Suspend ingredients and boil to dissolve. Adjust pH and autoclave 15 min at 121oC
| Basal Medium | |
|---|---|
| Tryptone | 5.0 g |
| Trehalose | 5.0 g |
| Yeast extract | 2.0 g |
| Sodium chloride (NaCl) | 3.0 g |
| Potassium chloride (KCl) | 2.0 g |
| Magnesium sulfate, heptahydrate (MgSO4 .7H2 O) |
0.2 g |
| Ferric chloride, hexahydrate (FeCl3.6H2) | 0.1 g |
| Bromothymol blue | 0.04 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 8.0 ± 0.2 | |
| Supplements | |
| Ethanol | 1.0 mL |
| Ampicillin | 20.0 mg |
| Sodium deoxycholate | 100 mg |
Complete Medium:
Add basal ingredients, except agar, to 1.0 L of water. Dissolve at room temperature and adjust to pH 8.0. Add agar and autoclave at 121oC for 15 min. Temper and add the additives. Mix well and dispense into Petri dishes. This medium can be stored for 2 months at 4oC.
| Basal Medium | |
|---|---|
| Methyl red | 0.10 g |
| Ethanol, 95% | 300 mL |
| Distilled water | 200 mL |
Complete Medium:
Dissolve methyl red in 300 mL ethanol and bring the volume to 500 mL with distilled water. Store under refrigeration.
| Basal Medium | |
|---|---|
| Pancreatic digest of casein | 15.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Tween 80TM (polysorbate 80) | 5.0 g |
| Enzymatic hydrolysate of soybean meal | 5.0 g |
| Lecithin | 0.7 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. Boil 1-2 min. Dispense into flasks or tubes and autoclave 15 min at 121oC. Pour into sterile Petri dishes or leave in tubes.
| Basal Medium | |
|---|---|
| Tryptose | 10.0 g |
| Proteose peptone no.3 | 5.0 g |
| Yeast extract | 3.0 g |
| Lactose | 12.5 g |
| Bile salts no.3 | 1.5 g |
| Sodium chloride (NaCl) | 5.0 g |
| Aniline blue | 0.1 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
Complete Medium:
Heat the mixture to boiling, temper to 50-55oC, and adjust the pH. Do not autoclave. Pour 15-20 mL into Petri plates. Plates keep for 4 weeks at 4oC.
* The formulation without rosolic acid was found to work better with the HGMF method for coliforms. If rosolic acid is added to the media, use 10 mL of a 1% solution in 0.2 N NaOH. Add after the other ingredients have been dissolved by heating and continue to boil for an additional minute. This supplement is also commercially available. Follow the manufacturer's instructions.
| Basal Medium: Mixture A | |
|---|---|
| Instant nonfat milk | 100 g |
| Distilled water | 500 mL |
| Basal Medium: Mixture B | |
| Nutrient broth | 12.5 g |
| Sodium chloride (NaCl) | 2.5 g |
| Agar | 15.0 g |
| Distilled water | 500 mL |
Complete Medium:
Autoclave mixtures separately, and temper to 50oC. Aseptically combine mixtures and pour 20.0 mL per plate.
| Modified CCDA Preston - see Campylobacter Agar with Charcoal and Deoxycholate (CCDA) |
| Basal Medium Solution 1 | |
|---|---|
| Peptone | 10.0 g |
| Beef extract | 5.0 g |
| Sodium chloride (NaCl) | 20.0 g |
| 1000x Dye stock solution* | 1.0 mL |
| Agar | 15.0 g |
| Distilled water | 900 mL |
| pH 7.6 | |
| Basal Medium Solution 2 | |
| Cellobiose | 10.0 g |
| Colistin | 400,000 units |
| Polymyxin B | 100,000 units |
| Distilled water | 100 mL |
| Supplement *1000x Dye solution | |
| Bromthymol blue | 4.0 g |
| Cresol red | 4.0 g |
| Ethanol, 95% | 100 mL |
Complete Medium:
Solution 1
Adjust pH to 7.6. Boil to dissolve agar and cool to 48-55oC.
*1000x Dye solution
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 litre of mCPC agar gives 40 mg bromthymol blue and 40 mg cresol red per litre.
Solution 2
Dissolve cellobiose in distilled water by heating gently. Cool. Add antibiotics.
