354
w. B. REDMOND et al.
Make three ten-fold serial dilutions of the filtrate into nutrient broth.
Using fresh cultures of the strain, or strains, of bacteria with which the
flasks were enriched make dilutions containing sufficient organisms to
produce an easily observable but slight turbidity (about 0-1-0.2 optical
density). The concentration will depend on the rate of growth of the organism. Inoculate 2 ml of bacterial suspension with 0.2 ml of each of the four
phage dilutions and spread about 0.5 ml on an agar plate (RVA-17) so as
to cover the entire surface. The exact amounts will vary depending on the
dryness of the plates. Incubate the plates at 36-37°C and examine daily for
cleared spots (plaques). On plates of rapidly growing bacteria, such as
ATCC 607, the plaques may disappear after one or two days due to the
rapid overgrowth of plaques by the bacteria. I n other instances, the plaques
may continue to enlarge for several days after appearing.
Isolate material from selected plaques with a sterile needle into small
amounts of nutrient broth. Make dilutions of each and replate as before
using also other strains of mycobacteria to determine lytic activity. Examine
each plate carefully for variant plaques and make individual isolations from
each type of plaque. Plaque variants may be in the form of large or small,
as well as clear or turbid plaques. There may be distinct differences in the
margin being either abrupt or gradual, or consisting of a distinct halo.
Phage isolated from each plaque type should be tested for lysis on several
host strains.
If no plaques are observed on the original plates with the dilutions used
test larger samples of the filtrate, e.g. 1 ml of filtrate mixed with 1 ml of
bacterial suspension. Also the undiluted filtrate may be spotted on lawns
of suitable bacterial strains.
Stool specimens, polluted water and other types of materials that are
contaminated with mycobacteria may produce phage when treated in a
similar manner. Material containing large numbers of mycobacteria may
yield phage without being enriched. However, sputum specimens rarely
have given rise to phage.
B. Phage propagation and suspensions
Once a phage strain is established on a susceptible host, it should be
propagated and a stock suspension obtained. Make several ten-fold serial
dilutions of a good phage suspension and spread each with its host as indicated above to determine the concentration necessary to produce just
complete lysis of the host. Using this concentration, inoculate several
plates and recover the phage when lysis is complete by flooding each plate
with 4 or 5 ml of nutrient broth. Allow to remain at room, or refrigerator,
temperature for 2-3 h. Add 2-3 ml of fresh nutrient broth to each plate
and agitate and scrape (mix) the lysed bacteria from the surface. Pipette into
w. B. REDMOND et al.
Make three ten-fold serial dilutions of the filtrate into nutrient broth.
Using fresh cultures of the strain, or strains, of bacteria with which the
flasks were enriched make dilutions containing sufficient organisms to
produce an easily observable but slight turbidity (about 0-1-0.2 optical
density). The concentration will depend on the rate of growth of the organism. Inoculate 2 ml of bacterial suspension with 0.2 ml of each of the four
phage dilutions and spread about 0.5 ml on an agar plate (RVA-17) so as
to cover the entire surface. The exact amounts will vary depending on the
dryness of the plates. Incubate the plates at 36-37°C and examine daily for
cleared spots (plaques). On plates of rapidly growing bacteria, such as
ATCC 607, the plaques may disappear after one or two days due to the
rapid overgrowth of plaques by the bacteria. I n other instances, the plaques
may continue to enlarge for several days after appearing.
Isolate material from selected plaques with a sterile needle into small
amounts of nutrient broth. Make dilutions of each and replate as before
using also other strains of mycobacteria to determine lytic activity. Examine
each plate carefully for variant plaques and make individual isolations from
each type of plaque. Plaque variants may be in the form of large or small,
as well as clear or turbid plaques. There may be distinct differences in the
margin being either abrupt or gradual, or consisting of a distinct halo.
Phage isolated from each plaque type should be tested for lysis on several
host strains.
If no plaques are observed on the original plates with the dilutions used
test larger samples of the filtrate, e.g. 1 ml of filtrate mixed with 1 ml of
bacterial suspension. Also the undiluted filtrate may be spotted on lawns
of suitable bacterial strains.
Stool specimens, polluted water and other types of materials that are
contaminated with mycobacteria may produce phage when treated in a
similar manner. Material containing large numbers of mycobacteria may
yield phage without being enriched. However, sputum specimens rarely
have given rise to phage.
B. Phage propagation and suspensions
Once a phage strain is established on a susceptible host, it should be
propagated and a stock suspension obtained. Make several ten-fold serial
dilutions of a good phage suspension and spread each with its host as indicated above to determine the concentration necessary to produce just
complete lysis of the host. Using this concentration, inoculate several
plates and recover the phage when lysis is complete by flooding each plate
with 4 or 5 ml of nutrient broth. Allow to remain at room, or refrigerator,
temperature for 2-3 h. Add 2-3 ml of fresh nutrient broth to each plate
and agitate and scrape (mix) the lysed bacteria from the surface. Pipette into