9 C.F.R. § 147.15 Laboratory procedure recommended for the bacteriological examination of mycoplasma reactors.15
Title 9 - Animals and Animal Products
15 Yoder, H. W., Jr., “Mycoplasmosis.” In: Isolation and Identification of Avian Pathogens. (Stephen B. Hitchner, Chairman, Charles H. Domermuth, H. Graham Purchase, James E. Williams.) 1980, pp. 40–42, Creative Printing Company, Inc., Endwell, NY 13760. (a) Turbinates, trachea, air sacs, sinuses, nasal passages, respiratory exudates, synovial fluid, eggs (including yolk, yolk sacs, membranes and allantoic fluid), should be directly sampled with sterile swabs. Aseptic techniques are very important as some organisms may not be suppressed by the antimicrobial agents used in this procedure. Tissue suspensions from large volumes are sometimes desirable from the sites listed above and occasionally from the oviduct and cloaca. Tissues should be ground or blended completely in 10 times their volume of Mycoplasma Broth Medium (MBM). (See paragraph (f) of this section.) Specimens submitted to referral laboratories in order of preference for recovery of the mycoplasma organisms are: (1) live birds, (2) refrigerated fresh tissues, (3) tissue specimens packed with dry ice. (b) Inoculate 5–10 ml of MBM with a swab, wire loop or 0.1 ml of the tissue suspension. When evidence of growth is observed (lowered pH or turbidity of broth) transfer each broth culture as needed to maintain the original isolates. Incubate tubes at 37 °C for at least 21 days before discarding as negative. When growth is first observed or if no growth occurs by the 4th or 5th day of incubation, inoculate broth culture onto a plate of Mycoplasma Agar Medium (MAM). (See paragraph (g) of this section.) Several cultures may be inoculated on one plate by using a wire loop or a cotton swab. Incubate plates 3–5 days at 37 °C in a high humidity chamber. If preferred, 5 percent CO2 may be added or a candle jar may be used. Tiny circular and translucent colonies with elevated centers are very suggestive of mycoplasma. Indirect lighting and a low power or dissecting microscope are recommended for observation of the colonies as they are rarely more than 0.2–0.3 mm in diameter. (c) Isolates must be serotyped. (1) Isolates may be shipped in MBM with ice packs if shipment will be in transit less than 2–3 days. Longer shipments require freezing of the MBM with dry ice, or shipping MAM slants at room temperature. Isolates must have indications of growth before shipment is made. (2) Isolates may be stored in MBM at −20 °C for 2–3 weeks, or they may be stored at −68 °C for several years. (d) Alternate method of culture: An overlay enrichment culture for fastidious and sensitive mycoplasma, especially for M. meleagridis should be included. (1) Pour 2–3 ml of MAM into a test tube and tilt the tube until a slant (approximately 45°) is obtained. Other containers are acceptable. (2) Overlay the slant with sufficient MBM, so that the media (including inoculum) covers the agar slope. (3) Inoculate the culture as indicated in paragraph (b) of this section. (4) Incubate and examine the overlay as indicated in paragraph (b) of this section. (e) Preparation of media components:16 16 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned. (1) Deionized distilled water suitable for cell culture fluids should be used. (2) All glassware should be carefully washed with a nonresidue detergent such as Alcojet and rinsed three times in tap water and twice in deionized distilled water.17 17 Alcojet is available from: Alconox, Inc., New York, NY 10003. (3) Thallium acetate in a 10 percent solution is added to an approximate final concentration of 1:4000; however, highly contaminated specimens may require a final concentration of 1:2000.18 18 Thallium acetate may be obtained from Fischer Scientific Company. (4) Mycoplasma Broth Base, dextrose, phenol red, and cysteine hydrochloride are added to deionized distilled water