VIII. METHODS FOR TYPING MYCOBACTERIA
351
Autoclave at 15 lb pressure for 20 min.
Solution B
Glucose (anhydrous)
Distilled water
5.0 g
50 ml
Autoclave at 15 lb pressure for 15 min.
Solution C
M g S 0 ~ . 7 H z 0 (1 M stock solution) (page 352)
1 ml
CaClz (1 M stock solution) (page 352)
0.5 ml
Fe3 (stock solution) (page 352)
2 ml
Water
485 ml
Autoclave.
Solution D. Sodium glutamate (stock solution) (page 352), 5-0 ml.
Solution E
Bovine albumin (Fraction V)
NaOH (2.5 N)
0-70 ml
Distilled water
100 ml
2.5 g
Sterilise by Seitz filtration.
Mix solutions A, B, C, and D after sterilising. Cool to 47"C, then add
solution E. Dispense into tubes, 10 ml/tube.
The preparation of the RVB-10 medium should follow the procedure as
given above. Due to the fact that several of the components produce some
interaction when autoclaved together this procedure has been found
necessary, The stock solutions may be kept in the refrigerator for long
periods of time when sterile. Serum may be a satisfactory substitute for the
bovine albumin in the RVB-10 medium, but should not be used in the agar
medium as serum has an inhibitory effect on phage action.
C. Nutrient broth
sions of phage and bacteria. I t may be made as follows:
Nutrient broth is a satisfactory medium for making and storing suspenNutrient broth
NaCl
CaC12 (1 M solution) (page 352)
Water (distilled)
8 g
5 g
1.0 ml
1 litre
Adjust to pH 6.8-7.0 with 2.5 N NaOH and sterilise by autoclaving at
15 Ib pressure for 20 min. Store in refrigerator.