IX. IDENTIFICATION OF MYCOPLASMAS
41 3
agglutination the deposit is finally suspended in formol saline of the
following composition (Klieneberger, 1938) :
0.2 M citric acid
0.15 ml
0.2 M NazHP04.12 HzO
1.85 ml
Formalin
0.25 ml
NaCl, 0.9%
to 100.00ml
The opacity of the suspension to be used for agglutination is suggested
by Edward (1950) as half of that of a Brown’s Opacity tube No. 1 (Wellcome
Reagents Ltd, Beckenham, Kent, England). Alternatively, suspensions can
be adjusted to give a 50% light transmittance at 650 nm in a spectrophotometer (Tully, 1963).
(b) Test procedure. The test is made in small test-tubes containing 0.1 ml
of serial two-fold dilutions of antiserum and 0.1 ml of antigen suspension.
Controls with saline and normal rabbit serum should be included. Tubes
are incubated in a water bath at 52-56°C. Agglutination is read after 4 and
6 h by the naked eye under a bright light, preferably in a dark room. The
endpoint of the titration is confirmed using a hand lens.
2. Slide agglutination
Several modifications of this alternative technique for direct agglutination have been described. The workability and relative merits of each of
these modifications are difficult to assess from the literature and the authors
must therefore withhold recommendation of any single procedure. According to Hromatka and Adler (1969) the sensitivity of the antigen is
maximal when suspended in citrate-phosphate buffer of pH 6-7, an
atmospheric pressure of 4 . 1 4 9 , and a molarity of 0.06-0-1. Reference
may be further made to Adler (1954, 1958), Adler and DaMassa (1968) and
Roberts (1970).
3. Comments
A major technical obstacle to the agglutination test is the tendency of
some antigen suspensions to autoagglutinate. A mere mechanical breaking
up of clumps and aggregates of particles is said to be more effective than
other methods tried for the preparation of homogeneous and stable
suspensions (Klieneberger, 1938). This was accomplished by Hromatka
and Adler (1969) by stirring the antigen suspension for 6 h at 4°C with a
magnetic stirrer and glass beads.
F. Complement fixation test
The test to be described is a two-dimensional one based on that designed
by Bradstreet and Taylor (1962) and Taylor (1967) for detecting virus
41 3
agglutination the deposit is finally suspended in formol saline of the
following composition (Klieneberger, 1938) :
0.2 M citric acid
0.15 ml
0.2 M NazHP04.12 HzO
1.85 ml
Formalin
0.25 ml
NaCl, 0.9%
to 100.00ml
The opacity of the suspension to be used for agglutination is suggested
by Edward (1950) as half of that of a Brown’s Opacity tube No. 1 (Wellcome
Reagents Ltd, Beckenham, Kent, England). Alternatively, suspensions can
be adjusted to give a 50% light transmittance at 650 nm in a spectrophotometer (Tully, 1963).
(b) Test procedure. The test is made in small test-tubes containing 0.1 ml
of serial two-fold dilutions of antiserum and 0.1 ml of antigen suspension.
Controls with saline and normal rabbit serum should be included. Tubes
are incubated in a water bath at 52-56°C. Agglutination is read after 4 and
6 h by the naked eye under a bright light, preferably in a dark room. The
endpoint of the titration is confirmed using a hand lens.
2. Slide agglutination
Several modifications of this alternative technique for direct agglutination have been described. The workability and relative merits of each of
these modifications are difficult to assess from the literature and the authors
must therefore withhold recommendation of any single procedure. According to Hromatka and Adler (1969) the sensitivity of the antigen is
maximal when suspended in citrate-phosphate buffer of pH 6-7, an
atmospheric pressure of 4 . 1 4 9 , and a molarity of 0.06-0-1. Reference
may be further made to Adler (1954, 1958), Adler and DaMassa (1968) and
Roberts (1970).
3. Comments
A major technical obstacle to the agglutination test is the tendency of
some antigen suspensions to autoagglutinate. A mere mechanical breaking
up of clumps and aggregates of particles is said to be more effective than
other methods tried for the preparation of homogeneous and stable
suspensions (Klieneberger, 1938). This was accomplished by Hromatka
and Adler (1969) by stirring the antigen suspension for 6 h at 4°C with a
magnetic stirrer and glass beads.
F. Complement fixation test
The test to be described is a two-dimensional one based on that designed
by Bradstreet and Taylor (1962) and Taylor (1967) for detecting virus