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Appendix G
Media Glossary
February 2006
Glossary of agars, broths and other reagents
Section T
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(PDF Version - 45 K)T1N0, T1N3 T1N2, T1N3 Medium - See Tryptone Salt Agar
| Basal Medium | |
|---|---|
| Tetracycline | 50. 0 mg |
| Gelatin phosphate diluent (see formula) | 25.0 mL |
Complete Medium:
Dissolve tetracycline in gelatin phosphate diluent. Store at 4oC up to 4 days. For longer storage, freeze in small portions. Avoid re-freezing.
| Basal Medium | |
|---|---|
| Peptone/proteose peptone | 5.0 g |
| Bile salts | 1.0 g |
| Calcium carbonate (CaCO3) | 10.0 g |
| Sodium thiosulphate (Na2S2O3) | 30.0 g |
| Distilled water | 1.0 L |
| pH 8.4 ± 0.2 | |
| Supplements | |
| Brilliant green dye | 1 g |
| Distilled water | 100 mL |
| Potassium iodide (KI) | 25.0 g |
| Iodine (I2) | 30.0 g |
| Distilled water | 100 mL |
Complete Medium:
Suspend ingredients of basal medium in 1.0 L of distilled water and heat to boiling. Cool to 45-50oC. Prepare the complete medium on the day of use. Aseptically add 1.0 mL of 1% brilliant green solution and 20.0 mL of iodine solution to 1L of basal medium. Mix thoroughly and aseptically dispense. Do not heat the medium after the addition of iodine.
Brilliant green solution (1% w/v): Dissolve brilliant green dye in 100 mL of distilled water.
Iodine solution: Suspend potassium iodide and iodine in 100 mL of distilled water. Do not heat. The iodine solution should be prepared well in advance because iodine is slow to dissolve.
| Basal Medium | |
|---|---|
| Yeast extract | 5.0 g |
| Proteose peptone | 5.0 g |
| Dextrose | 5.0 g |
| Dipotassium phosphate (K2HPO4) | 4.0 g |
| Agar | 20.0 g |
| Distilled water | 1.0 L |
| pH 5.0 ± 0.2 | |
Complete Medium:
Suspend ingredients in 1 L of distilled water. Heat to boiling to dissolve completely. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Peptone | 10.0 g |
| Yeast extract | 5.0 g |
| Sodium citrate | 10.0 g |
| Sodium thiosulphate.5 H2O (Na2S2O3•5 H2O) | 10.0 g |
| Sodium chloride (NaCl) | 10.0 g |
| Ox-gall powder | 5.0 g |
| Ferric citrate (FeC6H5O7) | 1.0 g |
| Sodium cholate | 3.0 g |
| Sucrose | 20.0 g |
| Thymol blue solution (0.2%) | 20.0 mL |
| Bromothymol blue solution (0.2%) | 20.0 mL |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 8.6 ± 0.2 | |
Complete Medium:
Suspend basal ingredients in 1 L of distilled water. Heat to boil to dissolve completely. DO NOT AUTOCLAVE. Cool to 45-50oC and dispense.
| Basal Medium | |
|---|---|
| Deoxyribonucleic acid (DNA) 1 | 0.3 g |
| Calcium chloride (CaCl2) | 1.1 mg |
| Sodium chloride (NaCl) | 10.0 g |
| Toluidine blue O2 | 0.08 g |
| Agar | 10.0 g |
| 0.05M Tris, pH 9.0 (see below) | 1.0 L |
Complete Medium:
Add all basal ingredients, except Toluidine blue O, to the Tris buffer and boil to dissolve the DNA and agar. Add Toluidine blue O to the solution and dispense in 50.0 to 100 mL volumes in flasks or bottles; seal tightly. Sterilization is not necessary. Store at room temperature in the dark. The medium is stable at room temperature for as long as 4 months and remains satisfactory even after several melting cycles.
Note:
1 All sources of DNA should be checked using a boiled supernatant of a 24 h BHI broth culture of TNase producing S. aureus. The DNA would be considered suitable if a DNA hydrolysis zone of ≥ 8.0 mm is observed with a 5.0 mL aliquot of the boiled supernatant after 4 h incubation at 35oC.
2 Toluidine blue O should also be checked against a suitable standard before being used with unknowns as the efficacy of the dye varies with the manufacturer.
| Basal Medium | |
|---|---|
| Peptone | 10.0 g |
| Beef extract | 10.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Yeast extract | 1.0 g |
| Trimethylamine N-Oxide (TMAO) dihydrate | 1.0 g |
| Distilled water | 1.0 L |
| Agar | 2.0 g |
| pH 7.5 ± 0.2 | |
Complete Medium:
Dissolve all ingredients, except agar, in 1 L of distilled water. Add agar and heat to dissolve. Dispense 4.0 mL into 13 x 100 mm screw-cap tubes. Autoclave at 121oC for 15 min. Cool with tubes upright.
| Basal Medium | |
|---|---|
| 2,3,5, Triphenyltetrazolium Chloride (TTC) | 1.0 g |
| Distilled water | 100.0 mL |
Complete Medium:
Suspend TTC in 100 mL of distilled water. Sterilize by filtration. Aseptically dispense into containers. Store refrigerated.
