[Code of Federal Regulations]
[Title 9, Volume 1]
[Revised as of January 1, 2002]
From the U.S. Government Printing Office via GPO Access
TITLE 9--ANIMALS AND ANIMAL PRODUCTS
CHAPTER I--ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF
PART 113--STANDARD REQUIREMENTS--Table of Contents
Sec. 113.26 Detection of viable bacteria and fungi except in live vaccine.
Each serial and subserial of biological product except live vaccines
shall be tested as prescribed in this section unless otherwise specified
by the Administrator. When cell lines, primary cells, or ingredients of
animal origin used in the preparation of a biological product are
required to be free of viable bacteria and fungi, they shall also be
tested as prescribed in this section.
(a) The media to be used shall be as follows:
(1) Fluid Thioglycollate Medium with 0.5 percent beef extract shall
be used to test for bacteria in biological products containing
clostridial toxoids, bacterins, and bacterin-toxoids.
(2) Fluid Thioglycollate Medium with or without 0.5 percent beef
extract shall be used to test for bacteria in biological products other
than clostridial toxoids, bacterins, and bacterin-toxoids.
(3) Soybean-Casein Digest Medium shall be used to test biological
products for fungi; provided, that Fluid Thioglycollate Medium without
beef extract shall be substituted when testing biological products
containing mercurial preservatives.
(b) Test procedure:
(1) Ten test vessels shall be used for each of two media selected in
accordance with paragraph (a)(1), (a)(2), or (a)(3) of this section.
Each test vessel shall contain sufficient medium to negate the
bacteriostatic or fungistatic activity in the inoculum as determined in
(i) When completed product is tested, 10 final container samples
from each serial and each subserial shall be tested. One ml from each
sample shall be inoculated into a corresponding individual test vessel
of culture medium: Provided, That, if each final container sample
contains less than 2 ml, one-half of the contents shall be used as
inoculum for each test vessel.
(ii) When cell lines, primary cells, or ingredients of animal origin
are tested, at least a 20 ml test sample from each lot shall be tested.
One ml shall be inoculated into each test vessel of medium.
(3) Incubation shall be for an observation period of 14 days at 30
deg.to 35 deg.C. to test for bacteria and 14 days at 20 deg.to 25
deg.C. to test for fungi.
(4) If the inoculum renders the medium turbid so that the absence of
growth cannot be determined by visual examination, subcultures shall be
made on the seventh to eleventh day from biological products prepared
from clostridial toxoids, bacterins, and bacterin-toxoids and the third
to seventh day for other biological products. Portions of the turbid
medium in amounts of not less than 1.0 ml. shall be transferred to 20 to
25 ml. of fresh medium, and incubated the balance of the 14-day period.
(c) Examine the contents of all test vessels for macroscopic
microbial growth during the incubation period. When demonstrated by
adequate controls to be invalid, the test may be repeated. For each set
of test vessels representing a serial or subserial in a valid test, the
following rules shall apply:
(1) If no growth is found in any test vessel, the serial or
subserial meets the requirements of the test.
(2) If growth is found in any test vessel, one retest to rule out
faulty technique may be conducted using 20 unopened final container
(3) If growth is found in any test vessel of the final test, the
serial, subserial, or ingredients to be used in the preparation of a
biological product, as the case may be, is unsatisfactory.
[35 FR 16039, Oct. 13, 1970, as amended at 37 FR 2430, Feb. 1, 1972; 39
FR 21042, June 18, 1974; 40 FR 758, Jan. 3, 1975; 40 FR 14084, Mar. 28,
1975; 56 FR 66784, Dec. 26, 1991]