156
A. SARAGEA ET AL.
into a tube containing 9 ml cystinated (0.001%) tryptic digest broth
(Pope Linggood) or heart broth and the liquid culture is shaken for 24 h
at 35°C. The following day, 1 ml of a 0.7 M sodium citrate solution is
added in order to obtain a final concentration of 0.07 M sodium citrate in
the medium (Gundersen and Henriksen, 1959). The addition of sodium
citrate will prevent readsorption of the liberated phage on to the bacteria.
After the addition of sodium citrate, the culture is shaken again for 1 h
at 35"C, with a view to obtaining the maximum yield of phage from the
lysogenic cells. The culture is then centrifuged at 3000 rev/min for 20-30
min and the decanted fluid is tested against a wide range of available
sensitive C. diphtheriae strains. T h e wider the range of sensitive strains
the better the chance of obtaining lysogenic phages.
Sensitive indicator strains of both schemes and even other multisensitive
strains are available for checking lysogeny.
2. Preparation of the stock phage in the sensitive (indicator) strains
Overnight cultures of the sensitive strains on Loeffler slants are
inoculated into 4-5 ml broth and incubated for 18 h at 35°C. Brain-heart
agar plates (as for phage typing) are flooded with the sensitive strains and
dried for 45 min. Every supernatant is dropped on to predetermined areas
recorded on the backs of the plates. The plates are incubated for 24 h at
35°C. If any degree of lysis occurs over the area of the spot, even isolated
plaques, they are cut out, inoculated into a tube with 5 m l broth, and
shaken overnight. After centrifugation and the addition of chloroform (for
2-3 h), the supernatant is again spotted on the sensitive strain. If propagation is successful, the suspension is titrated and checked for host range.
B. Induction of lysogenic phages by ultraviolet irradiation
An overnight fluid culture is diluted to an opacity of 0.3, shaken for 1 h,
and submitted to ultraviolet irradiation. The layer should not be deeper
than 2-3 mm. A distance of 76 cm and time of exposure of 135 s is used
with the lamps available in our laboratory, but preliminary experiments
will be needed to determine optimal conditions for other equipment. The
plate is shaken continuously during irradiation. The irradiated fluid is
transferred to a flask in the dark and again shaken for 3 h at 35°C before
centrifugation, chloroform treatment and testing lytic activity. As control,
a known lysogenic strain is always included to test the effectiveness of the
ultraviolet exposure (Barksdale and Pappenheimer, 1954).
A. SARAGEA ET AL.
into a tube containing 9 ml cystinated (0.001%) tryptic digest broth
(Pope Linggood) or heart broth and the liquid culture is shaken for 24 h
at 35°C. The following day, 1 ml of a 0.7 M sodium citrate solution is
added in order to obtain a final concentration of 0.07 M sodium citrate in
the medium (Gundersen and Henriksen, 1959). The addition of sodium
citrate will prevent readsorption of the liberated phage on to the bacteria.
After the addition of sodium citrate, the culture is shaken again for 1 h
at 35"C, with a view to obtaining the maximum yield of phage from the
lysogenic cells. The culture is then centrifuged at 3000 rev/min for 20-30
min and the decanted fluid is tested against a wide range of available
sensitive C. diphtheriae strains. T h e wider the range of sensitive strains
the better the chance of obtaining lysogenic phages.
Sensitive indicator strains of both schemes and even other multisensitive
strains are available for checking lysogeny.
2. Preparation of the stock phage in the sensitive (indicator) strains
Overnight cultures of the sensitive strains on Loeffler slants are
inoculated into 4-5 ml broth and incubated for 18 h at 35°C. Brain-heart
agar plates (as for phage typing) are flooded with the sensitive strains and
dried for 45 min. Every supernatant is dropped on to predetermined areas
recorded on the backs of the plates. The plates are incubated for 24 h at
35°C. If any degree of lysis occurs over the area of the spot, even isolated
plaques, they are cut out, inoculated into a tube with 5 m l broth, and
shaken overnight. After centrifugation and the addition of chloroform (for
2-3 h), the supernatant is again spotted on the sensitive strain. If propagation is successful, the suspension is titrated and checked for host range.
B. Induction of lysogenic phages by ultraviolet irradiation
An overnight fluid culture is diluted to an opacity of 0.3, shaken for 1 h,
and submitted to ultraviolet irradiation. The layer should not be deeper
than 2-3 mm. A distance of 76 cm and time of exposure of 135 s is used
with the lamps available in our laboratory, but preliminary experiments
will be needed to determine optimal conditions for other equipment. The
plate is shaken continuously during irradiation. The irradiated fluid is
transferred to a flask in the dark and again shaken for 3 h at 35°C before
centrifugation, chloroform treatment and testing lytic activity. As control,
a known lysogenic strain is always included to test the effectiveness of the
ultraviolet exposure (Barksdale and Pappenheimer, 1954).