406
E. A. FREUNDT ET AL.
4. Alternative methods
The original version of the G I test with antiserum incorporated in the
growth medium never obtained any wide use because of the relatively large
amounts of antiserum required. A serum-drop method was first described
by Herderschee (1963). The principle of this technique, that may deserve
more attention than has hitherto been paid to it, is to allow a drop of
antiserum to penetrate into a dried agar medium following inoculation of
part of the surface.? Attention should also be drawn to the development of
micromethods which utilise the cups of microtitre plastic plates as reservoirs
for the growth medium (Jensen, 1963; Campello et al., 1972).
C. Metabolism inhibition
1. Equipment and cleaning procedures
The metabolism inhibition (MI) test is performed in a 0.025 ml microtitre system manufactured by the Cooke Engineering Co., Alexandria,
Va., U.S.A., using Disposo Trays (IS-MRC-96) from Linbro Chemical
Co., Inc., New Haven, Connecticut. Equipment required includes micropipettes, microdiluters, absorbent “GO NO-GO” test pads, sealing tape
and a test reading mirror. Only trays with U-shaped wells, arranged in
eight horizontal rows, each with 12 wells, should be used.
The titrations should of course be performed in such a manner that
bacterial and fungal contamination is avoided. T o that end, the trays are
rinsed immediately before use in 70% ethanol for 5 min, and in deionised
water for 30 min, and air-dried. Reusable micropipettes are rinsed in saline
and distilled water, boiled for 5-10 min in distilled water and air-dried.
Stainless steel microdiluters are rinsed in saline, dried, rinsed again
in distilled water, dried and finally lightly flamed. Before reuse, microdiluters should be checked for accuracy of delivery by means of the “GO
NO-GO” test pads. Diluters that deliver exactly 0.025 ml, i.e. whose
liquid content just covers the circle on the test pad, are ready for use after
being gently flamed again. Those that do not deliver accurately should be
discarded unless thorough flaming, followed by immediate quenching,
results in readjustment.
2. Reagents
high titre (at least 64) and test media.
These include antigens, i.e. mycoplasma cultures, antisera of reasonably
t A modification of the serum-drop method that appears to be more sensitive
than other GI tests used today, and equally specific, was introduced very recently
(Meller, B. R. J. appl. Microbial. (1979). In press).
E. A. FREUNDT ET AL.
4. Alternative methods
The original version of the G I test with antiserum incorporated in the
growth medium never obtained any wide use because of the relatively large
amounts of antiserum required. A serum-drop method was first described
by Herderschee (1963). The principle of this technique, that may deserve
more attention than has hitherto been paid to it, is to allow a drop of
antiserum to penetrate into a dried agar medium following inoculation of
part of the surface.? Attention should also be drawn to the development of
micromethods which utilise the cups of microtitre plastic plates as reservoirs
for the growth medium (Jensen, 1963; Campello et al., 1972).
C. Metabolism inhibition
1. Equipment and cleaning procedures
The metabolism inhibition (MI) test is performed in a 0.025 ml microtitre system manufactured by the Cooke Engineering Co., Alexandria,
Va., U.S.A., using Disposo Trays (IS-MRC-96) from Linbro Chemical
Co., Inc., New Haven, Connecticut. Equipment required includes micropipettes, microdiluters, absorbent “GO NO-GO” test pads, sealing tape
and a test reading mirror. Only trays with U-shaped wells, arranged in
eight horizontal rows, each with 12 wells, should be used.
The titrations should of course be performed in such a manner that
bacterial and fungal contamination is avoided. T o that end, the trays are
rinsed immediately before use in 70% ethanol for 5 min, and in deionised
water for 30 min, and air-dried. Reusable micropipettes are rinsed in saline
and distilled water, boiled for 5-10 min in distilled water and air-dried.
Stainless steel microdiluters are rinsed in saline, dried, rinsed again
in distilled water, dried and finally lightly flamed. Before reuse, microdiluters should be checked for accuracy of delivery by means of the “GO
NO-GO” test pads. Diluters that deliver exactly 0.025 ml, i.e. whose
liquid content just covers the circle on the test pad, are ready for use after
being gently flamed again. Those that do not deliver accurately should be
discarded unless thorough flaming, followed by immediate quenching,
results in readjustment.
2. Reagents
high titre (at least 64) and test media.
These include antigens, i.e. mycoplasma cultures, antisera of reasonably
t A modification of the serum-drop method that appears to be more sensitive
than other GI tests used today, and equally specific, was introduced very recently
(Meller, B. R. J. appl. Microbial. (1979). In press).