366
w. B. REDMOND et al.
various protecting substrates, reported on satisfactory freeze-drying of 52
of 53 different mycobacteriophages in a medium of 5% sodium glutamate
and 0.5% gelatin. After propagation of the bacteriophages on their respective hosts on solid medium, the phage lysates were washed from the
surface of each plate with sterile distilled water in order to keep the salt
concentration as low as possible. (Salts decrease the freezing point of the
phage suspension thus causing foaming during the freeze-drying process.)
The lysates were filtered through a 0.45 pm membrane filter.
The filtered lysates were mixed with equal amounts of a solution of 10%
sodium glutamate (British Drug House Chemicals) and 1% gelatin
(Difco). One millilitre samples were dispensed into 2 ml glass ampoules,
which were subsequently aseptically attached to the ampoule holder of the
freeze drying apparatus. The holders were carefully rotated by hand in an
ethanol bath kept at -40°C by means of refrigeration to obtain a uniform
coating of frozen phage suspension on the walls of the ampoules. Subsequently the holders were attached to the freeze drying apparatus.
The drying process was a two-step procedure. The preliminary drying
was carried out by application of vacuum for 24 h until a vapour pressure of
8pm Hg was obtained. The water vapour was transported to a low temperature condenser ( - 60°C). Uncondensed vapours were drawn off through
F.G. 50 filters (American Air Filter Company, Louisville, Kentucky). I n
order to prevent the phage suspensions reaching the melting point too
early during the drying process, thus causing foaming, the ampoules
were refrigerated for the first 4-5 h by means of a CO2-ethanol mixture.
The first step was terminated by sterile aeration of the ampoules with
filtered air, after which they were removed and their necks flame-heated
and drawn to capillaries.
For the second step the ampoules were again attached. I n order to obtain
a pressure of less than 1 x 10-2 pm Hg, an oil diffusion pump in combination
with PzO5 absorption was used. This pressure was reached after 4-5h and
maintained for another 18 h.
The second step was completed by flame-sealing of the ampoules at the
capillary part keeping them still under vacuum. All ampoules were finally
checked for vacuum by means of an Edwards high frequency tester
(Model T2).
Titres determined after processing generally did not show more than a
ten-fold reduction. No changes in plaque morphology or lytic spectrum
were noted after reconstitution when compared with the original phage
preparation. The freeze-dried preparations could be stored in the dark at
room temperature for as long as four years or even longer (unpublished
results). Dubina et al. (1974) were able to confirm these results in work on
mycobacteriophage AGl.
w. B. REDMOND et al.
various protecting substrates, reported on satisfactory freeze-drying of 52
of 53 different mycobacteriophages in a medium of 5% sodium glutamate
and 0.5% gelatin. After propagation of the bacteriophages on their respective hosts on solid medium, the phage lysates were washed from the
surface of each plate with sterile distilled water in order to keep the salt
concentration as low as possible. (Salts decrease the freezing point of the
phage suspension thus causing foaming during the freeze-drying process.)
The lysates were filtered through a 0.45 pm membrane filter.
The filtered lysates were mixed with equal amounts of a solution of 10%
sodium glutamate (British Drug House Chemicals) and 1% gelatin
(Difco). One millilitre samples were dispensed into 2 ml glass ampoules,
which were subsequently aseptically attached to the ampoule holder of the
freeze drying apparatus. The holders were carefully rotated by hand in an
ethanol bath kept at -40°C by means of refrigeration to obtain a uniform
coating of frozen phage suspension on the walls of the ampoules. Subsequently the holders were attached to the freeze drying apparatus.
The drying process was a two-step procedure. The preliminary drying
was carried out by application of vacuum for 24 h until a vapour pressure of
8pm Hg was obtained. The water vapour was transported to a low temperature condenser ( - 60°C). Uncondensed vapours were drawn off through
F.G. 50 filters (American Air Filter Company, Louisville, Kentucky). I n
order to prevent the phage suspensions reaching the melting point too
early during the drying process, thus causing foaming, the ampoules
were refrigerated for the first 4-5 h by means of a CO2-ethanol mixture.
The first step was terminated by sterile aeration of the ampoules with
filtered air, after which they were removed and their necks flame-heated
and drawn to capillaries.
For the second step the ampoules were again attached. I n order to obtain
a pressure of less than 1 x 10-2 pm Hg, an oil diffusion pump in combination
with PzO5 absorption was used. This pressure was reached after 4-5h and
maintained for another 18 h.
The second step was completed by flame-sealing of the ampoules at the
capillary part keeping them still under vacuum. All ampoules were finally
checked for vacuum by means of an Edwards high frequency tester
(Model T2).
Titres determined after processing generally did not show more than a
ten-fold reduction. No changes in plaque morphology or lytic spectrum
were noted after reconstitution when compared with the original phage
preparation. The freeze-dried preparations could be stored in the dark at
room temperature for as long as four years or even longer (unpublished
results). Dubina et al. (1974) were able to confirm these results in work on
mycobacteriophage AGl.