[Code of Federal Regulations]
[Title 9, Volume 1]
[Revised as of January 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 9CFR113.28]

[Page 599-600]
 
                 TITLE 9--ANIMALS AND ANIMAL PRODUCTS
 
  CHAPTER I--ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF
                               AGRICULTURE
 
PART 113--STANDARD REQUIREMENTS--Table of Contents
 
Sec. 113.28 Detection of mycoplasma contamination.

   The heart infusion test, using heart infusion broth and heart
infusion agar, provided in this section shall be conducted when a test
for mycoplasma contamination is prescribed in an applicable Standard
Requirement or in the filed Outline of Production for the product.
   (a) Media additives provided in this paragraph shall be prepared as
follows:
   (1) DPN-Cysteine Solution:
   (i) Use Nicotinamide adenine dinucleotide (oxidized) and L-Cysteine
hydrochloride.
   (ii) Prepare 1 gram/100 milliliters (ml) purified water (1 percent
solution) of each. Mix the solutions together; the cysteine reduces the
DPN. Filter sterilize, dispense in appropriate amounts and store frozen
at -20 degrees centigrade.
   (2) Inactivated horse serum--horse serum which has been inactivated
at 56  deg.C for 30 minutes.
   (b) Heart infusion broth shall be prepared as provided in this
paragraph.
   (1) Dissolve in 970 ml of purified water, 25 grams of heart infusion
broth, 10 grams of proteose peptone No. 3, and either 5 grams of yeast
autolysate or 5 ml of fresh yeast extract.
   (2) Add the following:

1 percent tetrazolium chloride (ml)..........................        5.5
1 percent thallium acetate (ml)..............................        25
Penicillin (units)...........................................    500,000
Inactivated horse serum (ml).................................        100


   (3) Adjust pH to 7.9 with NaOH, filter sterilize, and dispense 100
ml aliquots into 125 ml flasks and store until needed.
   (4) Add 2 ml of DPN-Cysteine solution to each 100 ml of broth on day
of use.
   (c) Heart Infusion Agar shall be prepared as provided in this
paragraph.
   (1) Dissolve in 900 ml of purified water by boiling the following:

Heart infusion agar (g)......................................        25
Heart-infusion broth (g).....................................        10
Proteose peptone No. 3 (g)...................................        10
1 pct thallium acetate (ml)..................................        25


   (2) Cool the medium and adjust pH to 7.9 with NaOH.
   (3) Autoclave the medium.
   (4) Cool the medium 30 minutes in a 56 deg.C waterbath.
   (5) Dissolve 5 grams of yeast autolysate in 100 ml of distilled
water, filter sterilize, and add to the medium.
   (6) Add to the medium:

126 ml of inactivated horse serum
21 ml of DPN-Cysteine solution
525,000 units of Penicillin.
Dispense 10 ml aliquots into 60 x 15 mm disposable culture dishes or
petri dishes.

   (d) The test procedure provided in this paragraph shall be followed
when conducting the mycoplasma detection test.
   (1) Preparation of inoculum. Immediately prior to starting the test,
frozen liquid vaccine shall be thawed, and lyophilized vaccine shall be
rehydrated to the volume recommended on the label with mycoplasma
medium. In the case of a lyophilized biological product, e.g., 1,000
dose vial of poultry vaccine to be administered via the drinking water,
the vaccine shall be rehydrated to 30 ml with mycoplasma medium. In the
case of a cell line or a sample of primary cells, the inoculum shall
consist of the resuspended cells. Control tests shall be established as
provided in paragraph (d)(4) of this section.
   (2) Inoculation of plate. Plate 0.1 ml of inoculum on an agar plate
and make

[[Page 600]]

a short, continuous streak across the plate with a pipet. Tilt the plate
to allow the inoculum to flow over the surface.
   (3) Inoculation of flask of medium. Transfer 1 ml of the inoculum
into a flask containing 100 ml mycoplasma medium and mix thoroughly.
Incubate the flask at 33 to 37 deg.C for 14 days during which time, one
of four agar plates shall be streaked with 0.1 ml of material from the
incubating flask of inoculated medium on the 3d day, one on the 7th day,
one on the 10th day, and one on the 14th day post-inoculation.
   (4) Control tests shall be conducted simultaneously with the
detection test using techniques provided in paragraphs (d)(2) and (3) of
this section, except the inoculum for the positive control test shall be
selected mycoplasma cultures and the negative control test shall be
uninoculated medium from the same lot used in the detection test.
   (5) All plates shall be incubated in a high humidity, 4-6 percent
CO<INF>2</INF> atmosphere at 33  deg.to 37 deg.C for 10-14 days and
examined with a stereoscopic microscope at 35x to 100x or with a regular
microscope at 100x.
   (e) Interpretation of test results.
   (1) If growth appears on at least one of the plates in the positive
control test and does not appear on any of the plates in the negative
control test, the test is valid.
   (2) If mycoplasma colonies are found on any of the plates inoculated
with material being tested, the results are positive for mycoplasma
contamination.

[38 FR 29887, Oct. 30, 1973, as amended at 41 FR 6752, Feb. 13, 1976; 41
FR 32882, Aug. 6, 1976]