404
E. A. FREUNDT ET AL.
with 0.02 ml of antiserum using either a calibrated loop or a micropipette,
air-dried at room temperature and stored at 4°C. Discs can be stored at this
temperature for several months and possibly for several years. The test
is performed on a solid agar medium, approximately 4 mm deep and usually
of the composition used for isolating the test organism. The plates should
be free from surface moisture before use, for example, by leaving them in
the incubator at 37°C for 30 min. Mycoplasma cultures in broth medium
or suspensions of organisms containing about 105 colony forming units
(CFU)/ml are used as antigens. The importance of avoiding too large an
inoculum cannot be overemphasised. In order to ensure the optimal
density of colonies (well separated, though not too scattered) it is advisable
to use, in each test, three culture dilutions containing approximately 104,
105 and 106 CFU/ml, as determined by preliminary titrations.
(b) Test procedure. The plates are inoculated by the running-drop method.
One drop (0.01 ml) of the antigen is allowed to run down the surface of the
agar. When the fluid of the inoculum ‘has been absorbed by the growth
medium, the disc is placed in the centre of the inoculated area. Incubation
is performed in a moist atmosphere at 37°C until the appearance of
microscopically visible colonies. With some organisms this may be achieved
in about 48 h. If no growth has appeared by that time, incubation is
continued for a further 2448 h. Readings are made under a conventional
low-power microscope or a dissecting microscope. The zone of inhibition
is measured from the edge of the disc to the edge of the area of colonies.
Since the borderline between growth and absence of growth is unfortunately not always clear-cut, the extent of inhibition may be recorded in
terms of total, nearly total, or relative inhibition. A nearly total inhibition
is defined as a clear inhibition with less than ten “break through” colonies
appearing within the inhibition zone, and relative inhibition as a less
pronounced, although significant reduction in the size and/or number of
colonies round the disc. If identification is based solely upon the growth
inhibition test, a total or nearly total inhibition of at least 2 mm is required.
2. Agar well method
The test is performed as described above, except that wells filled with
antiserum are used instead of serum-impregnated discs (Black, 1973b).
Following inoculation of the plates by the running-drop method, a well
with, a diameter of 4 mm is punched out in the centre of the inoculated
area, and filled with about 0.05 ml of antiserum. The well is produced by
means of a stainless steel cylinder, the agar block being removed by gentle
suction.
E. A. FREUNDT ET AL.
with 0.02 ml of antiserum using either a calibrated loop or a micropipette,
air-dried at room temperature and stored at 4°C. Discs can be stored at this
temperature for several months and possibly for several years. The test
is performed on a solid agar medium, approximately 4 mm deep and usually
of the composition used for isolating the test organism. The plates should
be free from surface moisture before use, for example, by leaving them in
the incubator at 37°C for 30 min. Mycoplasma cultures in broth medium
or suspensions of organisms containing about 105 colony forming units
(CFU)/ml are used as antigens. The importance of avoiding too large an
inoculum cannot be overemphasised. In order to ensure the optimal
density of colonies (well separated, though not too scattered) it is advisable
to use, in each test, three culture dilutions containing approximately 104,
105 and 106 CFU/ml, as determined by preliminary titrations.
(b) Test procedure. The plates are inoculated by the running-drop method.
One drop (0.01 ml) of the antigen is allowed to run down the surface of the
agar. When the fluid of the inoculum ‘has been absorbed by the growth
medium, the disc is placed in the centre of the inoculated area. Incubation
is performed in a moist atmosphere at 37°C until the appearance of
microscopically visible colonies. With some organisms this may be achieved
in about 48 h. If no growth has appeared by that time, incubation is
continued for a further 2448 h. Readings are made under a conventional
low-power microscope or a dissecting microscope. The zone of inhibition
is measured from the edge of the disc to the edge of the area of colonies.
Since the borderline between growth and absence of growth is unfortunately not always clear-cut, the extent of inhibition may be recorded in
terms of total, nearly total, or relative inhibition. A nearly total inhibition
is defined as a clear inhibition with less than ten “break through” colonies
appearing within the inhibition zone, and relative inhibition as a less
pronounced, although significant reduction in the size and/or number of
colonies round the disc. If identification is based solely upon the growth
inhibition test, a total or nearly total inhibition of at least 2 mm is required.
2. Agar well method
The test is performed as described above, except that wells filled with
antiserum are used instead of serum-impregnated discs (Black, 1973b).
Following inoculation of the plates by the running-drop method, a well
with, a diameter of 4 mm is punched out in the centre of the inoculated
area, and filled with about 0.05 ml of antiserum. The well is produced by
means of a stainless steel cylinder, the agar block being removed by gentle
suction.