CONTROLLED//PROPIN//BASIC 

CVB-DAT-0120.02 Page 1 of 2 

Controlled by: APHIS Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture

Animal and Plant Health Inspection Service

Center for Veterinary Biologics

P. O. Box 844

Ames, IA 50010

1. Reagent Name: Mycoplasma synoviae

2. Strain or Source: BRP batch 1; European Directorate for the Quality of Medicines & 

HealthCare (EDQM); Strasbourg, France

3. Lot Number: 17Apr12-MS

4. Fill Date: April 17, 2012

5. Expiration Date: None

6. Intended Use: For use as a reference standard in the culture method to determine 

mycoplasma contamination of biological products. 

7. Instructions for Use: Vials of the culture should be rapidly thawed in a 35°-38°C water 

bath and immediately diluted in sterile saline (0.8-0.9% NaCl solution) to achieve the desired 

concentration of colony forming units (CFU); refer to Section 14. To maintain viability, culture 

dilutions must be inoculated onto medium within 10 minutes of preparation. 

8. Test of Reagent: Purity of this reagent was analyzed by the method described in 9 CFR 

113.27(d). The estimated titer of this reagent was determined empirically using standard plate 

count analysis. 

9. Container Size, Type, Weight, or Volume: 1.0 mL frozen culture

 10. Storage Conditions: -60°C or colder

11. CVB Technical Contact: Bacteriology Section, Center for Veterinary Biologics, (515) 

337-6100. 

12. Origin and Passage History: The strain was first isolated in France in 1999 from a flock 

of turkey broilers. The CVB acquired the strain from the EDQM in 2012. 

Precautions: This reagent must be handled in a manner consistent with Biosafety Level 2 

(BSL-2) standards.
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CVB-DAT-0120.02 Page 2 of 2 

Controlled by: APHIS Center for Veterinary Biologics, 515-337-6100 

13. Method of Preparation: The culture was diluted to a stock concentration recommended 

by the European Pharmacopoeia in Reference Standard Leaflet Y0000689 in mycoplasma 

medium supplemented with 15% glycerol. The diluted culture is stored at less than -60°C. 

14. Other: The estimated titer of this reagent is 37,147 CFU/mL. To achieve a use-dilution 

containing a target concentration of approximately 100 CFU/mL, prepare a 1:370 dilution in 

sterile saline. Begin by making a 1:9.25 dilution (0.5 mL culture + 4.125 mL saline) followed by 

a 1:4 dilution (1 mL [1:9.25 dilution] + 3 mL saline), and a 10-fold dilution. Subsequent 10-fold 

dilutions may be generated to produce 10 CFU/mL, 1 CFU/mL, and 0.1 CFU/mL solutions. To 

verify the concentration of the dilutions, plate 0.1 mL of each dilution on mycoplasma agar or 

Frey agar plates and incubate for 10-14 days in a humidified chamber containing 4-6% CO2 in 

air or 4-6% CO2 in nitrogen. Note: this strain grows best on Frey agar.

Reagent orders and feedback should be sent including phone number to the following email 

address: VS.DB.CVB.Reagent.Requests@usda.gov

Reagent orders forms (APHIS 2018) can be found on the CVB website. 

Restrictions: This reagent is intended as a one-time use reference standard for the 

mycoplasma contamination test. Culturing this organism with the intent to propagate a stock 

culture is not permitted.