IV. INVESTIGATION OF C. DIPHTHERIAE
153
and 16-22 on agravis tox- strain no. 9253 (Romania); phages nos. 14 and 15
on a gravis tox- strain no. 304 (Copenhagen).
The indicator strains are stored either freeze dried or on Loeffler slants,
by monthly transfer. They must be stored sealed in the dark at room
temperature. C. diphtheriae stock cultures must never be kept in the
refrigerator, since the organism dies in the cold within a few days.
(b) Preparation of phage stocks. With a view to starting from a single
isolated plaque 0.1 ml of the diluted phage (10-3-10-5) together with 0.2
ml of the indicator strain (a fresh culture of 13 h in broth) are added to
2 m l soft agar, spread over the surface of a brain-heart agar plate and
incubated overnight at 35°C. The next day single plaques are picked with
a sterile Pasteur pipette, dropped into 1-2 ml broth and stirred for 30-60 s
either mechanically or by hand. The supernatant obtained after centrifugation of this preparation is dropped on the propagating strain (fresh
culture) previously flooded on to the surface of brain-heart infusion agar.
The plates are incubated overnight at 35°C. The lytic areas are cut out
and dropped (10-15 areas) into 30-40 ml of broth and shaken for 5-6 h
overnight at 35°C. After centrifugation at 3000 rev/min for + h, the
supernatant containing the propagated phage is decanted and treated
with chloroform (5 dropslml).
The preparation is left for 2 h at room temperature and gently stirred
from time to time, then kept at 4'C until the following day when the
chloroform is discarded. The phage preparation is assayed for its proper
host range activity and for sterility and titrated in ten-fold dilutions
on its propagating strain. The RTD usually range from 10-2 to 10-4. For
phage typing 100 RTD are used. Each new phage stock is used only if it
shows the accepted host range.
2. The additional phage typing scheme
(a) Propagating strains for the additional typing phages are represented
by a series of 33 C. diphtheriae strains from different geographical areas.
Their record number and their origins are listed in the second vertical row
on the left of Fig. 15.
(b) Preparation of phage stocks. The phages are multiplied in the same way
as in the previously described method.
3. Isolation of new typing phages
(a) Phages of endogenous origin. New phage lysates are obtained from the
broth cultures of the supposed lysogenic strain by overnight shaking at
35°C. The following day, an hour before centrifugation sodium citrate
(0.07 M) is added to the lysates, followed by 1 h further shaking. Super-
153
and 16-22 on agravis tox- strain no. 9253 (Romania); phages nos. 14 and 15
on a gravis tox- strain no. 304 (Copenhagen).
The indicator strains are stored either freeze dried or on Loeffler slants,
by monthly transfer. They must be stored sealed in the dark at room
temperature. C. diphtheriae stock cultures must never be kept in the
refrigerator, since the organism dies in the cold within a few days.
(b) Preparation of phage stocks. With a view to starting from a single
isolated plaque 0.1 ml of the diluted phage (10-3-10-5) together with 0.2
ml of the indicator strain (a fresh culture of 13 h in broth) are added to
2 m l soft agar, spread over the surface of a brain-heart agar plate and
incubated overnight at 35°C. The next day single plaques are picked with
a sterile Pasteur pipette, dropped into 1-2 ml broth and stirred for 30-60 s
either mechanically or by hand. The supernatant obtained after centrifugation of this preparation is dropped on the propagating strain (fresh
culture) previously flooded on to the surface of brain-heart infusion agar.
The plates are incubated overnight at 35°C. The lytic areas are cut out
and dropped (10-15 areas) into 30-40 ml of broth and shaken for 5-6 h
overnight at 35°C. After centrifugation at 3000 rev/min for + h, the
supernatant containing the propagated phage is decanted and treated
with chloroform (5 dropslml).
The preparation is left for 2 h at room temperature and gently stirred
from time to time, then kept at 4'C until the following day when the
chloroform is discarded. The phage preparation is assayed for its proper
host range activity and for sterility and titrated in ten-fold dilutions
on its propagating strain. The RTD usually range from 10-2 to 10-4. For
phage typing 100 RTD are used. Each new phage stock is used only if it
shows the accepted host range.
2. The additional phage typing scheme
(a) Propagating strains for the additional typing phages are represented
by a series of 33 C. diphtheriae strains from different geographical areas.
Their record number and their origins are listed in the second vertical row
on the left of Fig. 15.
(b) Preparation of phage stocks. The phages are multiplied in the same way
as in the previously described method.
3. Isolation of new typing phages
(a) Phages of endogenous origin. New phage lysates are obtained from the
broth cultures of the supposed lysogenic strain by overnight shaking at
35°C. The following day, an hour before centrifugation sodium citrate
(0.07 M) is added to the lysates, followed by 1 h further shaking. Super-