208
T. BERGAN
at 37"C, most phage cultures have become transparent. At that time, the
culture is filtered through a membrane filter of 0.45 pm and kept as a stock
suspension. In preparations with secondary growth the titres are lower.
5 . Determination of strength and lytic spectra
The phage stock is diluted 10-1,10-2, etc., up to 10-9. It is important to
use new pipettes for each dilution to avoid carry-over. The plate is inoculated by flooding with a fresh 3-h bacterial culture, pipetting off the excess,
and drying at room temperature for 30min. The phage suspensions are
applied to the plates, incubated at 37°C for 7 h and then placed at 4°C until
the reactions are recorded on the following day. The highest dilution which
still has sufficient phage to induce confluent lysis on the host propagating
strain is recorded. This is termed the critical dilution (CD). The results
are recorded as follows:
CL = confluent lysis
OL = opaque lysis
SCL = semiconfluent lysis
+ + + = > 80 isolated plaques
+ + =40-80 isolated plaques
+ =20-40 isolated plaques
- = no plaques.
The lytic spectrum of the typing set is shown in Table VIII.
6. Typing procedure
Both bacterial incula and phage suspensions must be prepared with the
utmost consideration for meticulous repetition of details. Plates with the
strains to be typed are inoculated as described in Section VI . C, 5 of this
Chapter.
The phages are used at their routine test dilutions (RTD), which for
S. jlexneri is 3-5-fold more concentrated than CD (Table IX). As soon as
the plates have dried, 10 p1 volumes of each phage suspension (at RTD)
are applied to the agar surface with a multi-inoculator. Many inoculators
have been constructed. The model designed by Lidwell (1959) (Fig. 2),
which may be procured from Biddulph and Co. (Manchester) Ltd,
Commercial Street, Knott Mill, Manchester 15, England, is particularly
suitable.
It is essential that the phage drops have dried completely before the
plates are incubated, otherwise the phage number will increase in the drop
to render apparent inocula above RTD. After 20-30 min at room temperature without lids, the plates are immediately transferred to 37°C for 7 h
T. BERGAN
at 37"C, most phage cultures have become transparent. At that time, the
culture is filtered through a membrane filter of 0.45 pm and kept as a stock
suspension. In preparations with secondary growth the titres are lower.
5 . Determination of strength and lytic spectra
The phage stock is diluted 10-1,10-2, etc., up to 10-9. It is important to
use new pipettes for each dilution to avoid carry-over. The plate is inoculated by flooding with a fresh 3-h bacterial culture, pipetting off the excess,
and drying at room temperature for 30min. The phage suspensions are
applied to the plates, incubated at 37°C for 7 h and then placed at 4°C until
the reactions are recorded on the following day. The highest dilution which
still has sufficient phage to induce confluent lysis on the host propagating
strain is recorded. This is termed the critical dilution (CD). The results
are recorded as follows:
CL = confluent lysis
OL = opaque lysis
SCL = semiconfluent lysis
+ + + = > 80 isolated plaques
+ + =40-80 isolated plaques
+ =20-40 isolated plaques
- = no plaques.
The lytic spectrum of the typing set is shown in Table VIII.
6. Typing procedure
Both bacterial incula and phage suspensions must be prepared with the
utmost consideration for meticulous repetition of details. Plates with the
strains to be typed are inoculated as described in Section VI . C, 5 of this
Chapter.
The phages are used at their routine test dilutions (RTD), which for
S. jlexneri is 3-5-fold more concentrated than CD (Table IX). As soon as
the plates have dried, 10 p1 volumes of each phage suspension (at RTD)
are applied to the agar surface with a multi-inoculator. Many inoculators
have been constructed. The model designed by Lidwell (1959) (Fig. 2),
which may be procured from Biddulph and Co. (Manchester) Ltd,
Commercial Street, Knott Mill, Manchester 15, England, is particularly
suitable.
It is essential that the phage drops have dried completely before the
plates are incubated, otherwise the phage number will increase in the drop
to render apparent inocula above RTD. After 20-30 min at room temperature without lids, the plates are immediately transferred to 37°C for 7 h