For TSA-TCC, aseptically add 10 mL to 1 L of sterilized and cooled TSA and mix thoroughly. Dispense into petri plates.
For 0.1% TTC staining solution, add 10 mL of 1% TTC to 90 mL of distilled water and mix thoroughly.
| Basal Medium | |
|---|---|
| Beef extract | 3.0 g |
| Yeast extract | 3.0 g |
| Peptone | 15.0 g |
| Proteose peptone | 5.0 g |
| Lactose | 10.0 g |
| Sucrose | 10.0 g |
| Dextrose | 1.0 g |
| Ferrous sulfate (FeSO4•7H2O) | 0.2 g |
| Sodium chloride (NaCl) | 5.0 g |
| Sodium thiosulfate (Na2S2O3) | 0.3 g |
| Agar | 12.0 g |
| Phenol red | 0.024 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
Complete Medium:
Suspend basal ingredients in 1.0 L of distilled water and heat to boiling to dissolve completely. Dispense 8 mL into tubes and autoclave at 121oC for 15 min. Cool tubes in a slanted position to form deep butts.
| Basal Medium | |
|---|---|
| TSI agar dry basal ingredients | 65.0 g |
| Sodium chloride (NaCl) | 25.0 g |
| Distilled water | 1.0 L |
| pH 7.4 ± 0.2 | |
Complete Medium:
Suspend basal ingredients in 1.0 L of distilled water and heat to boiling to dissolve completely. Dispense 8 mL into tubes and autoclave at 121oC for 15 min. Cool tubes in a slanted position to form deep butts.
| Basal Medium | |
|---|---|
| Tris (Hydroxymethyl) Aminomethane (F.W. 121.14) | 6.06 g |
| Distilled water | 1.0 L |
| pH 9.0 | |
Complete Medium:
Dissolve Tris (Hydroxymethyl) Aminomethane into 1.0 L of distilled water. Adjust pH to 9.0 with 0.2N HCl using an electrode.
| Basal Medium | |
|---|---|
| Tris (Hydroxymethyl) Aminomethane | 2.42 g |
| Sodium chloride ( NaCl ) | 29.24 g |
| Distilled water | 1.0 L |
| pH 7.5 | |
Complete Medium:
Dissolve Tris (Hydroxymethyl) Aminomethane into 1.0 L of distilled water. Adjust pH to 9.0 with 0.2N HCl using an electrode.
| Basal Medium | |
|---|---|
| Gelatin | 1.0 g |
| Tris-Buffered Saline, pH 7.5 (see above) | 100 mL |
Complete Medium:
Heat TBS to 40oC. Add gelatin and stir to dissolve. Cool to room temperature before use.
| Basal Medium | |
|---|---|
| Gelatin | 3.0 g |
| Tris-Buffered Saline, pH 7.5 (see above) | 100 mL |
Complete Medium:
Heat TBS to 40oC. Add gelatin and stir to dissolve. Cool to room temperature before use.
| Basal Medium | |
|---|---|
| Tween 20 | 50 µL |
| Tris-Buffered Saline, pH 7.5 (see above) | 100 mL |
Complete Medium:
Suspend Tween 20 in 100 mL of TBS and stir to dissolve.
| Basal Medium | |
|---|---|
| Trypsin (1:250)* | 0.05 g |
| EDTA, disodium salt | 0.02 g |
| NaCl, 0.9% | 100 mL |
Complete Medium:
Dissolve trypsin and EDTA in the NaCl diluent. Filter sterilize.
Note: * 1 g Trypsin (1:250) will digest 250 g casein substrate under standard conditions.
| Basal Medium | |
|---|---|
| 1% Trypsin solution | |
| Trypsin | 200 mg |
| Distilled water | 20.0 mL |
| 1.4% Trypsin solution | |
| Trypsin | 280 mg |
| Gelatin phosphate | 20.0 mL |
Complete Medium:
1% solution : Dissolve 200 mg in 20.0 mL distilled water. Do not store.
1.4% solution : 280 mg in 20.0 mL gelatin phosphate. Filter through a 0.45 : m membrane. Use vacuum for filtration to effect partial deaeration of the filtrate.
