368
w. n. REDMOND et al.
certain extent by using two additional phage dilutions along with the R T D ;
for example, using 0.1 RTD, 1 R T D and ten-fold R T D for spotting on
each isolate to be typed. This requires more work, but its often valuable and
is recommended when questionable results are obtained.
The R T D is determined as follows: prepare a suspension of the bacterial
culture to be used (the propagating or the prototype strain) that will produce
a good, but not heavy lawn on the agar plate within one to three days. For
this purpose log-phase bacteria from broth culture (RVB-10) are preferable.
Dilute the bacterial culture into sterile saline, water or nutrient broth to
an optical density of about 0.2 (much more dilute for rapidly growing
strains). The concentration of the suspension will vary in indirect ratio to
the rate of growth of the individual strain. Determine this by previous
experiment. Inoculate an RVA-17 agar plate by flooding with 1-1-5 ml of
the suspension. Spread the suspension uniformly over the entire surface and
tilt the plate to drain the surplus inoculum to one side. Pipette off the surplus and allow the plate to dry in an incubator for 2-3 h (24 h in the case of
slowly growing organisms). Make eight (more if necessary) ten-fold serial
dilutions of the phage suspension into NB3 using a different pipette (1 ml
or 0.1 ml) for each transfer. Using the six highest dilutions, i.e. lO-3-lO-*,
spot 0.01 ml on marked areas of the inoculated plate. Incubate the plate
and examine daily for lysis. When maximal lysis has been attained determine the highest dilution which has produced complete lysis. Because of
the slowness of growth and lysis this may require three or four days.
This dilution is the RTD. Phage dilutions with smaller increments may
be used if necessary, e.g. if the 10-6 dilution produces a clear spot and the
10-7 dilution produces a slightly turbid spot, an intermediate dilution of
5 x 10-7 may be tested and used as the RTD, if it produces complete
lysis. Keep stock phage suspensions in the refrigerator and make fresh
R T D dilutions each day tests are made on unknown strains.
B. Phage and bacterial sources
Typing phages and their respective propagating strains of mycobacteria
may be obtained from the Rijks Instituut Voor de Volksgezondhera, Bilthoven, Netherlands, or they may be isolated and propagated locally as
indicated in Section 111.
C. Spotting with phage suspensions
With a small syringe and small gauge needle, or the Accu-drop assembly
sold by the Sylvania Company, Milburn, New Jersey, place a fraction of a
drop, 0-5-1 x 10-2, of each typing phage suspension diluted to the R T D on
each plate. Each plate should be marked to identify the position of the
droplets, or each plate should be placed on top of a circle of paper marked
w. n. REDMOND et al.
certain extent by using two additional phage dilutions along with the R T D ;
for example, using 0.1 RTD, 1 R T D and ten-fold R T D for spotting on
each isolate to be typed. This requires more work, but its often valuable and
is recommended when questionable results are obtained.
The R T D is determined as follows: prepare a suspension of the bacterial
culture to be used (the propagating or the prototype strain) that will produce
a good, but not heavy lawn on the agar plate within one to three days. For
this purpose log-phase bacteria from broth culture (RVB-10) are preferable.
Dilute the bacterial culture into sterile saline, water or nutrient broth to
an optical density of about 0.2 (much more dilute for rapidly growing
strains). The concentration of the suspension will vary in indirect ratio to
the rate of growth of the individual strain. Determine this by previous
experiment. Inoculate an RVA-17 agar plate by flooding with 1-1-5 ml of
the suspension. Spread the suspension uniformly over the entire surface and
tilt the plate to drain the surplus inoculum to one side. Pipette off the surplus and allow the plate to dry in an incubator for 2-3 h (24 h in the case of
slowly growing organisms). Make eight (more if necessary) ten-fold serial
dilutions of the phage suspension into NB3 using a different pipette (1 ml
or 0.1 ml) for each transfer. Using the six highest dilutions, i.e. lO-3-lO-*,
spot 0.01 ml on marked areas of the inoculated plate. Incubate the plate
and examine daily for lysis. When maximal lysis has been attained determine the highest dilution which has produced complete lysis. Because of
the slowness of growth and lysis this may require three or four days.
This dilution is the RTD. Phage dilutions with smaller increments may
be used if necessary, e.g. if the 10-6 dilution produces a clear spot and the
10-7 dilution produces a slightly turbid spot, an intermediate dilution of
5 x 10-7 may be tested and used as the RTD, if it produces complete
lysis. Keep stock phage suspensions in the refrigerator and make fresh
R T D dilutions each day tests are made on unknown strains.
B. Phage and bacterial sources
Typing phages and their respective propagating strains of mycobacteria
may be obtained from the Rijks Instituut Voor de Volksgezondhera, Bilthoven, Netherlands, or they may be isolated and propagated locally as
indicated in Section 111.
C. Spotting with phage suspensions
With a small syringe and small gauge needle, or the Accu-drop assembly
sold by the Sylvania Company, Milburn, New Jersey, place a fraction of a
drop, 0-5-1 x 10-2, of each typing phage suspension diluted to the R T D on
each plate. Each plate should be marked to identify the position of the
droplets, or each plate should be placed on top of a circle of paper marked