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SAM 900.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 900

Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid 

Thioglycollate Medium and Soybean-Casein Digest Medium using Bacillus 

subtilis Spores and Issatchenkia orientalis as the Indicator Organisms

Date: March 19, 2015

Number: SAM 900.04

Supersedes: SAM 900.03, March 7, 2012

Standard Requirement: 9 CFR 113.25

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

Approvals: /s/Larry R. Ludemann Date: 02Apr15

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: 03Apr15

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.
Center for Veterinary Biologics SAM 900.04

Testing Protocol Page 2 of 9

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

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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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 

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

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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1. Introduction

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

and Soybean-Casein Digest Medium (SCDM) 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.

Note: Trypticase Soy Broth (TSB) and SCDM are the same media formulation and the 

terms are used interchangeably throughout this document. 

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 20o- 25oC incubator 

2.1.3 HandyStep®electronic pipette

2.1.4 Laminar-flow Class II biosafety cabinet (BSC)

2.1.5 Vortex mixer

2.2 Reagents/supplies

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

2.2.1 Bacillus subtilis (ATCC #6633) or equivalent organism as specified in the 

current United States Pharmacopoeia (USP)

2.2.2 Issatchenkia orientalis (ATCC #6258) or equivalent organism as specified 

in the current USP

2.2.3 Soybean-Casein Digest Medium (SCDM) (National Centers for Animal 

Health (NCAH) Media #10423) (Appendix I) 
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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2.2.4 Fluid Thioglycollate Medium (FTM) (NCAH Media #10135) (Appendix 

II) 

2.2.5 Sterile pipettes

2.2.6 BRAND PD-TipTM Syringe Tips

2.2.7 4 x 4-inch sterile gauze pads

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

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

3.3.1 Bacillus subtilis stock culture is prepared according to the manufacturer’s 

instructions and titrated to determine colony forming unit (CFU) concentration.

3.3.2 Issatchenkia orientalis stock culture is prepared according to the 

manufacturer’s instructions and titrated to CFU concentration.
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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4. Performance of the Test

Test each batch of SCDM and FTM for growth promoting qualities using both B. subtilis and I.

orientalis as indicator organisms.

4.1 Thaw the frozen vials of indicator organism stock cultures in the BSC. Rehydrate 

lyophilized stock cultures with SCDM according to the reagent data sheet. Mix stock 

cultures thoroughly by vortexing immediately prior to use.

4.2 Prepare dilutions of the indicator organism stock cultures according to the reagent 

data sheet specifications. Mix the dilutions using a vortex mixer.

4.3 Prepare a sufficient volume of each working dilution for each indicator organism

(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 B. subtilis into each of ten 25 x 200-mm tubes containing 40.0

mL of SCDM. Change syringe tip and use to dispense 1.0 mL of the higher working 

dilution into each of ten 25 x 200-mm tubes containing 40.0 mL of FTM.

4.5 Change syringe tip and use to dispense 1.0 mL of the next lower working dilution 

of B. subtilis into each of ten 25 x 200-mm tubes containing 40.0 mL of SCDM. 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.

4.6 Repeat Sections 4.1 through 4.5 for the I. orientalis indicator organism. 

4.7 Incubate all tubes (40) containing the B. subtilis culture at 30o- 35oC and observe 

for growth of the organism throughout a 7-day incubation period.

4.8 Incubate all tubes (40) containing the I. orientalis culture at 20o- 25oC and observe 

for growth of the organism throughout a 14-day incubation period.

5. Interpretation of the Test Results

Growth is expected in at least 8 or more tubes inoculated with the lowest working dilution of 

each indicator organism and in greater than zero, but less than 8 tubes inoculated with the next 

higher working dilution of each indicator organism. 

5.1 If at least 8 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). 
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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5.2 If less than 8 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 the 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.

7.3 Kurtzman, C. P., C. J. Robnett, and E. Basehoar-Powers. 2008. Phylogenetic 

relationships among species of Pichia, Issatchenkia and Williopsis determined from 

multigene sequence analysis, and the proposal of Barnettozyma gen. nov., Lindnera gen. 

nov. and Wickerhamomyces gen. nov. FEMS Yeast Res 8:939-54. 

8. Summary of Revisions

Version .04

 The Bacteriology Section Leader has been updated.

 Deleted “formerly Candida krusei” from this document.

 Section 1 was rewritten for clarity.

Version .03

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

Biologics and to provide additional detail. One significant change was made that might impact 
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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the designation of the serial/lot final disposition of the test; the following changes were made to 

the document:

 The contact information has been updated.

 Candida krusei has been changed to the new name Issatchenkia orientalis and this 

document was updated to reflect the new name.

 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. This might change the outcome 

of the sterility test and could impact CVB’s overall disposition of the serial/lot on 

test.

 7.3: Reference added about Issatchenkia orientalis.

 Appendices: 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 Contact has been changed from Gerald Christianson to Sophia Campbell.

 1: Clarification on the use of this SAM and the media designations have been added.

 2: The list of equipment/instrumentation has been updated to reflect current 

procedures.

 3.3.3: The status of a valid Gram stain for B. subtilis has been clarified.

 3.3.5: A filtration step to remove beads has been added.

 3.4.2: The status of a valid Gram stain for C. krusei has been clarified.
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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 4.1: This section has been rewritten to provide further clarification of current 

procedures and to add an explanation on titration of each new lot of indicator 

organisms.

 4.2: This section has been rewritten to provide further clarification of current 

procedures.

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

 Appendices: The storage conditions have been added.
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Supplemental Assay Method for Testing Growth Promoting Qualities of Fluid Thioglycollate Medium 

and Soybean-Casein Digest Medium using Bacillus subtilis Spores and Issatchenkia orientalis as the Indicator 

Organisms

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Appendices – Media Formulations

Appendix I

NCAH Media #10423 

Trypticase Soy Broth (TSB) or Soybean-Casein Digest Medium (SCDM)

Trypticase Soy Broth 30 g

QH2O 1000 mL

Autoclave 20 minutes at 121oC. Media is stored at 20°- 25°C for up to 3 months.

TSB and SCDM are two names for the same media formulation from different 

media companies.

Appendix II

NCAH Media #10135 

Fluid Thioglycollate Medium (BBL)

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Mix and heat to boiling. Autoclave 20 minutes at 121°C. Media is stored at 20°-

25°C for up to 3 months.