VII. IDENTIFICATION OF ATYPICAL MYCOBACTERIA
331
types, three of which have been identified (Rynearson and Wolinsky, 1967;
Wolinsky and Rynearson, 1968; Jenkins et al., 1972).
M. triviale (Kubica et al., 1970) is saprophytic with non-pigmented
rough, slowly growing colonies resembling those of M . tuberculosis. In
contrast to M . tuberculosis it is niacin negative, has a very high catalase
activity, rapidly hydrolyses Tween-80, and is weakly positive in the threeday arylsulphatase test.
111. METHODOLOGY O F SEROTYPING ATYPICAL
MYCOBACTERIA
A. Culture of mycobacteria and preparation of suspensions
The mycobacterium is cultured in a tube of Tween 80-albumin medium
to obtain dispersed growth. 0.1 ml of ten-fold dilutions (10-2-10-6) of this
culture in 0.2% aqueous bovine albumin is inoculated on to oleic acidalbumin 7 H l l agar medium in Petri dishes (9 cm in diameter). The inoculum is spread over the agar surface by means of a bent capillary pipette
while the plate is revolving on a turntable. The pipette is sterilised by
dipping in alcohol and brief flaming. The inoculated plates are enclosed in
polyethylene bags and incubated in inverted position at 37°C ( M . marinum
is incubated at 30-33°C). The plates showing separate colonies (usually
10-5 and 10-6 dilutions) are examined for colony morphology and purity.
For serological study, the bacteria are collected from the surface of the
medium by emulsification in phosphate buffered saline at pH 7.0 containing 0.5% phenol (PPBS), the diluent being added slowly. The bacterial
suspensions, before being used in the agglutination test, are washed with
PPBS in order to eliminate soluble antigens. T o be suitable for agglutination the suspensions should have an optical density of 0.3 in tubes of
20 mm outer diameter, using the Coleman Junior Spectrophotometer at
525 nm. This density corresponds roughly to tube No. 2 of the McFarland
scale. Spontaneously agglutinating suspensions can sometimes be stabilised
by the addition of a heterologous rabbit serum at a dilution of 1 : 50.
Some rapid growers give unstable suspensions when grown at 37"C,
but give stable suspensions when grown at 30°C.
B. Preparation of type-specific antisera
Rabbits are injected intravenously twice weekly with 2 ml of the bacterial
suspension of a density similar to that used in the agglutination test. After
three days, following the fourth injection, the serum is titrated. If the
titre is insufficient, the rabbit is re-injected with 1 ml or less of the bacterial
suspension and the serum is retitrated after each of the subsequent injections because of the danger that the animal may die of anaphylactic shock.
331
types, three of which have been identified (Rynearson and Wolinsky, 1967;
Wolinsky and Rynearson, 1968; Jenkins et al., 1972).
M. triviale (Kubica et al., 1970) is saprophytic with non-pigmented
rough, slowly growing colonies resembling those of M . tuberculosis. In
contrast to M . tuberculosis it is niacin negative, has a very high catalase
activity, rapidly hydrolyses Tween-80, and is weakly positive in the threeday arylsulphatase test.
111. METHODOLOGY O F SEROTYPING ATYPICAL
MYCOBACTERIA
A. Culture of mycobacteria and preparation of suspensions
The mycobacterium is cultured in a tube of Tween 80-albumin medium
to obtain dispersed growth. 0.1 ml of ten-fold dilutions (10-2-10-6) of this
culture in 0.2% aqueous bovine albumin is inoculated on to oleic acidalbumin 7 H l l agar medium in Petri dishes (9 cm in diameter). The inoculum is spread over the agar surface by means of a bent capillary pipette
while the plate is revolving on a turntable. The pipette is sterilised by
dipping in alcohol and brief flaming. The inoculated plates are enclosed in
polyethylene bags and incubated in inverted position at 37°C ( M . marinum
is incubated at 30-33°C). The plates showing separate colonies (usually
10-5 and 10-6 dilutions) are examined for colony morphology and purity.
For serological study, the bacteria are collected from the surface of the
medium by emulsification in phosphate buffered saline at pH 7.0 containing 0.5% phenol (PPBS), the diluent being added slowly. The bacterial
suspensions, before being used in the agglutination test, are washed with
PPBS in order to eliminate soluble antigens. T o be suitable for agglutination the suspensions should have an optical density of 0.3 in tubes of
20 mm outer diameter, using the Coleman Junior Spectrophotometer at
525 nm. This density corresponds roughly to tube No. 2 of the McFarland
scale. Spontaneously agglutinating suspensions can sometimes be stabilised
by the addition of a heterologous rabbit serum at a dilution of 1 : 50.
Some rapid growers give unstable suspensions when grown at 37"C,
but give stable suspensions when grown at 30°C.
B. Preparation of type-specific antisera
Rabbits are injected intravenously twice weekly with 2 ml of the bacterial
suspension of a density similar to that used in the agglutination test. After
three days, following the fourth injection, the serum is titrated. If the
titre is insufficient, the rabbit is re-injected with 1 ml or less of the bacterial
suspension and the serum is retitrated after each of the subsequent injections because of the danger that the animal may die of anaphylactic shock.