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SAM 901.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 901

Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid 

Thioglycollate Medium with Beef Extract Using Clostridium chauvoei Spores 

as the Indicator Organism

Date: March 19, 2015

Number: SAM 901.04

Supersedes: SAM 901.03, March 9, 2012

Standard Requirement: 9 CFR 113.25

Contact: Sophia G. Campbell, (515) 337-7489

Approvals: /s/Larry R. Ludemann Date: 03Apr15

Larry R. Ludemann, Section Leader

Bacteriology 

/s/Byron E. Rippke Date: 03Apr15

Byron E. Rippke, Acting Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09Apr15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

with Beef Extract Using Clostridium chauvoei Spores as the Indicator Organism

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagent/control procedures

4. Performance of the Test

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References 

8. Summary of Revisions

Appendix
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

with Beef Extract Using Clostridium chauvoei Spores as the Indicator Organism

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1. Introduction

This Supplemental Assay Method (SAM) describes testing Fluid Thioglycollate Medium with 

Beef Extract (FTM/BE) for growth promoting qualities, as required in title 9, Code of Federal

Regulations (9 CFR), part 113.25(b). Each lot of media that is used for sterility testing of 

biological products (9 CFR 113.26 – 113.27) must be tested for growth promoting qualities.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 30o- 35oC incubator 

2.1.2 HandyStep®electronic pipette

2.1.3 Laminar-flow Class II biosafety cabinet (BSC)

2.1.4 Vortex mixer

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Clostridium chauvoei spores or equivalent organism as specified in the 

current United States Pharmacopoeia (USP)

2.2.2 Fluid Thioglycollate Medium with 0.5% Beef Extract (FTM/BE), National 

Centers for Animal Health (NCAH) Media #10227 (Appendix) 

2.2.3 Sterile pipettes

2.2.4 BRAND PD-TipTM Syringe Tips

2.2.5 Tubes, 25 x 200-mm, with sterile closures

2.2.6 4 x 4-inch sterile gauze pads
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Monitor temperature of incubators according to SOPs.

3.2.3 Turn on the BSC at least 30 minutes before starting work.

3.3 Preparation of reagent/control procedures

C. chauvoei stock culture is prepared according to the manufacturer’s instructions.

4. Performance of the Test

Test each batch of FTM/BE for growth promoting qualities using C. chauvoei as the 

indicator organism.

4.1 Thaw a frozen vial of C. chauvoei stock culture in the BSC. Mix stock culture

thoroughly by vortexing immediately prior to use.

4.2 Prepare dilutions of the C. chauvoei stock culture according to the reagent data 

sheet specifications. Mix the dilutions using a vortex mixer.

4.3 Prepare a sufficient volume of each working dilution of C. chauvoei (i.e., 25-30 

milliliter (mL) volume of each of the working dilutions).

4.4 Use a HandyStep pipette with a sterile syringe tip to dispense 1.0 mL of the 

higher working dilution of C. chauvoei into each of ten 25 x 200-mm tubes containing 

40.0 mL of FTM/BE. Change syringe tip and use to dispense 1.0 mL of the lower 

working dilution into each of ten 25 x 200-mm tubes containing 40.0 mL of FTM/BE. 
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

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4.5 Incubate all tubes (20) at 30o- 35oC and observe for growth of the organism 

throughout the 14-day incubation period.

5. Interpretation of the Test Results

Growth is expected in at least 9 or more tubes inoculated with the lowest working dilution of the 

indicator organism and in greater than zero, but less than 9 tubes inoculated with the next higher 

working dilution of the indicator organism.

5.1 If at least 9 of the tubes inoculated with the lower working dilution of a stock culture 

contain growth, the growth promoting quality of that medium is satisfactory (SAT). 

5.2 If less than 9 tubes inoculated with the lower working dilution of a stock culture have 

growth, then the growth promoting qualities of the media are in question and the test must be 

repeated. 

5.3 If a media’s growth promoting properties are still in question after a retest, the media 

must not be used and all tests conducted with this media lot must be considered no tests 

(NT).

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.25, U.S. Government Printing 

Office, Washington, DC.

7.2 The U.S. Pharmacopoeia, 1985, Vol. 21, pp 1151-1160, Mack Publishing Co., 

Easton, PA.

8. Summary of Revisions

Version .04

 The Bacteriology Section Leader has been updated.

 Sections 1-3 have been updated.
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

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Version .03

 The contact information has been updated.

 The table of contents has been updated.

 3.3: Renamed and updated to reflect current practices.

 4: This section has been updated to reflect current practices.

 5. The interpretation of test results has been clarified. 

 Appendix: Updated to include current media numbers.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 The document number has been changed from STSAM0901 to SAM 901.

 The Contact has been changed from Gerald Christianson to Sophia G. Campbell.

 1: Information to clarify the testing purpose has been added.

 2.1.4: Sterile glass tubes have been added to the equipment list.

 2.1.5: The class of biosafety cabinet to be used has been added.

 2.1.8: An anaerobic growth chamber for use in C. chauveoi propagation and spore 

harvest has been added.

 2.2: The list of reagents/supplies has been updated.

 3.1: Personnel qualifications have been clarified.

 3.3: Information on the growth of C. chauveoi culture and harvest of spore 

suspension has been added.

 4.1/4.2: These sections have been revised to clarify the procedures followed in 

testing.
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 5: The test interpretations have been clarified.

 Appendices: Media storage conditions have been added. 

 Appendix III: A temperature range has been added for the storage of Normal Saline.

 Appendix IV: This section has been added to provide the media recipe for beef 

infusion agar medium.

 Appendix V: This section has been added to provide the media recipe for 0.015M 

phosphate buffered saline pH 6.9.
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Fluid Thioglycollate Medium 

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Appendix

NCAH Media #10227

 Fluid Thioglycollate with Beef Extract

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Heat and add:

0.5% Beef Extract (Difco) 5 g

Bring to a boil and dispense. Autoclave 20 minutes at 121oC. Store at 20°- 25°C 

for no longer than 3 months.