400
E. A. FREUNDT ET AL.
to 7.3. The test medium is prepared by supplementing the basal medium
with 1% L-arginine (Appendix, Sections B, 2 (a) and (b)).
2. Test procedure
This is the same as that described for the glucose test, including similar
controls. A distinct alkaline shift in the pH of the test medium indicates a
positive reaction.
3. Comments
'The technical difficulties connected with the glucose test may also be
encountered with this test. Enzymatic pretreatment of the heart infusion
base is particularly important when testing strains that are both glucoseand arginine-positive, because the acid produced in conventional media
tends to mask the alkalisation caused by the hydrolysis of arginine.
The arginine test is based on the demonstration of the presence in some
mycoplasma species of the arginine dihydrolase pathway, which involves
arginine deiminase, ornithine transcarbamylase, and carbamyl phosphokinase, and results in the release of high-energy phosphate, C02 and NH3
as endproducts (Schimke and Barile, 1963; Barile et al., 1966).
E. Phosphatase activity
1. Test on solid medium
(a) Test medium. An agar medium, Bph, the specific substrate of which is
the sodium salt of phenolphthalein diphosphate, is used. It is important to
heat the serum and yeast extract incorporated in the medium at 60°C for
1 h in order to inactivate the enzymes in these components (Appendix,
Section B4).
(b) Test procedure. A plate is inoculated from the fluid stock culture by
the running drop technique. After incubating the inoculated agar plate
together with an uninoculated control for 7 days, the plates are tested by
flooding the surface with 5 N NaOH. T h e appearance within half a minute
of a red colour in and around the inoculated area indicates a positive
reaction.
2. Test in liquid medium
(a) Reagent. A 0.01% solution of the sodium salt of phenolphthalein
diphosphate is used.
(b) Testprocedure. A log phase culture of the test strain grown in 10-100 ml
of appropriate growth medium is centrifuged at about 20 000 x g for 1 h.
The pellet is washed in phosphate-buffered saline and resuspended in 1 ml
E. A. FREUNDT ET AL.
to 7.3. The test medium is prepared by supplementing the basal medium
with 1% L-arginine (Appendix, Sections B, 2 (a) and (b)).
2. Test procedure
This is the same as that described for the glucose test, including similar
controls. A distinct alkaline shift in the pH of the test medium indicates a
positive reaction.
3. Comments
'The technical difficulties connected with the glucose test may also be
encountered with this test. Enzymatic pretreatment of the heart infusion
base is particularly important when testing strains that are both glucoseand arginine-positive, because the acid produced in conventional media
tends to mask the alkalisation caused by the hydrolysis of arginine.
The arginine test is based on the demonstration of the presence in some
mycoplasma species of the arginine dihydrolase pathway, which involves
arginine deiminase, ornithine transcarbamylase, and carbamyl phosphokinase, and results in the release of high-energy phosphate, C02 and NH3
as endproducts (Schimke and Barile, 1963; Barile et al., 1966).
E. Phosphatase activity
1. Test on solid medium
(a) Test medium. An agar medium, Bph, the specific substrate of which is
the sodium salt of phenolphthalein diphosphate, is used. It is important to
heat the serum and yeast extract incorporated in the medium at 60°C for
1 h in order to inactivate the enzymes in these components (Appendix,
Section B4).
(b) Test procedure. A plate is inoculated from the fluid stock culture by
the running drop technique. After incubating the inoculated agar plate
together with an uninoculated control for 7 days, the plates are tested by
flooding the surface with 5 N NaOH. T h e appearance within half a minute
of a red colour in and around the inoculated area indicates a positive
reaction.
2. Test in liquid medium
(a) Reagent. A 0.01% solution of the sodium salt of phenolphthalein
diphosphate is used.
(b) Testprocedure. A log phase culture of the test strain grown in 10-100 ml
of appropriate growth medium is centrifuged at about 20 000 x g for 1 h.
The pellet is washed in phosphate-buffered saline and resuspended in 1 ml