Preparation of Selective Middlebrook- 7H9 Liquid Medium

Liquid media are used in the mycobacteriology laboratory for sub-culturing stock strains, for storing strains at - 80°C and other in-vitro tests. Mycobacteria tend to clump in a liquid medium. Therefore,  a wetting agent like Tween 80 must be included in the basal medium. It encourages more homogenous growth. Commercially available Middlebrook 7H9 media, along with growth supplement OADC, can be utilized for this purpose.

Steps in Preparation of Selective Middlebrook- 7H9 Liquid Medium

Salt solution: Disodium anhydrous hydrogen phosphate (Na₂HPO₄): 2. 5g 

                      Potassium dihydrogen orthophosphate (KH₂PO₄): 1. 0 g 

                      Ammonium sulphate ( NH4SO4) : 0. 5g 

                      L-sodium glutamate : 0. 5g 

                     Trisodium citrate (2H2O) : 0. 1g 

                      Pyridoxine hydrochloride: 1. 0 ml of 0. 1% aq. soln. 

                      Biotin : 1. 0 ml of 0. 05% aq. soln. 

                      Ferric ammonium citrate (green) : 0. 5 ml of 8% aq. solution 

                      Magnesium sulphate (MgSO4.7H2O): 1. 0 ml of 5% aq. solution 

                      Calcium chloride (CaCl2.2H2O): 1. 0 ml of 0. 05% aq. solution 

                     Zinc sulphate (ZnSO4.7H2O): 1. 0 ml of 0. 1% aq. solution 

                     Cupric sulphate (CuSO4.5H2O): 1. 0 ml of 0. 1% aq. solution

                     Tween-80, 10% (For obtaining dispersed cultures): 5. 0 ml (Or) Glycerol: 5. 0 ml Distilled water to 900 ml.

                     Mix well, distribute in 95 ml amounts and sterilize at 15 lbs/15 mins.

The salt solution can also be prepared by using Difco dehydrated powder.

Weigh 4. 7 g of the dehydrated base into a 2-litre flask, and add 900 ml distilled water and 0. 5 ml of Tween-80 or Glycerol. Mix well, distribute in 95 ml amounts and autoclave at 15 lbs/15 minutes.

Before use, to each 95 ml salt solution, aseptically add 5 ml sterile ADC (bovine albumin-dextrose-catalase) solution and mix well.

Distribute in 5-10 ml amounts in sterile universal containers, check sterility by overnight incubation at 37 ^oC and store in the cold.

ADC supplement

Bovine albumin, Fraction V: 10g. 

Glucose, A. R. (dextrose): 4g. 

Catalase : 3 mg* 

*Dissolve 30 mg catalase in 10 ml water by vigorous shaking and add 1 ml of this solution. 

Distilled water: 100ml.

Mix well and sterilize by Seitz filtration or membrane filtration.

Resource

      STANDARD OPERATING PROCEDURE FOR MYCOBACTERIOLOGY LABORATORY

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