VIII. METHODS FOR TYPING MYCOBACTERIA
367
The value of storage at temperatures of liquid nitrogen has been demonstrated by Clark et al. (1962), Clark and Klein (1966) and Carne and Greaves
(1974). Titres were reported constant for as long as five years (Clark and
Klein, 1966). The phages were frozen at controlled rate in three steps to
- 196°C and quickly thawed for use. As a protective additive 10% glycerol
was added to the lysates (Clark et al., 1962). Storage at - 196"C, however, is
limited by refrigeration space and shipping possibilities. Freeze-drying
permits storage and shipping at room temperature and therefore seems the
most useful method for long-term preservation of phages.
B. Mycobacteria
For storage and shipping of the bacterial hosts similar methods are
effective. Most mycobacterial strains keep well on slants at 4°C for several
months or years. This means of preservation is satisfactory for short-term
storage, but regular checks on viability and phage susceptibility remain
necessary. Other important characteristics (e.g. biochemical, immunological, virulence) must be checked to minimise selection of undesirable
mutants in subcultures. For long-term preservation, drying from the
unfrozen state was reported to provide stable stocks (Will et al., 1961).
Storage at - 70°C in Middlebrook 7H-9 liquid medium provided stocks
with 100% viability and stability of genetic characteristics for long periods
(Kim and Kubica, 1973). Freeze-drying of bacteria in various suspending
media (e.g. skimmed milk, 7.5% glucose, broth) is commonly used with
satisfactory results and is extensively reviewed by Lapage et al. (1970). It
may result in an initial kill of 40-50% as has been demonstrated for some
BCG preparations (Lind, 1967; Ungar, 1949). Storage and shipping without refrigeration are definite advantages of this method. The choice of
method will depend on local circumstances and requirements.
VI. PHAGE TYPING PROCEDURES ADAPTED TO T H E
MYCOBACTERIA
A. The routine test dilution (RTD)
The routine test dilution (RTD) has been defined as the highest dilution
of phage that will produce complete lysis of the propagating strain of bacteria. For typing M. tuberculosis a given prototype strain (see page 361) is
used to determine the RTD of each phage.
Despite some problems, the R T D method of phage typing appears to be
the most satisfactory procedure. The major difficulties encountered are due
to variations in susceptibility to the typing phages of bacterial strains from
different sources. Variations in the degree of lysis often make comparisons
between strains somewhat questionable. This problem has been solved to a
367
The value of storage at temperatures of liquid nitrogen has been demonstrated by Clark et al. (1962), Clark and Klein (1966) and Carne and Greaves
(1974). Titres were reported constant for as long as five years (Clark and
Klein, 1966). The phages were frozen at controlled rate in three steps to
- 196°C and quickly thawed for use. As a protective additive 10% glycerol
was added to the lysates (Clark et al., 1962). Storage at - 196"C, however, is
limited by refrigeration space and shipping possibilities. Freeze-drying
permits storage and shipping at room temperature and therefore seems the
most useful method for long-term preservation of phages.
B. Mycobacteria
For storage and shipping of the bacterial hosts similar methods are
effective. Most mycobacterial strains keep well on slants at 4°C for several
months or years. This means of preservation is satisfactory for short-term
storage, but regular checks on viability and phage susceptibility remain
necessary. Other important characteristics (e.g. biochemical, immunological, virulence) must be checked to minimise selection of undesirable
mutants in subcultures. For long-term preservation, drying from the
unfrozen state was reported to provide stable stocks (Will et al., 1961).
Storage at - 70°C in Middlebrook 7H-9 liquid medium provided stocks
with 100% viability and stability of genetic characteristics for long periods
(Kim and Kubica, 1973). Freeze-drying of bacteria in various suspending
media (e.g. skimmed milk, 7.5% glucose, broth) is commonly used with
satisfactory results and is extensively reviewed by Lapage et al. (1970). It
may result in an initial kill of 40-50% as has been demonstrated for some
BCG preparations (Lind, 1967; Ungar, 1949). Storage and shipping without refrigeration are definite advantages of this method. The choice of
method will depend on local circumstances and requirements.
VI. PHAGE TYPING PROCEDURES ADAPTED TO T H E
MYCOBACTERIA
A. The routine test dilution (RTD)
The routine test dilution (RTD) has been defined as the highest dilution
of phage that will produce complete lysis of the propagating strain of bacteria. For typing M. tuberculosis a given prototype strain (see page 361) is
used to determine the RTD of each phage.
Despite some problems, the R T D method of phage typing appears to be
the most satisfactory procedure. The major difficulties encountered are due
to variations in susceptibility to the typing phages of bacterial strains from
different sources. Variations in the degree of lysis often make comparisons
between strains somewhat questionable. This problem has been solved to a