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9 CFR Ch. I (1–1–10 Edition) § 113.29

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9 CFR Ch. I (1–1–10 Edition) § 113.29 § 113.29 9 CFR Ch. I (1–1–10 Edition) into 125 ml flasks and store until need- shall be streaked with 0.1 ml of mate- ed. rial from the incubating flask of inocu- (4) Add 2 ml of DPN-Cysteine solu- lated medium on the 3d day, one on the tion to each 100 ml of broth on day of 7th day, one on the 10th day, and one use. on the 14th day post-inoculation. (c) Heart Infusion Agar shall be pre- (4) Control tests shall be conducted pared as provided in this paragraph. simultaneously with the detection test (1) Dissolve in 900 ml of purified using techniques provided in para- water by boiling the following: graphs (d)(2) and (3) of this section, ex- Heart infusion agar (g) ........................................... 25 cept the inoculum for the positive con- Heart-infusion broth (g) .......................................... 10 Proteose peptone No. 3 (g) ................................... 10 trol test shall be selected mycoplasma 1 pct thallium acetate (ml) ..................................... 25 cultures and the negative control test shall be uninoculated medium from the (2) Cool the medium and adjust pH to same lot used in the detection test. 7.9 with NaOH. (3) Autoclave the medium. (5) All plates shall be incubated in a (4) Cool the medium 30 minutes in a high humidity, 4–6 percent CO2 atmos- ° ° 56 °C waterbath. phere at 33 to 37 C for 10–14 days and (5) Dissolve 5 grams of yeast autoly- examined with a stereoscopic micro- sate in 100 ml of distilled water, filter scope at 35x to 100x or with a regular sterilize, and add to the medium. microscope at 100x. (6) Add to the medium: (e) Interpretation of test results. (1) If growth appears on at least one 126 ml of inactivated horse serum of the plates in the positive control 21 ml of DPN-Cysteine solution 525,000 units of Penicillin. test and does not appear on any of the Dispense 10 ml aliquots into 60×15 mm dis- plates in the negative control test, the posable culture dishes or petri dishes. test is valid. (2) If mycoplasma colonies are found (d) The test procedure provided in on any of the plates inoculated with this paragraph shall be followed when material being tested, the results are conducting the mycoplasma detection positive for mycoplasma contamina- test. tion. (1) Preparation of inoculum. Imme- diately prior to starting the test, fro- [38 FR 29887, Oct. 30, 1973, as amended at 41 zen liquid vaccine shall be thawed, and FR 6752, Feb. 13, 1976; 41 FR 32882, Aug. 6, lyophilized vaccine shall be rehydrated 1976] to the volume recommended on the label with mycoplasma medium. In the § 113.29 Determination of moisture case of a lyophilized biological product, content in desiccated biological products. e.g., 1,000 dose vial of poultry vaccine to be administered via the drinking Methods provided in this section water, the vaccine shall be rehydrated must be used when a determination of to 30 ml with mycoplasma medium. In moisture content in desiccated biologi- the case of a cell line or a sample of cal products is prescribed in an appli- primary cells, the inoculum shall con- cable Standard Requirement or in the sist of the resuspended cells. Control filed Outline of Production for the tests shall be established as provided in product. Firms currently using meth- paragraph (d)(4) of this section. ods other than those provided in this (2) Inoculation of plate. Plate 0.1 ml section for determining the moisture of inoculum on an agar plate and make content in desiccated biological prod- a short, continuous streak across the ucts have until November 5, 2004 to up- plate with a pipet. Tilt the plate to date their Outlines of Production to be allow the inoculum to flow over the in compliance with this requirement. surface. (a) Final container samples of com- (3) Inoculation of flask of medium. pleted product shall be tested. The Transfer 1 ml of the inoculum into a weight loss of the sample due to drying flask containing 100 ml mycoplasma in a vacuum oven shall be determined. medium and mix thoroughly. Incubate All procedures should be performed in the flask at 33 to 37 °C for 14 days dur- an environment with a relative