402
E. A. FREUNDT ET AL.
1973; Woolcock et nl., 1973; Watanabe, 1975). The potential value of
these methods in the routine identification of mycoplasmas cannot be fully
assessed on the basis of currently availablc data.
G. Hydrolysis of aesculin and arbutin
1. Media and reagents
Cultivation is on conventional solid media. Filter paper discs, about
6 mm in diameter, are impregnated first with 0.02 ml of 5% (w/v) ferric
citrate; after 24 h 0.02ml of a 10% (w/v) solution of either aesculin or
arbutin is added to each disc.
2. Test procedure
The agar plate is inoculated with 0.01 ml of the stock culture by the
running-drop technique. Following absorption of the inoculum, a disc
containing ferric citrate plus either aesculin or arbutin is placed in the
centre of the inoculated area. Incubation is at 37"C, readings being performed daily for up to seven days. The appearance of a brownish colouration
of the culture streak, starting at the edge of the disc, indicates a positive
reaction.
3. Comments
The disc technique developed by G. Askaa (unpublished) for demonstrating hydrolysis of aesculin and arbutin has the practical advantage that
the test can be performed on a conventional growth medium without
incorporating test substrate and reagent (Williams and Wittler, 1971).
The fluoregenic test referred to in Section E, 3 has also been utilised for
demonstration of aesculin hydrolysis (Bradbury, 1977).
V. SEROLOGICAL METHODS I N IDENTIFICATION
A. Comments on antisera
No attempt will be made to discuss the methods that are used to prepare
reference antisera for classification or identification of mycoplasmas, since
these procedures do not differ essentially from those used for other microorganisms. The methods generally used in our respective laboratories for
production of immunising antigen and schemes of immunisation are
described by Hollingdale and Lemcke (1969) and Ern0 et al. (1973). However, a problem pertinent to mycoplasmas must be mentioned: the effect of
serum proteins adsorbed by mycoplasma antigens from the serum-supplemented media in which they are grown. Jf the immunising antigen is grown
in medium containing serum foreign to the animal which is to be immunised, the resulting antiserum will contain antibodies to the foreign
E. A. FREUNDT ET AL.
1973; Woolcock et nl., 1973; Watanabe, 1975). The potential value of
these methods in the routine identification of mycoplasmas cannot be fully
assessed on the basis of currently availablc data.
G. Hydrolysis of aesculin and arbutin
1. Media and reagents
Cultivation is on conventional solid media. Filter paper discs, about
6 mm in diameter, are impregnated first with 0.02 ml of 5% (w/v) ferric
citrate; after 24 h 0.02ml of a 10% (w/v) solution of either aesculin or
arbutin is added to each disc.
2. Test procedure
The agar plate is inoculated with 0.01 ml of the stock culture by the
running-drop technique. Following absorption of the inoculum, a disc
containing ferric citrate plus either aesculin or arbutin is placed in the
centre of the inoculated area. Incubation is at 37"C, readings being performed daily for up to seven days. The appearance of a brownish colouration
of the culture streak, starting at the edge of the disc, indicates a positive
reaction.
3. Comments
The disc technique developed by G. Askaa (unpublished) for demonstrating hydrolysis of aesculin and arbutin has the practical advantage that
the test can be performed on a conventional growth medium without
incorporating test substrate and reagent (Williams and Wittler, 1971).
The fluoregenic test referred to in Section E, 3 has also been utilised for
demonstration of aesculin hydrolysis (Bradbury, 1977).
V. SEROLOGICAL METHODS I N IDENTIFICATION
A. Comments on antisera
No attempt will be made to discuss the methods that are used to prepare
reference antisera for classification or identification of mycoplasmas, since
these procedures do not differ essentially from those used for other microorganisms. The methods generally used in our respective laboratories for
production of immunising antigen and schemes of immunisation are
described by Hollingdale and Lemcke (1969) and Ern0 et al. (1973). However, a problem pertinent to mycoplasmas must be mentioned: the effect of
serum proteins adsorbed by mycoplasma antigens from the serum-supplemented media in which they are grown. Jf the immunising antigen is grown
in medium containing serum foreign to the animal which is to be immunised, the resulting antiserum will contain antibodies to the foreign