248
T. BERGAN
phage dilution are added (i) 0.25 ml of the homologous propagating strains
(4-h broth culture agitated at 37"C), and (ii) 2 ml of melted soft agar at
45°C (heat block is suitable].
The ingredients are quickly mixed (air bubbles avoided) and the whole
volume spread on the surface of a 9 cm diameter nutrient agar plate. The
plates are incubated overnight at 37°C.
The RTD is determined on agar prepared by flooding with overnight
undiluted broth cultures (ZieschC and Rische use 3-h broth cultures) for
inoculation. After removal of excess liquid by suction, the plates are dried
at 37°C without lids for 15-30min. Ten-fold dilutions of the phages
(10-1-10-8) are spotted on the homologous host-propagating strains by the
help of the same nichrome coiled eye wire as is used in the multi-inoculator
typing apparatus.
Following phage application, the plates are dried at room temperature
and incubated at 37°C in the upright position for 5 h. RTD is adjusted by
making intermediate dilution steps between the points where decreasing
activity against the homologous strain is noticed. These intermediate
dilutions (e.g. 1 x 10-3; 0.75 x 10-3; 0.5 x 10-3; 0.25 x 10-3; 1 x 10-4) are
tested against a limited set of type strains with varying sensitivity, S.
sonnei lysotype standard strains 3,4, 5, 7, 22, 28, 53, 58, 61, 67, 68, and 96.
Before new preparations are used in routine typing, their lytic spectra
are checked by typing on all the 96 characterized types. The appearance of
the reactions on each strain is important. The reactions of some key strains
are shown in Table XV and Fig. 6. The maintenance (stability) of RTD is
checked at monthly intervals by repeat typing on the limited set of control
bacteria.
Reactions are scored as for S. Jlexneri. Common results with fresh preparations of phages and the relationship between RTD and CD are shown
in Table XVI.
The number of phages per millilitre necessary to give RTD varies with
plaque size. Hammarstrom (1949) demonstrated how plaque size compared
with the upper and lower number of plaques per reaction (Table XVII).
The RTD is not the same as the CD, although the two are closely related.
The number of plaque-forming units per millilitre varies with the type and
even production batch of the same brand of medium (Pruneda and Farmer,
1977).
6. Typing procedure
From agar plates (endo-plates are said to be better for selecting right
phase (ZieschC and Rische, 1973)), material from the edge of single, isolated
phase I1 colonies is transferred to two tubes of 4 ml nutrient broth. If a
strain shows evidence of differences in colony morphology, one broth
T. BERGAN
phage dilution are added (i) 0.25 ml of the homologous propagating strains
(4-h broth culture agitated at 37"C), and (ii) 2 ml of melted soft agar at
45°C (heat block is suitable].
The ingredients are quickly mixed (air bubbles avoided) and the whole
volume spread on the surface of a 9 cm diameter nutrient agar plate. The
plates are incubated overnight at 37°C.
The RTD is determined on agar prepared by flooding with overnight
undiluted broth cultures (ZieschC and Rische use 3-h broth cultures) for
inoculation. After removal of excess liquid by suction, the plates are dried
at 37°C without lids for 15-30min. Ten-fold dilutions of the phages
(10-1-10-8) are spotted on the homologous host-propagating strains by the
help of the same nichrome coiled eye wire as is used in the multi-inoculator
typing apparatus.
Following phage application, the plates are dried at room temperature
and incubated at 37°C in the upright position for 5 h. RTD is adjusted by
making intermediate dilution steps between the points where decreasing
activity against the homologous strain is noticed. These intermediate
dilutions (e.g. 1 x 10-3; 0.75 x 10-3; 0.5 x 10-3; 0.25 x 10-3; 1 x 10-4) are
tested against a limited set of type strains with varying sensitivity, S.
sonnei lysotype standard strains 3,4, 5, 7, 22, 28, 53, 58, 61, 67, 68, and 96.
Before new preparations are used in routine typing, their lytic spectra
are checked by typing on all the 96 characterized types. The appearance of
the reactions on each strain is important. The reactions of some key strains
are shown in Table XV and Fig. 6. The maintenance (stability) of RTD is
checked at monthly intervals by repeat typing on the limited set of control
bacteria.
Reactions are scored as for S. Jlexneri. Common results with fresh preparations of phages and the relationship between RTD and CD are shown
in Table XVI.
The number of phages per millilitre necessary to give RTD varies with
plaque size. Hammarstrom (1949) demonstrated how plaque size compared
with the upper and lower number of plaques per reaction (Table XVII).
The RTD is not the same as the CD, although the two are closely related.
The number of plaque-forming units per millilitre varies with the type and
even production batch of the same brand of medium (Pruneda and Farmer,
1977).
6. Typing procedure
From agar plates (endo-plates are said to be better for selecting right
phase (ZieschC and Rische, 1973)), material from the edge of single, isolated
phase I1 colonies is transferred to two tubes of 4 ml nutrient broth. If a
strain shows evidence of differences in colony morphology, one broth