348
w. B. REDMOND et al.
members of the same species could be shown. They further showed that
eight strains of M . tuberculosis, attenuated by repeated culture on Calmette’s
bile medium, were less susceptible to phage than the original virulent
strains even though the human strains H37Rv and H37Ra showed identical
susceptibility patterns. By 1963, sufficient information had been published
on the subject to permit a review article on mycobacteriophages (Redmond,
1963), but no mention of epidemiologic studies was made. I n this review
the phage GS4E was described regarding its isolation as a mutant from an
original phage obtained from garden soil. Phage GS4E was significant in
that it was recognised to lyse the M . tuberculosis strain H37Rv, but would
not lyse any of a group of strains of M. bovis. In the same year Murohashi
et al. (1963) reported the capacity of GS4E to subdivide the human strain
of mycobacteria. Little attention was given to these early reports, and most
investigators considered that mycobacteriophages which lysed one member
of a species would lyse all others. Thus, the continuing emphasis was on
isolating and characterising phages that would be useful in taxonomy and
in genetic studies of mycobacteria. However, as improved biochemical
techniques were developed that allowed a more precise characterisation of
mycobacteria, the expectation of utilising phage typing as a taxonomic tool
waned.
11. MEDIA
Probably the most important factor leading to the variations and diversity
of results obtained in early attempts to phage type mycobacteria was the
medium used in the tests. It soon became apparent from experiments
employing the same phages and bacteria in various laboratories that the
composition of the medium caused wide variations.
Several components commonly found in culture media have an inhibitory
action on phage. These may have little or no inhibitory effect on bacterial
growth. Probably the most important of these factors are those that interfere with adsorption of the phage and its subsequent introduction into the
bacterial cell. Many, or most, of the media that have been found to be quite
adequate for primary isolation of mycobacteria, e.g. Lowenstein-Jensen
( L J ) Medium, those used primarily in assaying for drug susceptibility
(Middlebrook’s 7H9-10), or others slightly modified for distinguishing
between growth characteristics of strains, have not been found to be
adequate for use in phage typing. A series of tests involving most of the
components used in these media were carried out in the Atlanta Laboratory
to determine the effect on phage activity. Details of these tests are to be
found in a publication by Redmond and Ward (1966). Calcium ions have
been found to be necessary for lysis. Citrate and phosphates are detrimental
because they tend to inactivate or to precipitate calcium. A high concentra-
w. B. REDMOND et al.
members of the same species could be shown. They further showed that
eight strains of M . tuberculosis, attenuated by repeated culture on Calmette’s
bile medium, were less susceptible to phage than the original virulent
strains even though the human strains H37Rv and H37Ra showed identical
susceptibility patterns. By 1963, sufficient information had been published
on the subject to permit a review article on mycobacteriophages (Redmond,
1963), but no mention of epidemiologic studies was made. I n this review
the phage GS4E was described regarding its isolation as a mutant from an
original phage obtained from garden soil. Phage GS4E was significant in
that it was recognised to lyse the M . tuberculosis strain H37Rv, but would
not lyse any of a group of strains of M. bovis. In the same year Murohashi
et al. (1963) reported the capacity of GS4E to subdivide the human strain
of mycobacteria. Little attention was given to these early reports, and most
investigators considered that mycobacteriophages which lysed one member
of a species would lyse all others. Thus, the continuing emphasis was on
isolating and characterising phages that would be useful in taxonomy and
in genetic studies of mycobacteria. However, as improved biochemical
techniques were developed that allowed a more precise characterisation of
mycobacteria, the expectation of utilising phage typing as a taxonomic tool
waned.
11. MEDIA
Probably the most important factor leading to the variations and diversity
of results obtained in early attempts to phage type mycobacteria was the
medium used in the tests. It soon became apparent from experiments
employing the same phages and bacteria in various laboratories that the
composition of the medium caused wide variations.
Several components commonly found in culture media have an inhibitory
action on phage. These may have little or no inhibitory effect on bacterial
growth. Probably the most important of these factors are those that interfere with adsorption of the phage and its subsequent introduction into the
bacterial cell. Many, or most, of the media that have been found to be quite
adequate for primary isolation of mycobacteria, e.g. Lowenstein-Jensen
( L J ) Medium, those used primarily in assaying for drug susceptibility
(Middlebrook’s 7H9-10), or others slightly modified for distinguishing
between growth characteristics of strains, have not been found to be
adequate for use in phage typing. A series of tests involving most of the
components used in these media were carried out in the Atlanta Laboratory
to determine the effect on phage activity. Details of these tests are to be
found in a publication by Redmond and Ward (1966). Calcium ions have
been found to be necessary for lysis. Citrate and phosphates are detrimental
because they tend to inactivate or to precipitate calcium. A high concentra-