Note: In the following media formulations the words tryptone, tryptic or trypticase peptone refer to a pancreatic digest of casein and the words Soya peptone, Soy or Phyto peptone refer to a papaïc digest of soybean meal.
| Basal Medium | |
|---|---|
| Trypticasea | 50.0 g |
| Peptone | 5.0 g |
| Glucose | 4.0 g |
| Yeast extract | 20.0 g |
| Sodium thioglycolate (C2H3O2SNa) | 1.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
a Manufactured by Oxoid as Special Peptone L72.
Complete Medium:
Suspend basal medium ingredients in 1.0 L of hot distilled water. Cool and adjust the pH. Dispense in 21.0 mL volumes into 20 x 150 mm screw-cap tubes. Autoclave at 121oC for 10 min. If stored in excess of 12 h, the medium must be deaerated (at 100oC for 10 min) and cooled before use.
| Basal Medium | |
|---|---|
| Tryptone | 15.0 g |
| Soya peptone | 5.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Agar | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Do not overheat. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 17.0 g |
| Soya peptone | 3.0 g |
| Dextrose | 2.5 g |
| Sodium chloride (NaCl) | 5.0 g |
| Dipotassium phosphate (K2HPO4 ) | 2.5 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Dispense and autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 17.0 g |
| Soya peptone | 3.0 g |
| Sodium chloride (NaCl) | 15.0 g |
| Dipotassium phosphate (K2HPO4) | 2.5 g |
| Glycerol | 240 mL |
| Distilled water | 760 mL |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Dispense and autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 17.0 g |
| Soya peptone | 3.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Fast green FCF | 0.25 g |
| Agar | 1 5.0 g |
| Dipotassium phosphate (K2HPO4) | 2.5 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 15.0 g |
| Soya peptone | 5.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Magnesium sulfate heptahydrate (MgSO47H2O) | 1.5 g |
| Agar | 1 5.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min.
Note: Tryptic soy agar is commercially available, to which MgSO4 A7H2O (1.5 g/L) can be added.
| Basal Medium | |
|---|---|
| Trypticase soy agar (see above) | 40.0 g |
| 1% TTC Solution (2,3,5-Triphenyltetrazolium Chloride) | 10 mL |
| Distilled water | 990 mL |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 990 mL of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min. Aseptically add 1% TTC solution and mix thoroughly. Pour into plates.
| Basal Medium | |
|---|---|
| Trypticase soy agar (see above) | 40.0 g |
| Yeast extract | 6.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Dispense into tubes or flasks and autoclave at 121oC for 15 min. Leave in tubes or pour into sterile Petri dishes.
| Basal Medium | |
|---|---|
| Trypticase soy broth (see above) | 30.0 g |
| Yeast extract | 6.0 g |
| Distilled water | 1.0 L |
| pH 7.3 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Dispense and autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 10.0 g |
| Distilled water | 1.0 L |
| pH to 7.0 ± 0.2 | |
Complete Medium:
Dissolve tryptone in 1.0 L of distilled water. Dispense in 5.0 mL portions and autoclave at 121oC for 15 min.
| Basal Medium | |
|---|---|
| Tryptone | 20.0 g |
| Bile salts no. 3 | 1.5 g |
| Agar | 15 g |
| Distilled water | 1.0 L |
| pH to 7.2 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min. Pour 15-20 mL per plate on a flat surface.
| Basal Medium | |
|---|---|
| Tryptone | 10.0 g |
| Sodium chloride (NaCl) | 0.0, 10.0, 30.0, 60.0, 80.0 or 100.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. For T1N0, add no NaCl; for T1N3, add 10 g NaCl (1% w/v NaCl); for T1N3, add 30 g NaCl (3% w/v NaCl), etc. Dispense into 16x125 mm screw-cap tubes. Tighten tubes to maintain the correct concentration in tube. Autoclave at 121oC for 15 min .
| Basal Medium | |
|---|---|
| Tryptone | 10.0 g |
| Sodium chloride (NaCl) | 10.0 g |
| Agar | 20.0 g |
| Distilled water | 1.0 L |
| pH 7.1 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min, cool and dispense.
Note: * For T1N0, T1N2, T1N3 use 10.0 g of tryptone and 0.0 g, 20.0 g and 30.0 g of NaCl respectively.
| Basal Medium | |
|---|---|
| Tryptose | 20.0 g |
| Sodium chloride (NaCl) | 5.0 g |
| Dextrose | 1.0 g |
| Agar (leave out of broth formula) | 15.0 g |
| Distilled water | 1.0 L |
| pH 7.2 ± 0.2 | |
Complete Medium:
Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling with constant stirring to dissolve all ingredients. Autoclave at 121oC for 15 min, cool and dispense. For agar, make generous slants.
