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SAM 902.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 902

Supplemental Assay Method for Testing Growth-Promoting Qualities of 

Brain Heart Infusion Agar using Bacillus subtilis Spores and Issatchenkia 

orientalis as Indicator Organisms

Date: February 25, 2015

Number: SAM 902.04

Supersedes: SAM 902.03, February 1, 2012

Standard Requirement: 9 CFR 113.25

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

Approvals: /s/Larry R. Ludemann Date: 09Mar15

Larry R. Ludemann, Section Leader

Bacteriology 

/s/Byron E. Rippke Date: 10Mar15

Byron E. Rippke, Acting Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 13Mar15

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 Brain Heart

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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 reagents/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 Brain Heart

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1. Introduction

This Supplemental Assay Method (SAM) describes testing Brain Heart Infusion Agar (BHIA) 

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 through 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 20o- 25oC incubator 

2.1.3 Thermo Scientific Finnpippette electronic pipette

2.1.4 Laminar-flow Class II biosafety cabinet (BSC)

2.1.5 Vortex mixer

2.1.6 Lab Armor® bead bath (set to 55°- 60°C) 

2.2 Reagents/supplies

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

2.2.1 Bacillus subtilis (American Type Culture Collection (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 Brain-Heart Infusion Agar (BHIA), National Centers for Animal Health

(NCAH) Media #10204 (Appendix I)
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Supplemental Assay Method for Testing Growth-Promoting Qualities of Brain Heart

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2.2.4 Soybean-Casein Digest Medium (SCDM), NCAH Media #10423

(Appendix II) 

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

different media companies.

2.2.5 Penicillinase Concentrate, 10,000,000 Kinetic (Kersey) units/mL (BBL

catalog number 211898)

2.2.6 Petri dishes, 100 x 15-mm

2.2.7 70% ethanol

2.2.8 4 x 4-inch sterile gauze pads

2.2.9 Sterile pipettes

2.2.10 Sterile pipet tips, 100-1000 μL

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 and waterbath according to SOPs.

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

3.2.4 Turn on the bead bath at least one day before use to allow the temperature 

to equilibrate. 
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3.3 Preparation of reagents/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.

3.3.3 A working solution of 100,000 Kinetic (Kersey) units of penicillinase/mL 

is prepared by adding 1.0 mL of the penicillinase concentrate (Section 2.2.5) 

into 99.0 mL of sterile water. 

3.3.4 Preparation of the BHIA medium: On the day of the test, melt the BHIA

in an autoclave for 30 minutes at 100°C. Place the bottles of melted media in the

bead bath. Do not begin testing until the agar has cooled to at least 60°C. The 

temperature of the bead bath should be approximately 60°C when the agar is 

ready. The BHIA supplemented medium is prepared by adding the penicillinase

working solution to the BHIA to yield 500 Kinetic (Kersey) units of 

penicillinase/mL of medium, just prior to pouring the medium into plates.

4. Performance of the Test

Test each batch of BHIA for growth promoting qualities using both B. subtilis and I. orientalis as

indicator organisms. 

4.1 Thaw the frozen vials of indicator organism stock culture 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 by vortex mixing.

4.3 For each indicator organism, use a Finnpippette electronic pipette with a sterile 

tip, to dispense into each of four sterile 15 x 100-mm petri dishes the volume of inoculum 

that will yield approximately100 CFU.

4.4 Pour 20-25 mL of BHIA supplemented with penicillinase into each of two plates

containing B. subtilis. Pour 20-25 mL of the new batch of BHIA without penicillinase 

into each of the other two plates containing B. subtilis.

4.5 Repeat Section 4.4 with the I. orientalis culture. 
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4.6 Incubate the four petri dishes containing B. subtilis culture at 30°- 35°C for 7 

days. Incubate the four petri dishes containing the I. orientalis culture at 20°- 25°C for 14

days.

4.7 On day 7, count the number of CFUs in the B. subtilis plates and record the counts

on the test worksheet. Average the counts for the two plates of BHIA supplemented with 

penicillinase separately from the two plates of BHIA without penicillinase.

4.8 On day 14, count the number of CFUs in the I. orientalis plates and record the 

counts on the test worksheet. Average the counts for the two plates of BHIA 

supplemented with penicillinase separately from the two plates of BHIA without 

penicillinase.

5. Interpretation of the Test Results

5.1 If the average colony count for each organism in plates supplemented with 

penicillinase is within ± 20% of the average colony count of that same organism in plates

without penicillinase, the growth-promoting quality of that batch of BHIA medium is 

satisfactory (SAT) and may be used for sterility testing. 

5.2 If the average colony count for each organism in plates supplemented with 

penicillinase is not within ± 20% of the average colony count of that same organism in 

BHIA without penicillinase, the growth-promoting quality of that batch of medium is

unsatisfactory (UNSAT) and is not acceptable for use in sterility testing. 

5.3 If any sterility tests have been conducted with unsatisfactory batches of BHIA,

those tests must be reported as no tests (NT) and repeated with a new satisfactory batch 

of BHIA media. 

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.
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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.

 Sections 2-3 have been updated and rewritten for clarity.

Version .03

 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.

 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.

 7.3: Reference added about Issatchenkia orientalis.

 Appendices: Updated to include current media numbers.

 Appendix III: Control limits are established according to standard operating 

procedures, so this section has been removed from this document. 

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 STSAM0902 to SAM 902.
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 The Contact has been changed from Gerald Christianson to Sophia G. Campbell.

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

 2.1.5: Petri dishes have been added to the equipment list.

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

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

 3.1: Personnel qualifications have been clarified

 3.2.2: This section has been revised to indicate additional equipment monitored.

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

testing.

 5: The test interpretations have been clarified.

 Appendices I & II: Media storage conditions have been added.

 The statistical tolerances table has been moved from the body of the document to 

Appendix III.
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Appendices - Media Formulations

Appendix I

Brain Heart Infusion Agar (BHIA) – NCAH Media #10204

Brain Heart Infusion Agar 52 g

QH2O 1000 mL 

Autoclave 20 minutes at 121oC. Store at 2°- 5°C for no longer than 3 months.

Appendix II

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

Media #10423

Trypticase Soy Broth 30 g

QH2O 1000 mL

Autoclave 20 minutes at 121oC. Store at 20°- 25°C for no longer than 3 months.

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

media companies.