Complete Medium:
Add Solution 2 to cooled Solution 1, mix, and dispense into Petri dishes. Final color, dark green to green-brown. This medium, like TCBS, is very inhibitory and does not require autoclaving. Medium may be stored 2 weeks at refrigeration temperatures.
| Basal Medium | |
|---|---|
| Tryptone | 20.0 g |
| Bile salts no. 3 | 1.5 g |
| Lactose | 5.0 g |
| Dipotassium phosphate (K2HPO4) | 4.0 g |
| Monopotassium phosphate (KH2PO4) | 1.5 g |
| Sodium chlloride (NaCl) | 5.0 g |
| Distilled water | 1.0 L |
| Supplements | |
| Novobiocin | 100 mg |
| Distilled water | 1.0 mL |
Complete Medium:
Add components, except novobiocin, to distilled water. Mix thoroughly and dispense into 225 mL aliquots. Autoclave at 121oC for 15 min. Let cool. Dispense in 225 mL aliquots. Add 50.0 µL of novobiocin (filter sterilized through a 0.45 um filter) solution to each sample tube just before addition of test sample.
Modified Fraser Broth (MFB) - see Fraser Broth
| Basal Medium | |
|---|---|
| Tryptone | 20.0 g |
| Bile Salts no. 3 | 1.12 g |
| Sodium chloride (NaCl) | 5.0 g |
| Sorbitol | 20.0 g |
| 4-Methylumbelliferyl- ß -D-glucuronide (MUG) (optional) | 0.10 g |
| 1.6% Bromocresol purple | 0.94 mL |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
| Supplements | |
| * 1% Cefsulodin (1.0 g/100 mL) | 1.0 mL |
| 0.1% Potassium Tellurite (0.1g/100mL) | 2.5 mL |
Complete Medium:
Suspend basal ingredients in 1.0 L of distilled water, with stirring to dissolve all ingredients (except agar) and adjust pH. Heat to boiling to dissolve agar. Autoclave 15 min at 121oC. Temper to 45- 50oC. Filter sterilize supplements, and add aseptically to the medium. Gently swirl medium (without adding air bubbles) to dispense supplements thoroughly. Aseptically dispense agar into petri dishes and allow to solidify. Dry plates in a laminar flow hood to remove excess moisture. Store prepared plates at 4oC up to two weeks.
* Cefsulodin is not needed if the enrichment broth EEB is used.
Note: The supplements are commercially-available. If purchasing reconstituted product (liquid form), ensure that the final concentration in the above media is correct. For mHC agar, Potassium Tellurite is 2.5 mg/L.
| Basal Medium | |
|---|---|
| Columbia blood agar base (depending on the brand) | 39.0 - 44.0 g |
| Agar | 2.0 g |
| Esculin | 1.0 g |
| Ferric ammonium citrate | 0.5 g |
| Lithium chloride | 15.0 g |
| Distilled water | 1.0 L |
| Supplements | |
| Moxalactam | 0.015 g |
| Colistin sulfate | 0.01 g |
| Distilled water | 10.0 mL |
Complete Medium:
MOX agar is a slight modification of the Listeria selective agar (Oxford Formulation).
Add ingredients, except supplement, to distilled water. Boil gently to dissolve. Autoclave at 121oC for 15 min. Cool rapidly to 46oC in a water bath. Add filter sterilized supplement to the basal medium, mix well and dispense.
| Basal Medium | |
|---|---|
| Peptone | 20.0 g |
| Yeast extract | 3.0 g |
| 2% Alcoholic solution of bromocresol purple | 2.0 mL |
| Distilled water | 1.0 L |
| Supplement | |
| Alaska seed peas | 8-10/tube |
Complete Medium:
Dissolve ingredients with gentle heating, if necessary, and dispense 19.0 mL portions into 20 x 150 mm screw-cap culture tubes containing 8-10 untreated Alaska seed peas. Sterilize for 30 min at 121oC or an equivalent. Normally the prepared sterilized media can be maintained unaffected under refrigeration (0oC to 4oC) for up to 4 weeks. Prior to use, the refrigerated medium should be deaerated at 100oC for 10 min. Cool to ambient temperature prior to use.
| Basal Medium | |
|---|---|
| Tryptose | 4.59 g |
| Casein hydrolysate | 4.59 g |
| Sodium chloride (NaCl) | 7.34 g |
| Monopotassium phosphate (KH2PO4) | 1.47 g |
| Agar | 2.7 g |
| Magnesium chloride (MgCl2) | 10.93 g |
| Malachite green oxalate | 0.037 g |
| Distilled water | 1.0 L |
| pH 5.2 ± 0.2 | |
| Supplements | |
| Novobiocin | 20.0 mg |
| Distilled water | 10.0 mL |
Complete Medium:
Add basal ingredients to distilled water. Heat to boiling to dissolve. Do not autoclave. Cool to 50oC and aseptically add the 10 mL of novobiocin solution. Mix well and dispense 18.0 mL into sterile Petri dishes. Plates must be completely dry (1-3 h) before replacing the lids. They can be left overnight at room temperature before being refrigerated. Prepared MSRV plates must be stored in an upright position. They can be stored for up to 2 weeks at refrigeration temperatures in the dark. Examine plates as soon as possible after removing from the incubator as false-positive motility reactions can develop at room temperature.
