[Code of Federal Regulations]
[Title 21, Volume 7]
[Revised as of April 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR610.30]

[Page 76-77]
 
                        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 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

[[Page 77]]

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]