356
w. B. REDMOND et al.
M. kansasii. Four phage strains, each with lytic activity on one of four
strains of bacteria, were inoculated into a flask containing medium free of
Tween-80 that had been inoculated with the four bacterial hosts and M .
kansasii. On day 5 or 6, subcultures were made and fresh suspensions of
bacteria added. This procedure was repeated four or five times following
which the mass was transferred to tubes and centrifuged. The supernataut liquid was filtered, using an ultrafine sintered glass filter. The
filtrate was diluted and each dilution mixed with a suspension of M.
kansasiiand spread on a RVA-17 plate. Also, small drops of each suspension
were placed on a lawn of M . kansasii. A few plaques were observed on two
of the plates. Phage was isolated from these plaques and plated on M.
kansasii as host. Repeated isolations and platings resulted in phage AG1
with good lytic action on this bacterium.
Adaptation by heavy inoculation of phage. Another adaptive
method that has been used with success is the heavy inoculum technique.
An undiluted phage suspension is inoculated in ratios of 0.1 to 10,0.5 to 10
and 1 to 10 (or more) into a good suspension of the resistant bacteria
desired to be lysed. Each mixture is then spread on an agar plate. This
procedure may result in non-specific lysis with no phage production. A few
clear plaques will indicate true phage lysis, and subsequent isolations of
phage from these plaques should produce good lytic activity.
Ultraviolet irradiation and other mutagenic agents. Phages
treated with ultraviolet irradiation either before applying to a bacterial
host or by irradiation of the infected bacteria is likely to produce variants
(mutants) with altered specificity. It has been indicated that irradiation of a
resistant bacterium followed by infection with a non-infecting phage is
likely to produce a phage with lytic action. In attempts of this type, heavy
inoculation of phage is found to produce best results. There are without
doubt mutant phages in all heavy suspensions of phage which may produce
lysis on a strain of bacteria resistant to the phage mass.
Phage recombinants. Since most bacteria are thought to be lysogenic,
most of which are carrying defective prophages not capable of producing
lysis, any phage to which the bacterium is resistant but which is capable of
entering the bacterial cell may combine with the prophage genome and
produce a recombinant phage that may be lytic. Two different phages
infecting one bacterium may result in a recombinant phage that differs
from each of the parent phages.
T o summarise these concepts of adaptation, it is possible that any heavy
suspension of phage may contain a few mutant phage particles capable of
lysing non-susceptible bacteria. Also many phages enter bacteria but are
incapable of producing lysis. These may combine with a phage genome
carried by the bacterium and result in a new lytic phage.
w. B. REDMOND et al.
M. kansasii. Four phage strains, each with lytic activity on one of four
strains of bacteria, were inoculated into a flask containing medium free of
Tween-80 that had been inoculated with the four bacterial hosts and M .
kansasii. On day 5 or 6, subcultures were made and fresh suspensions of
bacteria added. This procedure was repeated four or five times following
which the mass was transferred to tubes and centrifuged. The supernataut liquid was filtered, using an ultrafine sintered glass filter. The
filtrate was diluted and each dilution mixed with a suspension of M.
kansasiiand spread on a RVA-17 plate. Also, small drops of each suspension
were placed on a lawn of M . kansasii. A few plaques were observed on two
of the plates. Phage was isolated from these plaques and plated on M.
kansasii as host. Repeated isolations and platings resulted in phage AG1
with good lytic action on this bacterium.
Adaptation by heavy inoculation of phage. Another adaptive
method that has been used with success is the heavy inoculum technique.
An undiluted phage suspension is inoculated in ratios of 0.1 to 10,0.5 to 10
and 1 to 10 (or more) into a good suspension of the resistant bacteria
desired to be lysed. Each mixture is then spread on an agar plate. This
procedure may result in non-specific lysis with no phage production. A few
clear plaques will indicate true phage lysis, and subsequent isolations of
phage from these plaques should produce good lytic activity.
Ultraviolet irradiation and other mutagenic agents. Phages
treated with ultraviolet irradiation either before applying to a bacterial
host or by irradiation of the infected bacteria is likely to produce variants
(mutants) with altered specificity. It has been indicated that irradiation of a
resistant bacterium followed by infection with a non-infecting phage is
likely to produce a phage with lytic action. In attempts of this type, heavy
inoculation of phage is found to produce best results. There are without
doubt mutant phages in all heavy suspensions of phage which may produce
lysis on a strain of bacteria resistant to the phage mass.
Phage recombinants. Since most bacteria are thought to be lysogenic,
most of which are carrying defective prophages not capable of producing
lysis, any phage to which the bacterium is resistant but which is capable of
entering the bacterial cell may combine with the prophage genome and
produce a recombinant phage that may be lytic. Two different phages
infecting one bacterium may result in a recombinant phage that differs
from each of the parent phages.
T o summarise these concepts of adaptation, it is possible that any heavy
suspension of phage may contain a few mutant phage particles capable of
lysing non-susceptible bacteria. Also many phages enter bacteria but are
incapable of producing lysis. These may combine with a phage genome
carried by the bacterium and result in a new lytic phage.