V. UACTERIOPHAGE TYPING OF SHIC'EI.I,A
247
FIG. 5 (cont.). Shigella sonnei, phage y 66.
4. Propagation of phages
(a) Passage in broth. On day 3, one volume of bacteria in log-phase is
mixed with one volume of phage dilution (multiplicity of infection 1/200)
and incubated in a shaker at 37°C for 10 min. Thereafter 18 ml of prewarmed broth are added. After 4 h, the culture is lysed and the lysate is
incubated at 4°C overnight. The following day it is centrifuged at 10,000 x g
for 20 min at 4°C. The supernatant is filtered through a 0.22 p m Millipore
or equivalent filter. The filtrate represents the phage stock.
(b) Passage on agar. On day 1 , 5 ml of log phase bacteria and 5 ml of diluted
phages at a multiplicity of infection of 1/200 are mixed and incubated in a
shaker incubator at 37°C for 10 min when 90 ml nutrient agar with 0.6%
agar at 45°C are added. Portions of 8 ml are plated by spreading on top of
twelve 14 cm diameter plates containing 1.5% agar. The plates are
incubated at 37°C overnight.
The next day the plates are flooded by 10 ml of broth. The top layer is
scraped off, harvested by suction and subjected to a thorough shaking until
fairly homogeneous. Subsequently, the material is centrifuged and filtered
as above for broth passage.
5. Determination of strength and lytic spectra
For quantitation, 10-fold dilutions (with change of pipettes) are made in
nutrient broth, and assayed by agar overnight plates. 1'0 0.1 ml of each