first if autoclave sterilization is used.19 19 Mycoplasma Broth Base may be obtained from: (a) Product #M 33600, Gibco Diagnostics, 2801 Industrial Drive, Madison, WI 53711. (b) Product #3900–3212, Scott Laboratories, Inc., 8 Westchester Plaza, Elmsford, NY 10523. (5) Use sterile deionized distilled water to reconstitute penicillin. (6) Sterile serum should be inactivated by heating at 56 °C for 30 minutes. Swine serum may be used for M. gallisepticum, M. synoviae, M. gallinarum, and M. meleagridis isolation; however, horse serum is usually recommended for M. meleagridis isolation. (7) Phenol red should be prepared as a 1 percent solution. (8) NAD (beta nicotinamide adenine dinucleotide or coenzyme I) should be prepared as a 1 percent solution.20 20 NAD Grade III may be obtained from: Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. (9) Cysteine hydrochloride, prepared as a 1 percent solution, is used to reduce the NAD for M. synoviae growth. (10) A purified agar product such as Nobel (Special agar) is used in the MAM.21 21 Noble Agar may be obtained from: Difco Laboratories, Box 1058–A, Detroit, MI 48201. (11) Adjust the pH with NaOH. (12) Sterilization may be accomplished by two methods: (i) Filtration sterilization through a 0.20 micron filter is the recommended method. Aseptic techniques must be utilized. (ii) Autoclave sterilization at 120 °C, 15 pounds pressure (103 kPa), for 15 minutes may be used, if preferred, when following the procedure described in paragraph (e)(4) of this section. (13) Phenol red, dextrose, and NAD may be omitted when culturing for M. meleagridis and M. gallinarum. (14) When culturing for M. meleagridis from contaminated samples include 100 units/ml of Polymyxin B in MBM. (f) Mycoplasma Broth Medium (Frey) is prepared as follows: To 850–880 ml of deionized distilled water; Add: Thallium acetate (ml)—2.5 (1:4000) Potentially contaminated samples (ml)—5.0 (1:2000) Mycoplasma Broth Base (g)—22.5 Aqueous penicillin (units)—500,000 Sterile serum (ml)—120 to 150.0 Phenol red plus (ml)—2.5 NAD (ml)—12.5 Cysteine hydrochloride (ml)—12.5 Dextrose (g)—1.0–1.5 Adjust pH to 7.8 Filter sterilize (1) Broth may be stored at 4 °C for at least 2 weeks or at −40 °C for longer periods. (g) Mycoplasma Agar Medium (Frey) is prepared as follows: To 850–880 ml of deionized distilled water; Add: Mycoplasma Broth Base (g)—22.5 Adjust pH to 7.8 Purified agar (g)—12.0 Autoclave and cool in 45 °C water bath Thallium acetate (ml)—2.0; (1:4000) Sterile serum at 45 °C (ml)—150.0 Aqueous penicillin (units)—400,000 NAD (ml)—12.5 Cysteine hydrochloride (ml)—12.5 (1) Rotate flask gently and pour about 15 ml of media into each petri dish. (2) Stack petri dishes only 2–3 high in a 37 °C incubator up to 2 hours to remove excess moisture. (3) Wrap inverted plates in sealed bundles and store at 4 °C for not more than 15 days. (h) New component or media batches should be monitored to compensate for changes in formulation due to alterations of purity, concentration, preparation, etc. A known series of titrations from a single culture should be made on both new and old media. The media should be compared on the basis of growth, colony size, and numbers of colonies which develop.22 22 “Laboratory Procedures and Medium For The Isolation Of Mycoplasma From Clinical Materials.” Laboratory Diagnosis of Mycoplasma in Food Animals, Proceedings of Nineteenth Annual Meeting, The American Association of Veterinary Laboratory Diagnosticians, 1976, pp. 106–115, AAVLD, 6101 Mineral Point Road, Madison, WI 53705. [47 FR 21995, May 20, 1982, as amended at 57 FR 57343, Dec. 4, 1992; 59 FR 12805, Mar. 18, 1994; 61 FR 11524, Mar. 21, 1996; 65 FR 8019, Feb. 17, 2000]
Title 9: Animals and Animal Products
PART 147—AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN
Subpart B—Bacteriological Examination Procedure
§ 147.15 Laboratory procedure recommended for the bacteriological examination of mycoplasma reactors.15
