154
A. SARAGEA ET AL.
natants obtained after centrifugation of the lysates for 4 h at 3000 rev/min.
are tested by the spot method against a set of previously resistant C.
diphtheriae. If lytic activitjr occurs, the propagation of the lytic areas is
performed according to the methods used and described in the previous
section for obtaining phage stocks. These new phage preparations may
serve for further typing of non-typable local strains.
(b) Host restrictions and modiJcations of the highly virulent phage 951 “L”
are obtained from a lysate prepared from the 9.5 1 “L” C. diphtheriae strain
spotted on a newly chosen propagating strain from apparently non-typable
strains, as described by Maximescu and Saragea (1969). An overnight
shaken culture of the 12. diphtheriae 95 1 “L” strain in either Pope Linggood’s
broth or heart broth with 0.001% L-cystine, is centrifuged 1 h after
0-07 M sodium citrate has been added. The lytic activity of the lysate is
tested on a young culture (14 h) flooded on a brain-heart agar plate. After
overnight incubation at 35”C, even if isolated plaques occur, they should
be cut out, inoculated and propagated in nutrient broth (tryptic digest or
heart broth, with 0.001% L-cystine) for 24 h at 35°C in shaken cultures.
The following day the preparation is centrifuged, decanted and exposed
to chloroform (24 h in the refrigerator). The chloroform is discarded and
the new phage preparation titrated in ten-fold dilution steps and the hostrange activity tested on a large number of untypable strains isolated from
the same area or community. Sometimes, as pin-point, hardly visible
plaques could occur after the primary lysate has been spotted, areas
without visible plaques should be propagated and tested.
E. Conclusions
I n order to determine the phage type of a C. diphtheriae strain the two
phage typing schemes are used as follows: first, strains are tested with the
original set of phages; if non-typable, the additional set of phages or new
phage preparations may be used.
For practical and economic purposes, a restricted number of phages are
routinely used, namely: nos. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 19 and21
from the original scheme and 19 previously mentioned (Fig. 16) from the
additional scheme; for reasons of rapidity, both tests can be used simultaneously.
XI. PRACTICAL PROCEDURES OF PHAGE TYPING OF
C. ULCERANS AND C. OVIS
A. Basic set of typing phages
and a diphtherial phage restricted in a C. ovis strain (Fig. 26).
The basic set of typing phages is represented by six C. ulcerans phages
A. SARAGEA ET AL.
natants obtained after centrifugation of the lysates for 4 h at 3000 rev/min.
are tested by the spot method against a set of previously resistant C.
diphtheriae. If lytic activitjr occurs, the propagation of the lytic areas is
performed according to the methods used and described in the previous
section for obtaining phage stocks. These new phage preparations may
serve for further typing of non-typable local strains.
(b) Host restrictions and modiJcations of the highly virulent phage 951 “L”
are obtained from a lysate prepared from the 9.5 1 “L” C. diphtheriae strain
spotted on a newly chosen propagating strain from apparently non-typable
strains, as described by Maximescu and Saragea (1969). An overnight
shaken culture of the 12. diphtheriae 95 1 “L” strain in either Pope Linggood’s
broth or heart broth with 0.001% L-cystine, is centrifuged 1 h after
0-07 M sodium citrate has been added. The lytic activity of the lysate is
tested on a young culture (14 h) flooded on a brain-heart agar plate. After
overnight incubation at 35”C, even if isolated plaques occur, they should
be cut out, inoculated and propagated in nutrient broth (tryptic digest or
heart broth, with 0.001% L-cystine) for 24 h at 35°C in shaken cultures.
The following day the preparation is centrifuged, decanted and exposed
to chloroform (24 h in the refrigerator). The chloroform is discarded and
the new phage preparation titrated in ten-fold dilution steps and the hostrange activity tested on a large number of untypable strains isolated from
the same area or community. Sometimes, as pin-point, hardly visible
plaques could occur after the primary lysate has been spotted, areas
without visible plaques should be propagated and tested.
E. Conclusions
I n order to determine the phage type of a C. diphtheriae strain the two
phage typing schemes are used as follows: first, strains are tested with the
original set of phages; if non-typable, the additional set of phages or new
phage preparations may be used.
For practical and economic purposes, a restricted number of phages are
routinely used, namely: nos. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 19 and21
from the original scheme and 19 previously mentioned (Fig. 16) from the
additional scheme; for reasons of rapidity, both tests can be used simultaneously.
XI. PRACTICAL PROCEDURES OF PHAGE TYPING OF
C. ULCERANS AND C. OVIS
A. Basic set of typing phages
and a diphtherial phage restricted in a C. ovis strain (Fig. 26).
The basic set of typing phages is represented by six C. ulcerans phages