CONTROLLED//PROPIN//BASIC 

CVB-DAT-0134.04 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

1800 Dayton Avenue

Ames, IA 50010 

1. Reagent Name: Clostridium perfringens type C (beta) toxin 

2. Strain or Source: Not applicable.

3. Lot Number: IRP 624 

4. Fill Date: October 29, 2014 

5. Expiration Date: February 28, 2029 

6. Intended Use: IRP 624 serves as the standard toxin when conducting C. perfringens

type C toxin-neutralization tests in mice. 

7. Instructions for Use: IRP 624 diluted 1:75 is considered the standard toxin dilution 

when conducting toxin-neutralization tests in mice as outlined in title 9, Code of Federal 

Regulations (9 CFR), section 113.111, and 9 CFR 113.454. The standard toxin dilution is 

prepared by adding 0.5 mL of IRP 624 to 4.5 mL of peptone diluent (1.0% peptone, 0.25% 

sodium chloride, pH 7.2). The toxin is further diluted to 1:75 by adding 1.0 mL of the 1:10 

dilution to 6.5 mL of diluent. A volume of 0.5 mL of the toxin diluted 1:75 and 0.5 mL of diluent 

is equivalent to 10 Lo doses. A volume of 0.9 mL of toxin diluted 1:75 and 0.1 mL of diluent is 

equivalent to 10 L+ doses. C. perfringens type C (beta) toxin IRP 624 diluted 1:10 is stable when 

stored at-60°C or lower.

8. Test of Reagent: Determination of test dose of toxin - The 10 Lo and 10 L+ doses were 

established by injecting mice intravenously with 0.2 mL of a mixture of varying amounts of IRP 

624 combined with 10 International Units (IU) of C. perfringens beta antitoxin. The 10 Lo dose 

for C. perfringens type C (beta) toxin neutralization test is the largest amount of toxin which can 

be mixed with 10 IU of beta antitoxin and not cause death in injected mice within 24 hours. The 

10 L+ dose for C. perfringens type C (beta) toxin neutralization test is the smallest amount of 

toxin which can be mixed with 10 IU of beta antitoxin and cause death in at least 80% of injected 

mice within 24 hours. 

Determination of LD50 in mice - Female white Swiss mice weighing 16-20 g were injected 

intravenously with 0.2 mL of IRP 624 diluted in peptone diluent. The toxin was found to contain 

104.3247 mouse lethal dose fifty (LD50) per mL.

Precautions: This reagent does not present a hazard to laboratory personnel who work with the 

toxin provided good fundamental laboratory techniques are followed.
CONTROLLED//PROPIN//BASIC 

CVB-DAT-0134.04 Page 2 of 2 

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

Determination of toxin type - The toxin type was confirmed by performing toxin neutralization 

tests in mice. The mice were injected intravenously with mixtures of IRP 624 and C. perfringens

type A, B, C, or D antitoxin. All of the mice died within 24 hours except those receiving 

mixtures containing type B or C antitoxin. 

Sterility test - The toxin was tested for sterility and found to be free of viable bacteria and fungi 

according to the procedures outlined in 9 CFR 113.26. 

9. Container Size, Type, Weight, or Volume: 1.5-mL glass vials containing 0.8 mL of 

toxin. 

10. Storage Conditions: Store at -70°± 10°C. 

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

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

12. Origin and Passage History: C. perfringens type C (beta) culture #4414, used to 

produce IRP 624, was obtained from Coopers Animal Health, Inc., on July 28, 1975. The 

number of passages is unknown. 

13. Method of Preparation: Culture #4414 was grown in a 14-liter New Brunswick 

fermentor containing media consisting of N-Z case, proteose peptone, and yeast extract. Actively 

growing culture was aseptically added to the fermentor and incubated at 35°C for approximately 

4 hours. The culture was centrifuged at 10,000 x g for 60 minutes. The supernatant was passed 

through a Millipore filtration unit containing a 0.2-μm membrane. The filtrate was further 

processed using a Millipore pellicon cassette system containing a high volume ultra-filter. The 

concentrated toxin was adjusted to pH 6.6 and passed through a sterile Millipore filtration unit 

containing a 0.22-mm membrane. Sterile glycerol was added to the product at a final 

concentration of 15% v/v. 

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