VIII. METHODS FOR TYPING MYCOBACTERIA
353
Mix the two above suspensions, when cooled addt mixture no 1, 100 ml;
mixture no. 2, 100 ml; mixture no. 4, 50 ml; Na glutamate (stock), 5.0 ml;
bovine albumin mixture, 100 ml.
To make RVA-17: add 8-0 ml glycerol, 2.0 ml ZC1 (stock) to 150 ml
water; autoclave.
Dissolve 11.0 g agar in 500 ml water by warming. Add 1 ml Mg4
(stock), 1 ml CaClz (1 M) (stock), and 1 ml Fe3 (stock). Autoclave for
20 min at 15 lb pressure. Mix the above two solutions.
Add mixture no. 1, 100 ml; mixture no. 3, 100 ml; mixture no. 4, 50 ml;
5 ml Na glutamate (stock); and oleic acid albumin, 100 ml. Mix all solutions so as to maintain temperature at 4643°C when oleic acid albumin is
added. Do not allow agar to solidify. Dispense into Petri dishes.
111. BACTERIOPHAGE
A. Isolation of phages
Mycobacterial phages appear to be widespread and can be isolated with
little difficulty. Soil, human excreta, polluted water and various biopsy
specimens, as well as other sources, have yielded phages on each of several
occasions. It has been necessary in most instances to enrich soil samples
with one or more strains of mycobacteria in order to obtain lytic plaques
(Cater and Redmond, 1963; Takeya and Yoshimura, 1957).
The procedure is as follows. T o approximately SO g of soil sample add
50 ml of medium (NB3 or RVB-10 without Tween-SO) and 10 ml of culture
of a strain of mycobacterium that is likely to serve as host or indicator strain
to the phage. ATCC 607 has been used extensively. It is easily grown and
readily available. Repeat the addition of 10 ml of the same culture at
intervals of one to two times each week for four or five weeks, keeping the
flask stoppered with a porous substance and incubate at 37°C. The contents
of the flask should be thoroughly mixed at least each time bacteria are
added. Organisms washed from solid medium containing Malachite green
(or other inhibiting substance) should not be used. Two or more strains of
mycobacteria may be used for enrichment and in this case each bacterial
strain should be used for testing for phage. About one week after the last
enrichment, add 25-SO ml of nutrient broth to the flask, mix thoroughly
and centrifuge to eliminate the debris. Pipette off the supernateant liquid
and filter through a sintered glass filter or membrane filter with 0.45 pm
porosity. Seitzfiltersretainmost of the phage. This phage filtrate may be stored
in the refrigerator at 4°C for some length of time for subsequent testing.
t Mix so as to avoid bacterial contamination. Dispense into caltnre tubes,
7.5 or 10 ml as desired.
353
Mix the two above suspensions, when cooled addt mixture no 1, 100 ml;
mixture no. 2, 100 ml; mixture no. 4, 50 ml; Na glutamate (stock), 5.0 ml;
bovine albumin mixture, 100 ml.
To make RVA-17: add 8-0 ml glycerol, 2.0 ml ZC1 (stock) to 150 ml
water; autoclave.
Dissolve 11.0 g agar in 500 ml water by warming. Add 1 ml Mg4
(stock), 1 ml CaClz (1 M) (stock), and 1 ml Fe3 (stock). Autoclave for
20 min at 15 lb pressure. Mix the above two solutions.
Add mixture no. 1, 100 ml; mixture no. 3, 100 ml; mixture no. 4, 50 ml;
5 ml Na glutamate (stock); and oleic acid albumin, 100 ml. Mix all solutions so as to maintain temperature at 4643°C when oleic acid albumin is
added. Do not allow agar to solidify. Dispense into Petri dishes.
111. BACTERIOPHAGE
A. Isolation of phages
Mycobacterial phages appear to be widespread and can be isolated with
little difficulty. Soil, human excreta, polluted water and various biopsy
specimens, as well as other sources, have yielded phages on each of several
occasions. It has been necessary in most instances to enrich soil samples
with one or more strains of mycobacteria in order to obtain lytic plaques
(Cater and Redmond, 1963; Takeya and Yoshimura, 1957).
The procedure is as follows. T o approximately SO g of soil sample add
50 ml of medium (NB3 or RVB-10 without Tween-SO) and 10 ml of culture
of a strain of mycobacterium that is likely to serve as host or indicator strain
to the phage. ATCC 607 has been used extensively. It is easily grown and
readily available. Repeat the addition of 10 ml of the same culture at
intervals of one to two times each week for four or five weeks, keeping the
flask stoppered with a porous substance and incubate at 37°C. The contents
of the flask should be thoroughly mixed at least each time bacteria are
added. Organisms washed from solid medium containing Malachite green
(or other inhibiting substance) should not be used. Two or more strains of
mycobacteria may be used for enrichment and in this case each bacterial
strain should be used for testing for phage. About one week after the last
enrichment, add 25-SO ml of nutrient broth to the flask, mix thoroughly
and centrifuge to eliminate the debris. Pipette off the supernateant liquid
and filter through a sintered glass filter or membrane filter with 0.45 pm
porosity. Seitzfiltersretainmost of the phage. This phage filtrate may be stored
in the refrigerator at 4°C for some length of time for subsequent testing.
t Mix so as to avoid bacterial contamination. Dispense into caltnre tubes,
7.5 or 10 ml as desired.