MTB Culture Medium.docx

Middlebrook 7H9 liquid medium (TB 7H9)ComponentPer LDistilled water897.5 mlsMiddlebrook 7H9 powder4.7g100% glycerol2 mls100% Tween 800.5 mls1. Mix the above reagents together aliquot as required (either 9 mls into 50ml conical tubes or 90mls in bottles) and autoclave 121C for 10 min.2. When cool add either 1 or 10 mls, respectively, of Albumin-dextrose-catalase (ADC).3. Incubate two aliquots at 37C for one week to detect contamination.Often Oleic-Albumin-Dextrose-Catalase (OADC) is used in the culture media to save stocking both ADC and OADC.top of pageMiddlebrook 7H10 Solid Medium (TB 7H10)ComponentPer LPer 400mlDistilled water895 mls358 mlsMiddlebrook 7H10 powder19 g7.6 g100% glycerol5 mls2 mls1. Add the above reagents together and autoclave 121C for 10 min. Mix when removed from the autoclave and cool to 50C.2. Add 100mls or 40ml, respectively, of Oleic-Albumin-Dextrose-Catalase (OADC) and antibiotics (if required) and pour 20 ml plates.3. Store plates at 4C and use within a month (plates containing some antibiotics may need to be used more rapidly).4. Incubate two plates at 37C for one week to detect contamination.top of pageMiddlebrook 7H11 Solid Medium (TB 7H11)ComponentPer LPer 400mlDistilled water895 mls358 mlsMiddlebrook 7H11 powder21 g8.4 g100% glycerol5 mls2 mls1. Add the above reagents together and autoclave 121C for 10 min. Mix when removed from the autoclave and cool to 50C.2. Add 100 mls or 40 mls, respectively, of Oleic-Albumin-Dextrose-Catalase (OADC) and antibiotics (if required) and pour 20 ml plates.3. Store plates at 4C and use within a month (plates containing some antibiotics may need to be used more rapidly).4. Incubate two plates at 37C for one week to detect contamination.top of pageAlbumin-dextrose-catalase (ADC)ComponentPer LDistilled water1 LNaCl8.5 gBSA fraction V50 gD-glucose20 gCatalase (beef)30 mg1. Dissolve NaCl and BSA in 900 mls of the distilled water, add D-Glucose and catalase and make up to a final volume of 1 ltr with distilled water.2. Spin at 1,500 x g ( 3,000 rpm on Econo spin, Sorvall instrument, Dupont with rotor H13223, located in the Media Making Unit) for 30 min to precipitate insoluble material.3. Sterilize the clarified solution by filtration through a 0.22 m-pore membrane and aliquot in 40 ml volumes.4. Incubate a sample at 37C overnight to detect possible contamination.5. Store at 4C for up to 4 months.top of pageOleic -Albumin-Dextrose-Catalase (OADC)ComponentPer LDistilled water1 LNaCl8.5 gBSA fraction V50 gD-glucose20 gCatalase (beef)40 mgOleic Acid (0.89 g/ml)0.56 ml1. Dissolve NaCl and BSA in 900 mls of the distilled water, add D-Glucose, catalase, and oleic acid and make up to a final volume of 1 ltr with distilled water.2. Spin at 1,500 x g ( 3,000 rpm on Econo spin, Sorvall instrument, Dupont with rotor H13223) for 30 min to precipitate insoluble material.3. Sterilize the clarified solution by filtration through a 0.22 m-pore membrane and aliquot in 40 ml volumes.4. Incubate a sample at 37C overnight to detect possible contamination.5. Store at 4C for up to 4 months.Alternative to making ADC and OADC is to purchase it pre-made.Difco makes it and Bioscientific Pty Ltd, or PLP distributes it.top of pageComponents of Middlebrook 7H10 and 7H11 base mediaComponent7H10(g per L)7H11(g per L)Ammonium sulphate0.50.5Monopotassium phosphate1.51.5Disodium phosphate1.51.5Sodium citrate0.40.4Magnesium sulphate0.0250.05Calcium chloride0.0005Zinc sulphate0.001Copper sulphate0.001L-glutamic acid (sodium salt)0.50.5Ferric ammonium citrate0