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SAM 928.07

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 928

Supplemental Assay Method for Detection of Extraneous Bacteria and Fungi 

in Live Bacterial Vaccines and Master Seed Bacteria Samples

Date: July 26, 2018

Number: SAM 928.07

Supersedes: SAM 928.06, October 27, 2016

Standard Requirement: 9 CFR, Part 113.27(b) and (d)

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

Mindy J. Toth, (515) 337-6242

Approvals:

/s/Larry R. Ludemann Date: 31Jul18

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 31Jul18

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

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.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 02Aug18 

 Linda S. Snavely Date

 Quality Management Program Assistant
Center for Veterinary Biologics SAM 928.07

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Supplemental Assay Method for Detection of Extraneous Bacteria and Fungi in Live Bacterial Vaccines and 

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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

3.4 Preparation of the samples

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 Detection of Extraneous Bacteria and Fungi in Live Bacterial Vaccines and 

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1. Introduction

This Supplemental Assay Method (SAM) describes the test procedures used to detect viable 

extraneous bacteria and fungi in live bacterial vaccines and master seed bacteria (MSB) samples, 

as per title 9, Code of Federal Regulations (9 CFR), part 113.27(b) and (d). Extraneous viable 

bacteria or fungi are detected by comparison to an appropriate positive control by macroscopic 

evaluation on differential media and microscopic observation.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Microscope 

2.1.2 Laminar-flow Class II biosafety cabinet (BSC)

2.1.3 30°- 35°C incubator

2.1.4 20°- 25°C incubator

2.2 Reagents/supplies

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 Trypticase Soy Broth (TSB) (National Centers for Animal Health (NCAH) 

Media #10423) (Appendix I) 

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

II) 

2.2.5 Trypticase Soy Agar (TSA), NCAH Media #10487 (Appendix III)

2.2.6 MacConkey Agar (MC), NCAH Media #10217 (Appendix IV)

2.2.7 Triple Sugar Iron Agar (TSIA), NCAH Media #10406 (Appendix V)
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2.2.8 70% ethanol

2.2.9 Sterile water in serum vials

2.2.10 Lab coat or sterile sleeves and gloves

2.2.11 4 x 4-inch sterile gauze pads

2.2.12 Sterile syringes with needles

2.2.13 Vacutainer® needles

2.2.14 Sterile pipettes, individually packaged

2.2.15 Disposable inoculating loops

2.2.16 Microscope slides 

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 Turn on the BSC one hour prior to testing.

3.2.3 Monitor the temperature of incubators, freezers, and coolers according to 

SOPs.
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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 Positive control stock cultures are prepared according to the 

manufacturers’ instructions and must be equivalent to the live bacterial product 

and/or the MSB being tested. The positive controls are used as a comparison to

the organism(s) in the product. 

3.3.4 Dilution of Preservative Screening (Eleventh Vessel Positive Control): 

For each serial of live bacterial vaccine tested, inoculate an additional container of 

medium for each incubation temperature with 0.2 mL of sample and

approximately 100 CFU of the appropriate indicator organism (Sections 2.2.1 and

2.2.2). This control is used to confirm the ratio of inoculum to medium that will 

result in sufficient dilution of the product to prevent bacteriostatic and fungistatic 

activity according to 9 CFR 113.25(d).

3.3.5 Maximum Medium Volume Limits per Vessel: The maximum volume of 

medium per vessel used will not exceed 500 mL. Volumes greater than 500 mL 

will be divided evenly into two vessels and the volume of inoculum will be 

divided accordingly.

3.3.6 Negative Controls: Incubate 10 test vessels of each medium to confirm the 

sterility according to 9 CFR 113.25(c). Negative control test vessels are incubated 

with the serial test vessels at each temperature for the duration of the test.

3.4 Preparation of the samples

3.4.1 Samples to be tested are live bacterial vaccines and MSB. Ten vials of 

final product and a minimum of 4.0 mL MSB are required for sterility testing. 

3.4.2 For products without accompanying diluent, rehydrate product with sterile 

purified water with the volume specified on the product label or in the Outline of 

Production (OP).

3.4.3 Follow Section V.A. of the OP for final product(s) to determine the 

volume of media per test vessel. Order a sufficient volume of media to 

accommodate the test vessels, positive controls, and negative controls. 
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4. Performance of the Test

4.1 Label the media test vessels to identify the serials and/or lots tested. Conduct all 

testing procedures in the BSC.

4.2 Disinfect the interior surfaces of the BSC with 70% ethanol. Disinfect the tops of 

the samples with a 4 x 4-inch gauze pad soaked in 70% ethanol.