humid- ing which time, one of four agar plates ity less than 45 percent. The equipment 686 VerDate Nov<24>2008 12:41 Mar 16, 2010 Jkt 220028 PO 00000 Frm 00696 Fmt 8010 Sfmt 8010 Y:\SGML\220028.XXX 220028 cprice-sewell on DSK89S0YB1PROD with CFR Animal and Plant Health Inspection Service, USDA § 113.31 necessary to perform the test is as fol- (6) Calculate the percentage of mois- lows: ture in the original sample as follows: (1) Cylindrical weighing bottles with (B¥C)/(B¥A) × (100) = Percentage of re- airtight glass stoppers. sidual moisture, where: (2) Vacuum oven equipped with vali- A = tare weight of weighing bottle dated thermometer and thermostat. A B¥A = weight of sample before drying suitable air-drying device should be at- B¥C = weight of sample after drying tached to the inlet valve. (3) Balance, accurate to 0.1 mg (rated (7) The results are considered satis- precision ±0.01mg). factory if the percentage of residual (4) Desiccator jar equipped with phos- moisture is less than or equal to the phorous pentoxide, silica gel, or equiv- manufacturer’s specification. alent. [68 FR 57608, Oct. 6, 2003] (5) Desiccated vaccine in original sealed vial. Sample and control should § 113.30 Detection of Salmonella con- be kept at room temperature in their tamination. original airtight containers until use. The test for detection of Salmonella (b) Test procedure: contamination provided in this section (1) Thoroughly cleaned and labeled shall be conducted when such a test is sample-weighing bottles with stoppers prescribed in an applicable Standard should be allowed to dry at 60 ±3 °C Requirement or in the filed Outline of under vacuum at less than 2.5 kPa. Production for the product. (i) Transfer hot bottles and stoppers (a) Samples shall be collected from into the desiccator and allow to cool to the bulk suspension before room temperature. bacteriostatic or bactericidal agents (ii) After bottles have cooled, insert have been added. When tissue culture stoppers and weigh and record the products are to be tested, 1 ml of tissue weights of the bottles as ‘‘A.’’ extract used as the source of cells or 1 (iii) Return weighing bottles to the ml of the minced tissue per se shall be desiccator. tested. (2) Remove the sample container (b) Five ml of the liquid vaccine sus- seal. pension shall be used to inoculate each (i) Using a spatula, break up the sam- 100 ml of liquid broth medium (tryptose ple plug and transfer the required and either selenite F or tetrathionate). amount of sample to the previously The inoculated media shall be incu- tared weighing bottle. bated 18–24 hours at 35–37 °C. (ii) Insert the stopper and weigh and (c) Transfers shall be made to either record the weights of the weighing bot- MacConkey agar or Salmonella- tles as ‘‘B.’’ Shigella agar, incubated for 18–24 hours (3) Place the weighing bottle with the and examined. stopper at an angle in the vacuum (d) If no growth typical of Salmonella oven. Set the vacuum to < 2.5 kPa and is noted, the plates shall be incubated the temperature to 60 ±3 °C. an additional 18–24 hours and again ex- (4) After a minimum of 3 hours of amined. drying time, turn off the vacuum pump (e) If suspicious colonies are ob- and allow dry air to bleed into the oven served, further subculture on suitable until the pressure inside the oven is media shall be made for positive identi- equalized with the prevailing atmos- fication. If Salmonella is found, the pheric pressure. bulk suspension is unsatisfactory. (5) While the bottle is still warm, re- [38 FR 29888, Oct. 30, 1973] place the stopper in its normal position and transfer the weighing bottle to the § 113.31 Detection of avian lymphoid desiccator. leukosis. (i) Allow a minimum of 2 hours for The complement-fixation test for de- the weighing bottle to cool to room tection of avian lymphoid leukosis pro- temperature or for its weight to reach vided in this section shall be conducted equilibrium. on all biological products containing (ii) Weigh, and record the weight as virus which has been propagated in ‘‘C.’’ substrates of chicken origin: Provided, 687 VerDate Nov<24>2008 12:41 Mar 16, 2010 Jkt 220028 PO 00000 Frm 00697 Fmt 8010 Sfmt 8010 Y:\SGML\220028.XXX 220028 cprice-sewell on DSK89S0YB1PROD with CFR.
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