IX. IDENTIFICATION OF MYCOPLASMAS
399
culture or with a drop of liquid culture. The inoculated tubes are incubated
aerobically together with controls consisting of uninoculated bg and bl
tubes. A positive reaction is indicated by a colour change from red to
orange or yellow in the inoculated test medium, but not in any of the
controls. If the same colour change is observed in either the bl medium or
in the uninoculated bg medium the result is recorded as equivocal or
negative.
(c) Comments. The use of media less complex than those normally used for
growth is intended to prevent, as far as possible, any decrease of p H
resulting from metabolic activities other than catabolism of glucose. For
example, the fall in pH that occurs during incubation of M. bovkenitalium,
which is known to be a non-fermenter of glucose, has been shown to be
greater in the presence of yeast extract (Edward and Razin, 1974). The
enzymatic removal of pre-existing amounts of glucose and arginine assists
further the standardisation of the test, thereby enabling more clear-cut and
unequivocal results to be obtained. Since, unfortunately, not all strains
will grow in the standard test medium, appropriate enrichments may be
required. Whatever medium is used, the importance of including in the
test system the two controls, (i) uninoculated medium with glucose, and
(ii) inoculated medium without glucose, should be strongly emphasised.
Determination of the number of colony forming units occurring during
growth may also be required to ensure that there are sufficient organisms to
produce a definite change of colour. I n our experience about 107 CFU/ml
are needed to produce such a change, which means that a negative result
associated with a peak number of 106 CFU/ml or less is unreliable.
2. Alternative methods
The relatively simple methods described above for determination of
catabolism of glucose may, as indicated already, be unsuitdble or give
ambiguous results with some strains. More sensitive and specific tests are
available, based on (i) determination of glucose disappearance by the
glucose oxidase reaction, (ii) determination of acid fermentation products
from radioactive glucose, or (iii) determination of hexokinase activity as an
indicator of the presence of the glycolytic pathway. Although these methods
are probably too laborious as part of the routine identification procedure,
the interested reader is referred to a WHO Working Document prepared
by Edward and Razin (1974) describing these techniques.
D. Arginine hydrolysis
1. Test medium
The basal medium, bz, is identical with b l except that the pH is adjusted
399
culture or with a drop of liquid culture. The inoculated tubes are incubated
aerobically together with controls consisting of uninoculated bg and bl
tubes. A positive reaction is indicated by a colour change from red to
orange or yellow in the inoculated test medium, but not in any of the
controls. If the same colour change is observed in either the bl medium or
in the uninoculated bg medium the result is recorded as equivocal or
negative.
(c) Comments. The use of media less complex than those normally used for
growth is intended to prevent, as far as possible, any decrease of p H
resulting from metabolic activities other than catabolism of glucose. For
example, the fall in pH that occurs during incubation of M. bovkenitalium,
which is known to be a non-fermenter of glucose, has been shown to be
greater in the presence of yeast extract (Edward and Razin, 1974). The
enzymatic removal of pre-existing amounts of glucose and arginine assists
further the standardisation of the test, thereby enabling more clear-cut and
unequivocal results to be obtained. Since, unfortunately, not all strains
will grow in the standard test medium, appropriate enrichments may be
required. Whatever medium is used, the importance of including in the
test system the two controls, (i) uninoculated medium with glucose, and
(ii) inoculated medium without glucose, should be strongly emphasised.
Determination of the number of colony forming units occurring during
growth may also be required to ensure that there are sufficient organisms to
produce a definite change of colour. I n our experience about 107 CFU/ml
are needed to produce such a change, which means that a negative result
associated with a peak number of 106 CFU/ml or less is unreliable.
2. Alternative methods
The relatively simple methods described above for determination of
catabolism of glucose may, as indicated already, be unsuitdble or give
ambiguous results with some strains. More sensitive and specific tests are
available, based on (i) determination of glucose disappearance by the
glucose oxidase reaction, (ii) determination of acid fermentation products
from radioactive glucose, or (iii) determination of hexokinase activity as an
indicator of the presence of the glycolytic pathway. Although these methods
are probably too laborious as part of the routine identification procedure,
the interested reader is referred to a WHO Working Document prepared
by Edward and Razin (1974) describing these techniques.
D. Arginine hydrolysis
1. Test medium
The basal medium, bz, is identical with b l except that the pH is adjusted