VIII. METHODS FOR TYPING MYCOBACTERIA
3 69
to designate the positions of the droplets. Handle the plate carefully so as
to avoid spreading and coalescence of the droplets. Place in the incubator
at 36°C. Read results after 24 h and daily thereafter for as long as necessary,
up to 7 days. The final reading on rapidly growing bacteria can be made
after one or two days; for slowly growing pathogenic bacteria five to seven
days are necessary for full development of the phage activity.
D. Reading and reporting of the results
The results of the phage activity should be recorded daily. The following
designations are recommended. Complete lysis + + + + ; nearly complete
lysis + + +; lysis with slight turbidity + -I-; individual plaques +
(number may be indicated as +(16)); no lysis -. For routine reporting
of results of phage typing a simpler form is frequently used as follows;
complete or nearly complete lysis + + + ; partial lysis + + ; few plaques + ;
no lysis -.
E. Methods for typing M . tuberculosis
Slight modifications of the techniques have been found beneficial in
subtyping strains of M . tuberculosis. The media and methods for growth of
the bacterial cultures and the propagation of the phages are the same as
indicated in the previous Section. In order to obtain homogeneous growth
of the cultures to be tested, each strain should be transferred two or more
times in RVB-10 broth until a well-dispersed suspension is obtained.
When the cultures have reached readily visible and dispersed growth,
10ml should be centrifuged and resuspended in NB3 so as to give a
turbidity corresponding to the number 3 or 4 tube on the McFarland Scale.
Spread 1 ml amounts on the surface of RVA-17 plates and allow to dry,
with the cover on, in the incubator for 24 h. Spot with 0.01 ml of each
phage using three dilutions, 0.10 x RTD, 1 x RTD and 10 x RTD. Titration of the stock phages should be made periodically and not more than
14 days before their use.
T A B L E IV
Proposed division of phage type A using auxiliary phages
Subtype
A.
Ax
Type A lysed by not more than one auxiliary phage
Type A lysed by two or more auxiliary phages
~~~
~
Adapted from Rado et ul. (1975).
13
3 69
to designate the positions of the droplets. Handle the plate carefully so as
to avoid spreading and coalescence of the droplets. Place in the incubator
at 36°C. Read results after 24 h and daily thereafter for as long as necessary,
up to 7 days. The final reading on rapidly growing bacteria can be made
after one or two days; for slowly growing pathogenic bacteria five to seven
days are necessary for full development of the phage activity.
D. Reading and reporting of the results
The results of the phage activity should be recorded daily. The following
designations are recommended. Complete lysis + + + + ; nearly complete
lysis + + +; lysis with slight turbidity + -I-; individual plaques +
(number may be indicated as +(16)); no lysis -. For routine reporting
of results of phage typing a simpler form is frequently used as follows;
complete or nearly complete lysis + + + ; partial lysis + + ; few plaques + ;
no lysis -.
E. Methods for typing M . tuberculosis
Slight modifications of the techniques have been found beneficial in
subtyping strains of M . tuberculosis. The media and methods for growth of
the bacterial cultures and the propagation of the phages are the same as
indicated in the previous Section. In order to obtain homogeneous growth
of the cultures to be tested, each strain should be transferred two or more
times in RVB-10 broth until a well-dispersed suspension is obtained.
When the cultures have reached readily visible and dispersed growth,
10ml should be centrifuged and resuspended in NB3 so as to give a
turbidity corresponding to the number 3 or 4 tube on the McFarland Scale.
Spread 1 ml amounts on the surface of RVA-17 plates and allow to dry,
with the cover on, in the incubator for 24 h. Spot with 0.01 ml of each
phage using three dilutions, 0.10 x RTD, 1 x RTD and 10 x RTD. Titration of the stock phages should be made periodically and not more than
14 days before their use.
T A B L E IV
Proposed division of phage type A using auxiliary phages
Subtype
A.
Ax
Type A lysed by not more than one auxiliary phage
Type A lysed by two or more auxiliary phages
~~~
~
Adapted from Rado et ul. (1975).
13