IX. IDENTIFICATION OF MYCOPLASMAS
409
(iii) Endpoint control: add 0.2 ml (eight drops) of test medium with
the pH of the desired endpoint to four wells. (Not included for
tetrazolium reduction tests.)
(7) Seal the plate with tape and incubate at an appropriate temperature.
(8) Read the test when the antigen control has changed approximately
0.5 pH units, i.e. when it shows the same colour as the “endpoint controls”.
Tetrazolium reduction readings are made when the antigen control wells
show a red precipitate. The endpoint is recorded as the highest serum
dilution that prevents a colour change from occurring.
D. Immunofluorescence
1. Equipment
At the FAO/WHO Collaborating Centre for Animal Mycoplasmas,
immunofluorescence tests are carried out by the plate epi-immunofluorescence (epi-IMF) procedure of Del Giudice et al. (1967). Microscopy
is performed with a Zeiss Standard (RA) microscope equipped with an
Osram HBQ mercury lamp and an incident illumination attachment,
allowing illumination of the specimen from above and thereby avoiding
the transmission of exciting ultraviolet light through the agar and the
plastic plate or glass slide. Two exciting filters, BG 12/4 and BG 3/4
(Zeiss), and a barrier filter No. 47 (Zeiss) are employed. An aperture
diaphragm is inserted between the source of light and the exciting filters.
2. Reagents
The test is performed with either unfixed mycoplasma colonies or hot
water-fixed impressions of colonies as antigen. Following inoculation of an
appropriate agar medium with approximately 105 CFU/ml the plates are
incubated until the colonies are clearly visible under the microscope but
preferably before they are fully grown.
When using unfixed agar colonies, agar blocks about 0-25 cm2 in size
are cut in different geometric patterns to facilitate their identification
and placed on slides with the colonies uppermost. For the hot waterfixation technique, agar blocks are transferred to slides with the colony
side downward. The slides are placed at an angle of 45” in a bath with
distilled water at 80°C. The temperature of the water is maintained at
80°C until the blocks drop off, which usually occurs after 30 to 60 s. The
slides are then immediately washed in distilled water and air-dried.
For the direct immunofluorescence technique, the antisera are conjugated
according to Nairn (1968). The globulin is precipitated with 40% saturated
ammonium sulphate. Conjugation with fluorescein isothiocyanate (FITC)
I (Sigma) is performed at 25°C and pH 9.5 in phosphate buffer, adding one
409
(iii) Endpoint control: add 0.2 ml (eight drops) of test medium with
the pH of the desired endpoint to four wells. (Not included for
tetrazolium reduction tests.)
(7) Seal the plate with tape and incubate at an appropriate temperature.
(8) Read the test when the antigen control has changed approximately
0.5 pH units, i.e. when it shows the same colour as the “endpoint controls”.
Tetrazolium reduction readings are made when the antigen control wells
show a red precipitate. The endpoint is recorded as the highest serum
dilution that prevents a colour change from occurring.
D. Immunofluorescence
1. Equipment
At the FAO/WHO Collaborating Centre for Animal Mycoplasmas,
immunofluorescence tests are carried out by the plate epi-immunofluorescence (epi-IMF) procedure of Del Giudice et al. (1967). Microscopy
is performed with a Zeiss Standard (RA) microscope equipped with an
Osram HBQ mercury lamp and an incident illumination attachment,
allowing illumination of the specimen from above and thereby avoiding
the transmission of exciting ultraviolet light through the agar and the
plastic plate or glass slide. Two exciting filters, BG 12/4 and BG 3/4
(Zeiss), and a barrier filter No. 47 (Zeiss) are employed. An aperture
diaphragm is inserted between the source of light and the exciting filters.
2. Reagents
The test is performed with either unfixed mycoplasma colonies or hot
water-fixed impressions of colonies as antigen. Following inoculation of an
appropriate agar medium with approximately 105 CFU/ml the plates are
incubated until the colonies are clearly visible under the microscope but
preferably before they are fully grown.
When using unfixed agar colonies, agar blocks about 0-25 cm2 in size
are cut in different geometric patterns to facilitate their identification
and placed on slides with the colonies uppermost. For the hot waterfixation technique, agar blocks are transferred to slides with the colony
side downward. The slides are placed at an angle of 45” in a bath with
distilled water at 80°C. The temperature of the water is maintained at
80°C until the blocks drop off, which usually occurs after 30 to 60 s. The
slides are then immediately washed in distilled water and air-dried.
For the direct immunofluorescence technique, the antisera are conjugated
according to Nairn (1968). The globulin is precipitated with 40% saturated
ammonium sulphate. Conjugation with fluorescein isothiocyanate (FITC)
I (Sigma) is performed at 25°C and pH 9.5 in phosphate buffer, adding one