VIII. METHODS FOR TYPING RTYCOBACTERIA
355
sterile tubes. Wash the surface with 1 ml of broth and add to the tube.
Centrifuge and remove the supernatant liquid with a sterile pipette.
Filter using a sintered glass or membrane filter to remove all the
bacteria. Store temporarily at 4°C. Make separate suspensions for each
phage variant that has been isolated.
C. Phage assay
Each suspension should be assayed to determine the number of plaque
forming units (PFU). Make eight ten-fold serial dilutions into nutrient
broth. Add 0.2 ml of each of the three highest dilutions to 2 ml of the host
bacteria diluted and suspended in nutrient broth. Spread 0-5 ml of each
suspension on one RVA-17 plate. Do not remove excess. Incubate at 37°C
and observe daily for lytic plaques. When there is optimal development of
the plaques, make accurate counts of plates containing approximately
100-200 distinct plaques. Multiply the number by the dilution factor for
this plate. This is the number of active phage particles (PFU), per millilitre
of the stock suspension. Label, giving the concentration in PFU of each
tube, seal and place in refrigerator at 4°C. With these standardised
stock suspensions, comparisons can be made with various phages and on
different host bacteria and they may be used, after determining the
routine test dilution (RTD) in phage typing of mycobacterial strains
(see below).
D. Phage mutants and adaptation to new hosts
Enrichment cultures. Although in a few cases phage has been found
in soil or other samples without enrichment with mycobacteria, this is a
rare occurrence. The logical assumption when phage is isolated from
enrichment mixtures is that a few phages were present in the sample and
that the number was increased through lysing of the bacteria that were
added. Variations in the methods and conditions of the experiments and
variations in the activity of the phages isolated make it very difficult to
evaluate the factors that may be active in these procedures. The consensus
is that adaptation and variation takes place in enrichment cultures (Redmond, 1963).
A procedure somewhat similar to soil enrichment has been used by
various workers in attempts to adapt phage to lyse certain bacteria not
previously susceptible. Some have been successful, some not. Many
workers think that adaptation to new hosts is a very likely means of
obtaining specific phage strains for typing purposes (Piguet, 1960; Hnatko,
1953).
This procedure has been used to adapt phage to lyse one of the strains of
355
sterile tubes. Wash the surface with 1 ml of broth and add to the tube.
Centrifuge and remove the supernatant liquid with a sterile pipette.
Filter using a sintered glass or membrane filter to remove all the
bacteria. Store temporarily at 4°C. Make separate suspensions for each
phage variant that has been isolated.
C. Phage assay
Each suspension should be assayed to determine the number of plaque
forming units (PFU). Make eight ten-fold serial dilutions into nutrient
broth. Add 0.2 ml of each of the three highest dilutions to 2 ml of the host
bacteria diluted and suspended in nutrient broth. Spread 0-5 ml of each
suspension on one RVA-17 plate. Do not remove excess. Incubate at 37°C
and observe daily for lytic plaques. When there is optimal development of
the plaques, make accurate counts of plates containing approximately
100-200 distinct plaques. Multiply the number by the dilution factor for
this plate. This is the number of active phage particles (PFU), per millilitre
of the stock suspension. Label, giving the concentration in PFU of each
tube, seal and place in refrigerator at 4°C. With these standardised
stock suspensions, comparisons can be made with various phages and on
different host bacteria and they may be used, after determining the
routine test dilution (RTD) in phage typing of mycobacterial strains
(see below).
D. Phage mutants and adaptation to new hosts
Enrichment cultures. Although in a few cases phage has been found
in soil or other samples without enrichment with mycobacteria, this is a
rare occurrence. The logical assumption when phage is isolated from
enrichment mixtures is that a few phages were present in the sample and
that the number was increased through lysing of the bacteria that were
added. Variations in the methods and conditions of the experiments and
variations in the activity of the phages isolated make it very difficult to
evaluate the factors that may be active in these procedures. The consensus
is that adaptation and variation takes place in enrichment cultures (Redmond, 1963).
A procedure somewhat similar to soil enrichment has been used by
various workers in attempts to adapt phage to lyse certain bacteria not
previously susceptible. Some have been successful, some not. Many
workers think that adaptation to new hosts is a very likely means of
obtaining specific phage strains for typing purposes (Piguet, 1960; Hnatko,
1953).
This procedure has been used to adapt phage to lyse one of the strains of