408
E. A. FREUNDT ET AL.
with either glucose ( l x ) , arginine (lye), urea (0*45%), or 2,3,5-triphenyl
tetrazolium chloride (0.04%). In the first three media Phenol red (0.002%)
is used as an indicator of the pH change resulting from growth and metabolism, but in the last, reduction of tetrazolium itself produces a colour
change. Since there is no single medium that will support the growth of all
mycoplasmas, a formula for a standard growth medium cannot be given. It
should be remembered, moreover, that the requirements for a medium to
be used for the MI test may be even more exacting than for a mere growth
medium. The outcome of the MI test is not simply a matter of growth; the
enzymes required for the specific metabolic activity on which the test is
based also need to be produced in sufficient quantity to allow a clearly
visible colour change to take place. I n the Appendix is given the composition
of four media referred to as BG, BA, B T and SU. The base of the first three
is a modified Hayflick medium (B) that is suitable for the growth of a great
many Mycoplasma or Acholeplasma species. Medium SU is used for ureaplasmas. The initial pH of BG and B T is 7-8, of BA and SU 7-3 and 6.8,
respectively.
In order to provide a convenient method of judging the pH changes that
occur during incubation of the test system, controls are included which
consist of the test medium, BG, BA or SU, the p H of which is adjusted to
the desired endpoint, viz. 7.3, 7.8 and 7.3, respectively.
3 . Test proper
Serum is inactivated (30 min at 56°C) and a primary dilution of 1 : 10 is
made.
(1) Add 0.025 ml (one drop) of test medium to 12 wells, using one
horizontal row for the titration of each serum.
(2) Add 0.025 ml of the primary dilution of serum (1 : 10) by microdiluter,
to the first well.
(3) With the microdiluter in the first well prepare two-fold serial dilutions
of serum, rotating the diluter about 25 times in each well and transferring
0.025 ml to the next well. From the last well the microdiluter is discharged
on to a test pad. This also serves as a check on the delivery volume.
(4) Add 0.05 ml (two drops) of the appropriate dilution of antigen
(100-1000 CCU) to each well.
(5) Add 0.125 ml (five drops) of test medium to each well.
(6) One horizontal row is reserved for controls:
(i) Medium control: add 0.2ml (eight drops) of test medium to
four wells.
(ii) Antigen control: add 0.15 ml (six drops) of test medium+0*05 ml
(two drops) of diluted antigen to four wells.
E. A. FREUNDT ET AL.
with either glucose ( l x ) , arginine (lye), urea (0*45%), or 2,3,5-triphenyl
tetrazolium chloride (0.04%). In the first three media Phenol red (0.002%)
is used as an indicator of the pH change resulting from growth and metabolism, but in the last, reduction of tetrazolium itself produces a colour
change. Since there is no single medium that will support the growth of all
mycoplasmas, a formula for a standard growth medium cannot be given. It
should be remembered, moreover, that the requirements for a medium to
be used for the MI test may be even more exacting than for a mere growth
medium. The outcome of the MI test is not simply a matter of growth; the
enzymes required for the specific metabolic activity on which the test is
based also need to be produced in sufficient quantity to allow a clearly
visible colour change to take place. I n the Appendix is given the composition
of four media referred to as BG, BA, B T and SU. The base of the first three
is a modified Hayflick medium (B) that is suitable for the growth of a great
many Mycoplasma or Acholeplasma species. Medium SU is used for ureaplasmas. The initial pH of BG and B T is 7-8, of BA and SU 7-3 and 6.8,
respectively.
In order to provide a convenient method of judging the pH changes that
occur during incubation of the test system, controls are included which
consist of the test medium, BG, BA or SU, the p H of which is adjusted to
the desired endpoint, viz. 7.3, 7.8 and 7.3, respectively.
3 . Test proper
Serum is inactivated (30 min at 56°C) and a primary dilution of 1 : 10 is
made.
(1) Add 0.025 ml (one drop) of test medium to 12 wells, using one
horizontal row for the titration of each serum.
(2) Add 0.025 ml of the primary dilution of serum (1 : 10) by microdiluter,
to the first well.
(3) With the microdiluter in the first well prepare two-fold serial dilutions
of serum, rotating the diluter about 25 times in each well and transferring
0.025 ml to the next well. From the last well the microdiluter is discharged
on to a test pad. This also serves as a check on the delivery volume.
(4) Add 0.05 ml (two drops) of the appropriate dilution of antigen
(100-1000 CCU) to each well.
(5) Add 0.125 ml (five drops) of test medium to each well.
(6) One horizontal row is reserved for controls:
(i) Medium control: add 0.2ml (eight drops) of test medium to
four wells.
(ii) Antigen control: add 0.15 ml (six drops) of test medium+0*05 ml
(two drops) of diluted antigen to four wells.