VIII. METHODS FOR TYPING MYCOBACTERIA
365
review of the effects of various substances on phages in general is presented
by Adams (1959). Tween-80 in concentrations normally used in mycobacterial culture media has no effect on the stability of the mycobacteriophages, but interferes with adsorption to the hosts (Bowman, 1958;
Sellers et al., 1962). Low temperatures favour stability. In a good suspending medium, most mycobacteriophages remain stable for months or years
at 4"C, although titres will decrease gradually. At room temperature,
many phages lose their activity within a few days, although stability may be
increased by keeping the phages on small agar plugs sealed in vials (Redmond and Ward, 1966). Thus storage at 4°C in broth appears to be the
most simple method for short-term preservation of phages in most instances, provided that viability and other important characters are regularly
checked.
For long-term preservation (for two years or more), drying from the
unfrozen or frozen state (freeze-drying) and freezing at ultra-low temperatures (near -196°C) have been applied. Although drying of phages on
filter paper discs from the unfrozen state was reported as quite unsuccessful
by Clark (1962), rapid drying of mycobacteriophages in small volumes of
heart infusion broth gave satisfactory results according to Will et al.
(1961). The mycobacteriophages survived well and retained all their
characteristics upon reconstitution after storage in the dark, or even at
room temperature. It is well known that great differences in sensitivity to
drying and freezing do exist among various phages. Campbell-Renton
(1941) even proposed using these differences as a basis for the classification
of phages. Freeze-drying of phages has been performed by a number of
workers with more or less satisfactory results. Although Clark (1962)
reported high losses on freeze drying of various phages, he later mentioned
the routine use of this technique for distribution of bacteriophages by the
American Type Culture Collection (Clark, 1970). Successful freeze-drying
depends on such factors as suspending medium, cooling rate and drying
temperature as demonstrated by Davies and Kelly (1969), who developed
a satisfactory freeze-drying method for a corynebacteriophage that was
extremely labile in suspension at 4°C and even at -25°C. The method,
using a solution containing 20% peptone, 10% sucrose, and 2% sodium
glutamate as a protective substrate, has been successfully applied to a
series of other corynebacteriophages by Carne and Greaves (1974). Murohashi et al. (1960,1969) freeze-dried high concentrations (109-1011 PFU/ml)
of eight mycobacteriophages in their original propagation medium (trypticase-soy-broth) without any additional protecting substances. Inactivation
by freeze-drying varied from lO-SO%. Inactivation during storage of the
freeze-dried phages at 5°C for more than six years caused no more than a
three-fold reduction in active particles. Engel et al. (1974), comparing
365
review of the effects of various substances on phages in general is presented
by Adams (1959). Tween-80 in concentrations normally used in mycobacterial culture media has no effect on the stability of the mycobacteriophages, but interferes with adsorption to the hosts (Bowman, 1958;
Sellers et al., 1962). Low temperatures favour stability. In a good suspending medium, most mycobacteriophages remain stable for months or years
at 4"C, although titres will decrease gradually. At room temperature,
many phages lose their activity within a few days, although stability may be
increased by keeping the phages on small agar plugs sealed in vials (Redmond and Ward, 1966). Thus storage at 4°C in broth appears to be the
most simple method for short-term preservation of phages in most instances, provided that viability and other important characters are regularly
checked.
For long-term preservation (for two years or more), drying from the
unfrozen or frozen state (freeze-drying) and freezing at ultra-low temperatures (near -196°C) have been applied. Although drying of phages on
filter paper discs from the unfrozen state was reported as quite unsuccessful
by Clark (1962), rapid drying of mycobacteriophages in small volumes of
heart infusion broth gave satisfactory results according to Will et al.
(1961). The mycobacteriophages survived well and retained all their
characteristics upon reconstitution after storage in the dark, or even at
room temperature. It is well known that great differences in sensitivity to
drying and freezing do exist among various phages. Campbell-Renton
(1941) even proposed using these differences as a basis for the classification
of phages. Freeze-drying of phages has been performed by a number of
workers with more or less satisfactory results. Although Clark (1962)
reported high losses on freeze drying of various phages, he later mentioned
the routine use of this technique for distribution of bacteriophages by the
American Type Culture Collection (Clark, 1970). Successful freeze-drying
depends on such factors as suspending medium, cooling rate and drying
temperature as demonstrated by Davies and Kelly (1969), who developed
a satisfactory freeze-drying method for a corynebacteriophage that was
extremely labile in suspension at 4°C and even at -25°C. The method,
using a solution containing 20% peptone, 10% sucrose, and 2% sodium
glutamate as a protective substrate, has been successfully applied to a
series of other corynebacteriophages by Carne and Greaves (1974). Murohashi et al. (1960,1969) freeze-dried high concentrations (109-1011 PFU/ml)
of eight mycobacteriophages in their original propagation medium (trypticase-soy-broth) without any additional protecting substances. Inactivation
by freeze-drying varied from lO-SO%. Inactivation during storage of the
freeze-dried phages at 5°C for more than six years caused no more than a
three-fold reduction in active particles. Engel et al. (1974), comparing