4.3 Rehydrate each vial of product, if needed, using a syringe or a Vacutainer® needle 

with accompanying sterile diluent or sterile water.

4.4 Inoculate 0.2 mL rehydrated liquid or thawed vaccine or MSB into 10 vessels

containing TSB and 10 vessels containing FTM. Swirl the vessel to distribute the product

in the medium. Inoculate two TSA plates with one loopful of culture (approximately 10 

μL) from each vial of product or MSB and streak for isolation; incubate one plate per 

incubation temperature. Smear one drop of culture on a glass slide and prepare for Gram 

staining and microscopic examination according to SOP.

4.5 Inoculate 0.2 mL of rehydrated or thawed vaccine into one additional vessel of 

TSB and FTM to serve as the 11th vessel positive controls (see Section 3.3.4). The 

sample may be obtained from any of the ten product containers or from an eleventh vial 

of product. Place the 11th vessels to the side and continue with the testing session.

Note: The 11th vessel positive controls are only used with live bacterial vaccines. 

4.6 Conduct Sections 4.3 through 4.5 for all vaccine serial(s) and Sections 4.3 

through 4.4 for MSB tested.

4.7 Conduct Sections 4.3 through 4.4 using the positive control(s) corresponding to 

the product being tested (see Section 3.3.3), except inoculate only two vessels per 

medium type. 

4.8 Following the sterility portion of the test, prepare the 11th vessel control 

organisms in the BSC designated for this purpose (see Sections 3.3.1 and 3.3.2).

4.8.1 Inoculate approximately 100 CFU of the appropriate indicator organism 

into the vessels prepared in Section 4.5 and swirl the vessel to distribute the 

organism in the medium. 

4.8.2 Following inoculation of the 11th test vessels with the indicator organisms, 

inoculate two TSA plates per indicator organism with a representative volume of 

inoculum. These plates serve to demonstrate that the appropriate number of viable 

organisms were added to the test vessel.
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4.9 Incubate test vessels containing FTM and half the inoculated TSA plates into a 

30°- 35°C incubator. Incubate test vessels containing TSB and the other TSA plates into a

20°- 25°C incubator. Incubate the TSA plates up to 3 days. Incubate test vessels, negative

controls, and positive controls for 14 days.

4.10 Following incubation, examine the TSA plates.

4.10.1 Look for abnormal growth and make slides of each colony type and Gram 

stain according to SOP. Compare colonial and cellular morphology to those of the 

positive control and record the results.

4.10.2 Count the colonies of 11th vessel indicator organisms and the control 

plates.

4.11 Following incubation, examine the TSB and FTM cultures for atypical microbial 

growth. If growth of extraneous microorganisms cannot be reliably determined by visual

examination, make a slide for microscopic examination and subculture for isolation. 

4.11.1 Select three or more culture vessels per test serial per incubation

temperature for further examination. 

4.11.2 Smear one drop of culture on a glass slide and prepare for Gram staining 

and microscopic examination according to SOP. 

4.11.3 Subculture the same test vessels onto MC, TSA, and TSIA slant. Incubate 

at the appropriate temperature for 3 days.

4.12 Compare colony morphology of the test sample to the positive control on MC and 

TSA cultures. Any differences may be further investigated by Gram staining and 

microscopic examination. Compare the TSIA reaction of the test sample to the positive 

control. Compare the cellular morphology of the test sample to those of the positive 

control. 

5. Interpretation of the Test Results

5.1 The criteria for a valid test must be met or the test is considered invalid or a no 

test (NT). Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory (UNSAT) sterility result for that product. Supervisor discretion 

is required for no test results of MSB.

5.1.1 There must be no growth in the Negative Control vessels.

5.1.2 The positive control produces expected growth.
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5.1.3 For final products tested for dilution of preservative at the Center for 

Veterinary Biologics, the TSA plates containing B. subtilis must contain an 

average count of 81-112 CFU and the TSA plates containing I. orientalis must 

contain an average count of 76-124 CFU. 

Note: A range of approximately 100 CFU should be determined at each biologics 

manufacturer facility for each new positive control lot.

5.1.4 For final products tested for dilution of preservative, growth of the 

indicator organism must be observed in the 11th vessel positive control. If there is 

a lack of indicator organism growth in the 11th vessel positive control(s) in a final 

product, the serial will be evaluated utilizing a complete dilution of preservative 

study according to 9 CFR 113.25(d). If it is found that the volume of media listed 

in the OP for sterility testing permits interference in the dilution of preservative

test, the test will be considered UNSAT and the sterility test will be reported as 

NT. 

