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Preparation of Selective Middlebrook- 7H9 Liquid Medium
Learning ObjectivesDescribe steps in media preparation
Liquid media are used in the mycobacteriology laboratory for sub-culturing stock strains, for storing strains at - 80°C and other in-vitro tests.
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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Assessment
Question |
Answer 1 |
Answer 2 |
Answer 3 |
Answer 4 |
Correct answer |
Correct explanation |
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Part of Pre-test |
Part of Post-te |
Liquid media are used for which of the following?
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Sub-culturing stock strains for storing strains at - 80°C | To perform in-vitro tests | Only option 1 | Both options 1 & 2 | Both options 1 & 2 |
Liquid media are used in the mycobacteriology laboratory for sub-culturing stock strains, for storing strains at - 80°C and other in-vitro tests.
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