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W. B. SCHAEFER
reduce an agglutinin titre 16-fold or more are considered to possess the
homologous antigen. An eight-fold reduction of titre is interpreted to
indicate that the strain is closely related to, but not quite identical with,
the homologous antigen. A two- to four-fold reduction of titre and no
reduction of titre is considered to indicate non-identity.
After termination of the agglutination test the tubes undergo routine
cleaning procedure. They are kept in a phenolic disinfectant solution overnight, autoclaved, washed and dried for future use.
Several modifications of the agglutination test have been proposed. (1)
a slide agglutination test (Engel and Beerwald, 1970)) (2) a “simplified”
agglutination test using volumes of antisera and antigens of 0.1 instead of
0.5 ml and only a single antiserum for each type (Reznikov and Leggo,
1972), (3) an agglutination inhibition test for the differentiation of M.
avium from M. intracellulare (Richards and Eacret, 1972)) (4) a micromethod for serotyping strains of the M . avizim complex (Thoen et al.,
1975).
F. Preparation of absorbed antisera
Because of cross-reactions between mycobacterial species (Schaefer,
1967), absorptions have to be carried out in the following way: 5-8 ml nonabsorbed undiluted antiserum is mixed with 1 ml of the sediment of a
strain of each coagglutinating type. The mixture is incubated at 4°C overnight and centrifuged at 5 rev/min for 10 min. The serum is decanted and
its homologous and heterologous titres are determined by using suspensions
of two homologous and two heterologous strains as the antigens. If the
serum is not yet type-specific the absorption is repeated. The antiserum is
used at the lowest dilution at which it is type-specific. Table I1 indicates
the coagglutinating types for 23 serotypes of M. avium, M . intracellulare
and M . scrofulaceum.
IV. CHEMISTRY OF T H E TYPE-SPECIFIC ANTIGENS
The specific antigens of the atypical mycobacteria can be extracted from
the bacteria by boiling methanol. The bulk of the antigen is contained in
the acetone-soluble fraction of the methanol extract (Schaefer, 1964). The
antigens are also soluble in phenol (Wayne, 1971a, b).
Work in progress on the purification of the specific antigens (Brokl,
Schaefer and Goren, unpublished) indicates that the antigens of M.
intracellulare serotype 8 are very likely peptidoglycolipids, whereas those
from M . sxulgai and M . kansasii may be lipo-oligosaccharides containing
only a small portion of a nitrogenous component. The specific substances
are obtained from methanol extracts by a combination of solvent parti-
W. B. SCHAEFER
reduce an agglutinin titre 16-fold or more are considered to possess the
homologous antigen. An eight-fold reduction of titre is interpreted to
indicate that the strain is closely related to, but not quite identical with,
the homologous antigen. A two- to four-fold reduction of titre and no
reduction of titre is considered to indicate non-identity.
After termination of the agglutination test the tubes undergo routine
cleaning procedure. They are kept in a phenolic disinfectant solution overnight, autoclaved, washed and dried for future use.
Several modifications of the agglutination test have been proposed. (1)
a slide agglutination test (Engel and Beerwald, 1970)) (2) a “simplified”
agglutination test using volumes of antisera and antigens of 0.1 instead of
0.5 ml and only a single antiserum for each type (Reznikov and Leggo,
1972), (3) an agglutination inhibition test for the differentiation of M.
avium from M. intracellulare (Richards and Eacret, 1972)) (4) a micromethod for serotyping strains of the M . avizim complex (Thoen et al.,
1975).
F. Preparation of absorbed antisera
Because of cross-reactions between mycobacterial species (Schaefer,
1967), absorptions have to be carried out in the following way: 5-8 ml nonabsorbed undiluted antiserum is mixed with 1 ml of the sediment of a
strain of each coagglutinating type. The mixture is incubated at 4°C overnight and centrifuged at 5 rev/min for 10 min. The serum is decanted and
its homologous and heterologous titres are determined by using suspensions
of two homologous and two heterologous strains as the antigens. If the
serum is not yet type-specific the absorption is repeated. The antiserum is
used at the lowest dilution at which it is type-specific. Table I1 indicates
the coagglutinating types for 23 serotypes of M. avium, M . intracellulare
and M . scrofulaceum.
IV. CHEMISTRY OF T H E TYPE-SPECIFIC ANTIGENS
The specific antigens of the atypical mycobacteria can be extracted from
the bacteria by boiling methanol. The bulk of the antigen is contained in
the acetone-soluble fraction of the methanol extract (Schaefer, 1964). The
antigens are also soluble in phenol (Wayne, 1971a, b).
Work in progress on the purification of the specific antigens (Brokl,
Schaefer and Goren, unpublished) indicates that the antigens of M.
intracellulare serotype 8 are very likely peptidoglycolipids, whereas those
from M . sxulgai and M . kansasii may be lipo-oligosaccharides containing
only a small portion of a nitrogenous component. The specific substances
are obtained from methanol extracts by a combination of solvent parti-