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CVB-DAT-0137.03 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: Clostridium chauvoei Spore Preparation

2. Strain or Source: Not Applicable

3. Lot Number: IRP 631 

4. Fill Date: October 16, 2015 

5. Expiration Date: February 28, 2029 

6. Intended Use: IRP 631 serves as the standard challenge material for use in evaluating the 

potency of biological products containing C. chauvoei.

7. Instructions for Use: IRP 631 diluted 1:37,000 is considered the challenge dilution for 

conducting C. chauvoei potency tests in guinea pigs as outlined in title 9, Code of Federal 

Regulations (9 CFR), section 113.106. A 1:100 dilution is prepared by adding 1.0 mL of well mixed 

IRP 631 to 99.0 mL of sterile 0.85% sodium chloride (NaCl) solution. The spore suspension is 

further diluted to 1:3700 by adding 1.0 mL of the diluted spore suspension (1:100) into 36.0 mL of 

sterile 0.85% NaCl solution. The final challenge dilution (1:37,000) is prepared by adding 3.0 mL of 

well-mixed 1:3700 dilution to 27 mL of 7.5% calcium chloride (CaCl2

. 2H2O) solution.

8. Test of Reagent:

Determination of culture purity – IRP 631 was tested for purity and found to be a pure culture of 

C. chauvoei based on 9 CFR 113.27 testing. 

Determination of spore preparation LD50 – Guinea pigs weighing 500 to 600 grams were 

injected intramuscularly with 0.5 mL of IRP 631 diluted in 7.5% CaCl2 

. 2H2O solution. The 

guinea pig LD50 was calculated by the method of Reed and Muench and found to be 1:3,680,000 

per 0.5 mL of spore suspension. 

9. Container Size, Type, Weight, or Volume: Five-mL glass vials containing 1.5 mL of 

spore suspension. 

 10. Storage Conditions: Store at <-60°C.

Precautions: Personnel must take precautions against being stuck with needles or cut with 

sharp instruments contaminated with C. chauvoei spores. 
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CVB-DAT-0137.03 Page 2 of 2 

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

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

(515) 337-6100 or FAX (515) 337-7673.

12. Origin and Passage History: Clostridium chauvoei IRP 631 was prepared from 

Clostridium chauvoei IRP 206, which was prepared from C. chauvoei Lot F obtained from the 

American Cyanamid Company in 1962. The number of times the culture was passed prior to 

being sent to the Center for Veterinary Biologics was unknown.

13. Method of Preparation: Clostridium chauvoei spores were cultivated on the surface of a 

beef infusion agar medium in 500-mL Erlenmeyer flasks. The flasks were incubated in an 

anaerobic chamber containing 85% nitrogen (N2), 10% hydrogen (H2), and 5% carbon dioxide 

(CO2) at 35°C for 2 to 4 days than 25°- 29°C for 4 days before spores were harvested by washing 

the agar surface with sterile 0.015 M phosphate buffered saline, pH 6.9. The spore suspension 

was then mixed with an equal volume of sterile glycerol. 

14. Other: None

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

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

Reagent order forms (APHIS Form 2018) can be found on the Biologics Forms page of the CVB 

website.