3 72
w. B. REDMOND et al.
the bacteria and agar, and pour the mixture on to the hard basal oleic
albumin agar. Gently rotate the Petri dish to ensure even distribution of the
soft-agar over the surface of the hard basal medium. Place the Petri dishes
on a flat level surface to allow the soft-agar to cool and harden. After allowing the soft-agar to thoroughly harden, place the inoculated plates in a
37°C incubator, in the inverted position, and incubate overnight. This
incubation period allows the surface of the medium to dry sufficiently to
prevent “running” of the drops of the phage lysates. The dried plates are
then spotted with the RTDs of the respective test phages as recommended.
The 7H9 broth cultures of M . tuberculosis may be used between the
tenth and thirteenth days of incubation. All isolates of M. tuberculosis
utilise Tween-80, but ’very slowly and with variation among strains;
therefore, 7H9 broth cultures less than ten days old should not be used.
This procedure will circumvent the occasional problem with the interference of Tween-80 with the adsorption of phage which may be encountered if younger cultures are used.
G. Reproducibility of phage typing results
Statistical evaluations of results reported by five of the WHO Study
Group Laboratories have been made and reported by Rado et al. (1975). I n
this study 100 strains of M. tuberculosis were typed, the same phages and
techniques being used in each laboratory. The tests were repeated in a
second study one year later.
It was found that the strains could be assigned to phage types A and B
with better than 98% reliability. In both studies there was 100% agreement
among all laboratories with one phage, M T P H 2. Although the reproducibility of type C strains (total less than ten strains) was below SOYo,
improvements in methods appeared to justify continuation of this as a
major type.
Subdivision of type A strains by the auxiliary phages showed less agreement among the five laboratories. The results of the final study and analysis
on the 100 strains were in agreement 70.4% of the time. It is apparent that
phages with greater specificity will make it possible to increase the reproducibility in this group.
VII. LYSOGENY
Lysogeny in the mycobacteria appears to be very wide-spread, if not
universal. Recent reports by Sula et al. (1973), Jones (1973), Mankiewicz
(1961), Buraczewska et al. (1971), Murohashi et al. (1959) and others have
revealed phage in bovine, atypical group 111, and M . fortuitum strains
isolated from human or animal sources. The lysogenic condition affects the
w. B. REDMOND et al.
the bacteria and agar, and pour the mixture on to the hard basal oleic
albumin agar. Gently rotate the Petri dish to ensure even distribution of the
soft-agar over the surface of the hard basal medium. Place the Petri dishes
on a flat level surface to allow the soft-agar to cool and harden. After allowing the soft-agar to thoroughly harden, place the inoculated plates in a
37°C incubator, in the inverted position, and incubate overnight. This
incubation period allows the surface of the medium to dry sufficiently to
prevent “running” of the drops of the phage lysates. The dried plates are
then spotted with the RTDs of the respective test phages as recommended.
The 7H9 broth cultures of M . tuberculosis may be used between the
tenth and thirteenth days of incubation. All isolates of M. tuberculosis
utilise Tween-80, but ’very slowly and with variation among strains;
therefore, 7H9 broth cultures less than ten days old should not be used.
This procedure will circumvent the occasional problem with the interference of Tween-80 with the adsorption of phage which may be encountered if younger cultures are used.
G. Reproducibility of phage typing results
Statistical evaluations of results reported by five of the WHO Study
Group Laboratories have been made and reported by Rado et al. (1975). I n
this study 100 strains of M. tuberculosis were typed, the same phages and
techniques being used in each laboratory. The tests were repeated in a
second study one year later.
It was found that the strains could be assigned to phage types A and B
with better than 98% reliability. In both studies there was 100% agreement
among all laboratories with one phage, M T P H 2. Although the reproducibility of type C strains (total less than ten strains) was below SOYo,
improvements in methods appeared to justify continuation of this as a
major type.
Subdivision of type A strains by the auxiliary phages showed less agreement among the five laboratories. The results of the final study and analysis
on the 100 strains were in agreement 70.4% of the time. It is apparent that
phages with greater specificity will make it possible to increase the reproducibility in this group.
VII. LYSOGENY
Lysogeny in the mycobacteria appears to be very wide-spread, if not
universal. Recent reports by Sula et al. (1973), Jones (1973), Mankiewicz
(1961), Buraczewska et al. (1971), Murohashi et al. (1959) and others have
revealed phage in bovine, atypical group 111, and M . fortuitum strains
isolated from human or animal sources. The lysogenic condition affects the