5.2 Master Seed Bacteria (MSB)

5.2.1 If extraneous growth is detected in any test vessel of the initial test for 

MSB, one retest (RT) may be conducted using new unopened vials of MSB. If 

extraneous growth is found in any test vessel of the RT, the MSB is UNSAT.

5.2.2 MSB that exhibit no extraneous growth are considered satisfactory (SAT). 

5.3 Live Bacterial Vaccines

5.3.1 If extraneous growth is detected in 2 or 3 of the 20 test vessels of the 

initial test, one RT may be conducted using 20 new unopened samples. Conduct

the RT in the same manner as Sections 4.1 through 4.13 and omit the 11th vessel 

test, unless necessary. If a RT is not conducted within 21 days, the product is 

UNSAT by the results determined in the initial test. 

5.3.2 If no extraneous growth is detected in 19 or 20 test vessels of the initial 

test, or 39 or 40 test vessels of the RT, the serial is considered SAT.

5.3.3 If extraneous growth is found in 4 or more of the 20 test vessels of the 

initial test, or 2 or more of the 40 test vessels in the RT, the serial is UNSAT. 

5.4 If a MSB or live bacterial vaccine is found UNSAT, acquire a 3-4 mL sample of 

the contaminated culture, label the tube with sample identification, and freeze at -65°C or 

lower. The contaminant will also be identified by Matrix-Assisted Laser 

Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).
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6. Record and Report of Test Results

Record and report test results as described by SOPs.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.27(b) and (d), U.S. Government 

Printing Office, Washington, DC.

7.2 The U.S. Pharmacopoeia, Mack Publishing Co., Eaton, PA, Vol. 21, 1985, pp 

1151-1160.

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 genera novel, Lindnera 

genera novel and Wickerhamomyces genera novel. FEMS Yeast Res 8:939-54. 

7.4 Center for Veterinary Biologics Notice 12-21.

8. Summary of Revisions

Version .07

 Updated Sections 3 through 5.

Version .06

 The contact information, the Bacteriology Section Leader, and CVB-PEL Director

have been updated.

 Updated Sections 4.9 and 5.1-5.3.

 Appendix III-V: Updated media storage limits.

Version .05

 Reformatted Section 5 to clarify the test interpretation for MSB and live bacterial 

vaccines

 Clarified interpretation of test results for consistency.
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Version .04

 Revised Section 4.4 to include gram staining the sample directly from the vial.

Version .03

 Revised to include dilution of preservative requirements cited in CVB Notice 12-21.

 Sections 2-6 have been updated to reflect current practices.

 7.3: Reference added for name change of Candida krusei to Issatchenkia orientalis.

Version .02

 The document number has been changed from STSAM0928 to SAM 928.

 The Contact has been changed from Dolores Strum to Sophia Campbell and Alaina 

Ingebritson. 

 1: Information regarding use of this SAM for Master Seed Bacteria has been added.

 2.1: The Bunsen burner has been removed from the list of equipment that is needed 

for the test.

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

 3.1: Personnel qualifications have been clarified.

 3.3.3/3.3.4: These sections have been rewritten for clarification.

 3.4.2: This section has been rewritten for clarification.

 3.4.3: This section has been added to indicate that the volume of media cited in the 

Outline of Production is used for the testing.

 4: This section has been revised to clarify the procedures currently used.

 5: This section has been revised to clarify the interpretation of the test results.

 Appendices: Media storage conditions have been added.
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Appendices

Appendix I

NCAH Media #10423

Trypticase Soy Broth (TSB)

Trypticase Soy Broth 30 g

QH2O 1000 mL

Autoclave 20 minutes at 121°C. Store at 20°- 25°C for up to 3 months.

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. Store at 20°- 25°C for 

up to 3 months.

Appendix III

NCAH Media #10487

Trypticase Soy Agar (TSA)

Trypticase Soy Agar (BBL) 40 g

QH2O 1000 mL

Autoclave 20 minutes at 121°C. Store at 2°- 5°C for up to 6 months.
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Appendix IV

NCAH Media #10217

MacConkey Agar

MacConkey Agar (BBL) 50 g

QH2O 1000 mL

Autoclave 20 minutes at 121°C. Store at 2°- 5°C for up to 6 months.

Appendix V

NCAH Media #10406

Triple Sugar Iron Agar

Triple Sugar Iron Agar Slants (TSIA) 65 g

QH2O 1000 mL

Autoclave 15 minutes at 121°C. Slant tubes at a 20° angle. Store at 2°- 5°C for up 

to 6 months.