Supplement: Dissolve the novobiocin in water, filter sterilize. Dispense in appropriate amounts and store frozen.
| Modified Sorbitol MacConkey Agar (TCCSMAC) | |
| Basal medium (Sorbitol MacConkey Agar) See Note below | |
| Peptone | Difco: 15.5 g [Oxoid: 20 g] |
| Proteose peptone | Difco: 3.0 g [Oxoid: 0 g] |
| Sorbitol | 10.0 g |
| Bile salts No 3 | 1.5 g |
| Sodium Chloride (NaCl) | 5.0 g |
| Neutral red | 0.03 g |
| Crystal violet | 0.001 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.1 ± 0.2 | |
| Supplements | |
| *1% Cefsulodin (1 .0 g/100mL) | 1.0 mL |
| Cefixime (1.0 mg/mL) | 50.0 µL |
| 0.1% Potassium Tellurite (0.1g/100mL) | 2.5 mL |
Complete Medium:
Suspend basal ingredients in 1.0 L of distilled water, with stirring to dissolve all ingredients (except agar) and adjust pH. Heat to boiling to dissolve agar. Autoclave 15 min at 121oC. Temper to 45- 50oC. Filter sterilize supplements, and add aseptically to the medium. Gently swirl medium (without adding air bubbles) to dispense supplements thoroughly. Aseptically dispense agar into petri dishes and allow to solidify. Dry plates in a laminar flow hood to remove excess moisture. Store prepared plates at 4oC up to two weeks.
* Cefsulodin is not needed if the enrichment broth EEB is used.
The supplements are commercially-available. If purchasing reconstituted product (liquid form), ensure that the final concentration in the above media is correct. For TCCSMAC agar, Potassium Tellurite is 2.5 mg/L and Cefixime is 0.050 mg/L.
| Basal Medium | |
|---|---|
| Tryptic Soy Broth or Tryptone Soya Broth | 30.0 g |
| Bile salts no. 3 | 1.5 g |
| Dipotassium phosphate (K2HPO4) | 1.5 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
| Supplement Novobiocin Solution (100 mg/mL) | |
| Novobiocin (sodium salt) | 100 mg |
| Distilled water | 1.0 mL |
Complete Medium:
Dissolve ingredients, except novobiocin, in distilled water. Heat to boiling and dispense. Autoclave for 15 min at 121oC. After cooling store at 4oC. Add 0.2 mL of novobiocin solution before use.
Supplement:
Dissolve novobiocin. Filter sterilize, using 0.2 µm filter and syringe. May be stored several months in a dark bottle at 4oC.
| Basal Medium | |
|---|---|
| Beef extract | 3.0 g |
| Pancreatic digest of gelatin | 10.0 g |
| NaCl | 5.0 g |
| Agar | 4.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Suspend ingredients in water and mix thoroughly. Heat with frequent agitation and boil 1 min to dissolve completely. Dispense and sterilize at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Pancreatic digest of gelatin | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Beef extract | 3.0 g |
| Agar | 3.5 g |
| TTC | 0.05 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Suspend ingredients in water and mix thoroughly. Heat with frequent agitation and boil 1 min to dissolve completely. Dispense and sterilize at 121oC for 15 min. This medium should be stored in an upright position at 4oC and protected from the light. Under these conditions the medium has a shelf life of 26 weeks from the date of manufacture.
Note: This medium can be prepared by adding TTC to the regular commercially available motility test medium
| Basal Medium | |
|---|---|
| Agar | 12.0 g |
| L-Lysine HCl | 5.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Sodium thiosulfate | 5.0 g |
| Yeast extract | 2.0 g |
| Magnesium sulfate heptahydrate (MgSO4.7H2O) |
1.5 g |
| Lactose | 1.25 g |
| Sucrose | 1.25 g |
| Xylose | 1.25 g |
| Ferric ammonium citrate | 0.80 g |
| Phenol red | 0.08 g |
| Nalidixic acid | 0.037 g |
| Kanamycin | 8.0 mg |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Add components to distilled water and bring the volume to 1.0 L. Mix thoroughly. Gently heat and bring to a boil. Autoclave for 15 min at 121oC and dispense.
MR-VP medium - See Buffered Glucose Broth and Buffered Glucose Salt Broth (Vibrio)
| Basal Medium | |
|---|---|
| Beef, infusion from | 300 g |
| Casamino acids, technical | 17.5 g |
| Soluble starch | 1.5 g |
| Agar | 17.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend ingredients in distilled water. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min. Cool and dispense.
* When used for halophilic Vibrio spp. add NaCl to a final concentration of 2-3%.
Mueller-Hinton Agar with Blood (commercially available)
Complete Medium:
Add 50.0 mL of sterile defibrinated horse blood per 950 mL of Mueller-Hinton agar (final 5% blood concentration)
