[Code of Federal Regulations]
[Title 21, Volume 7]
[Revised as of April 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR610.30]

[Page 73-74]
 
                        TITLE 21--FOOD AND DRUGS
 
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN 
                          SERVICES (CONTINUED)
 
PART 610--GENERAL BIOLOGICAL PRODUCTS STANDARDS--Table of Contents
 
                          Subpart D--Mycoplasma
 
Sec. 610.30  Test for Mycoplasma.


    Except as provided otherwise in this subchapter, prior to 
clarification or filtration in the case of live virus vaccines produced 
from in vitro living cell cultures, and prior to inactivation in the 
case of inactivated virus vaccines produced from such living cell 
cultures, each virus harvest pool and control fluid pool shall be tested 
for the presence of Mycoplasma, as follows:

    Samples of the virus for this test shall be stored either (1) 
between 2 and 8  deg.C for no longer than 24 hours, or (2) at -20  deg.C 
or lower if stored for longer than 24 hours. The test shall be performed 
on samples of the viral harvest pool and on control fluid pool

[[Page 74]]

obtained at the time of viral harvest, as follows: No less than 2.0 ml. 
of each sample shall be inoculated in evenly distributed amounts over 
the surface of no less than 10 plates of at least two agar media. No 
less than 1.0 ml. of sample shall be inoculated into each of four tubes 
containing 10 ml. of a semisolid broth medium. The media shall be such 
as have been shown to be capable of detecting known Mycoplasma and each 
test shall include control cultures of at least two known strains of 
Mycoplasma, one of which must be M. pneumoniae. One half of the plates 
and two tubes of broth shall be incubated aerobically at 36  deg.C 
1  deg.C and the remaining plates and tubes shall be 
incubated anaerobically at 36  deg.C 1  deg.C in an 
environment of 5-10 percent CO2 in N2. Aerobic 
incubation shall be for a period of no less than 14 days and the broth 
in the two tubes shall be tested after 3 days and 14 days, at which 
times 0.5 ml. of broth from each of the two tubes shall be combined and 
subinoculated on to no less than 4 additional plates and incubated 
aerobically. Anaerobic incubation shall be for no less than 14 days and 
the broth in the two tubes shall be tested after 3 days and 14 days, at 
which times 0.5 ml. of broth from each of the two tubes shall be 
combined and subinoculated onto no less than four additional plates and 
incubated anaerobically. All inoculated plates shall be incubated for no 
less than 14 days, at which time observation for growth of Mycoplasma 
shall be made at a magnification of no less than 300x. If the Dienes 
Methylene Blue-Azure dye or an equivalent staining procedure is used, no 
less than a one square cm. plug of the agar shall be excised from the 
inoculated area and examined for the presence of Mycoplasma. The 
presence of the Mycoplasma shall be determined by comparison of the 
growth obtained from the test samples with that of the control cultures, 
with respect to typical colonial and microscopic morphology. The virus 
pool is satisfactory for vaccine manufacture if none of the tests on the 
samples show evidence of the presence of Mycoplasma.

[38 FR 32056, Nov. 20, 1973, as amended at 63 FR 16685, Apr. 6, 1998]