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SAM 918.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 918

Supplemental Assay Method for Bacterial Count of Pasteurella multocida, 

Avian Isolates, Vaccines

Date: February 21, 2023

Number: SAM 918.06

Supersedes: SAM 918.05, February 15, 2017

Standard Requirement: 9 CFR Part 113.70(c)(2)

Contact: Email: Methodsrequest.notification@usda.gov

 Phone: Center for Veterinary Biologics, 515-337-6100

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 Bacterial Count of Pasteurella multocida, Avian Isolates, Vaccines

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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 sample

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 Bacterial Count of Pasteurella multocida, Avian Isolates, Vaccines

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1. Introduction

This Supplemental Assay Method (SAM) establishes the titration method for the analysis of

Pasteurella multocida, avian isolates, vaccines to determine the colony-forming units (CFU) in 

final container samples, per title 9, Code of Federal Regulations (9 CFR), part 113.70(c)(2). 

This method uses Tryptose Broth (TB) as a diluent and Tryptose Agar (TA) plates with 5% 

bovine blood for determining CFUs.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name 

listed below.

2.1.1 Vortex mixer

2.1.2 Colony counter

2.1.3 HandyStep® electronic pipette

2.1.4 35°± 2°C incubator

2.1.5 Laminar-flow Class II biosafety cabinet (BSC)

2.2 Reagents/supplies

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

below.

2.2.1 Tryptose broth (TB) (Appendix I), National Centers for Animal Health 

(NCAH) Media #10404

2.2.2 Tryptose agar (TA) with 5% bovine blood (Appendix II), NCAH Media 

#10218 or as stated in the Outline of Production (OP) from the biologics 

manufacturer. 

2.2.3 P. multocida reference culture (American Type Culture Collection 

[ATCC] #11039)

2.2.4 70% ethanol
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2.2.5 Sterile water in serum vials

2.2.6 Inoculum spreader

2.2.7 Sterile syringes and needles

2.2.8 Sterile pipettes, individually packaged

2.2.9 Sterile culture tubes 

2.2.10 Lab coat and gloves

2.2.11 4 x 4-inch sterile gauze pads

2.2.12 Test tube rack 

2.2.13 Sharps container

2.2.14 Pipetting aid

2.2.15 Micropipettors, 100-μL to 1.0-mL

2.2.16 Pipette tips, 100-μL to 1-mL

2.2.17 BRAND PD-TipTM Syringe Tips

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).
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3.2.2 Monitor temperature of incubators, freezers, and coolers according to 

SOPs.

3.2.3 Turn on BSC at least 30 minutes prior to testing.

3.2.4 Label all plates with sample number or name, vial number, and dilution 

series. Label 3 plates per dilution series for each serial.

3.3 Preparation of reagents/control procedures 

3.3.1 Warm the samples and reference culture to 20°- 25°C before rehydrating 

to the appropriate volume.

3.3.2 P. multocida reference stock culture is prepared according to the 

manufacturers’ instructions.

3.3.3 Negative and Positive Controls: Incubate 1 uninoculated plate of TA with 

5% bovine blood and 1 plate inoculated with sterile diluent used in testing with 

test sample plates as negative control plates. P. multocida reference culture 

(positive control) is diluted the same as the test samples, but plated depending on 

the titer found in Section 3.3.2.

3.3.4 Store plates used for making counts at refrigerator temperature. Plates to 

be used for counts are placed in a 35°± 2°C incubator overnight prior to use or 

allowed to dry in a BSC before use. At the time of use, plates are no more than 6 

months old.

3.4 Preparation of the sample

Samples are P. multocida vaccines and/or combination products containing this fraction. 

Sterile purified water in serum vials, in volumes specified on the product label or in the 

firm’s OP, are used for samples that are not accompanied by a diluent.

4. Performance of the Test

4.1 Remove 2 vials (or the number of vials stated in the OP for testing) of product to 

be tested and 1 vial of P. multocida reference stock culture from the freezer or cooler 

storage and allow sample to warm to room temperature.

4.2 Disinfect the caps with 70% ethanol. Rehydrate the vials and allow the contents to 

reconstitute for at least 5 minutes. Shake the vials by inversion until thoroughly mixed.
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 4.3 Prepare a tenfold dilution series of the product by setting up a rack of sterile 

culture tubes and pipetting 9.0 mL of tryptose broth into each tube using a HandyStep 

pipette with a sterile syringe tip. Label the tubes 10-1 to 10-x as needed.

4.4 Transfer 1 mL of sample from Section 4.2 into the first tube of tryptose broth by 

using a micropipettor with a sterile tip. Cap the tube and vortex. The dilution series is 

continued by using a new sterile tip to transfer a 1.0 mL sample from this tube to the tube 

labeled 10-2. Repeat this method using a new sterile tip for each transfer until the required 

number of serial tenfold dilutions (refer to the firm’s OP) is attained. 

4.5 Deposit 0.1 mL of the sample from the last 3 dilution points of the dilution series 

for the product onto the surface of media in Section 2.2.2 using a micropipettor with a 

sterile tip. 

4.6 Use a sterile inoculum spreader to evenly distribute the inoculum over the surface 

of the agar medium. 

4.7 Repeat Sections 4.4 through 4.6 with the second vial of product. 

4.8 Prepare 3 plates of media as in Sections 4.5 through 4.6 from each of 3 reference 

control dilutions as determined from Section 3.3.2. 

4.9 Invert all plates and incubate at 35°± 2°C for 24 ± 4 hours. After incubation, 

count plates from each series that contain 30 to 300 CFUs. Determine the mean CFU per 

dose for the number of vials tested using the calculation listed below. 

 (Average Count) x (mL Used to Rehydrate) = CFU/dose 

(Dilution Used) x (mL Plated) x (Number of Doses) 

5. Interpretation of the Test Results

5.1 If on the initial test the CFU per dose is equal to or exceeds the required minimum 

as written in the firm’s OP, the serial or subserial is satisfactory (SAT) for bacterial count 

without additional testing.

5.2 If on the initial test the CFU per dose is less than the required minimum as written 

in the firm’s OP, the serial or subserial results are inconclusive (INC) and may be retested 

using double the amount of new vaccine samples used in the initial test, provided that if 

the retest (RT) is not conducted within 21 days, the serial or subserial is unsatisfactory 

(UNSAT) by the results determined in the first test. Compare the firm’s OP method to 

this SAM when retesting the vials. If on the RT, the average count of the doubled vaccine 
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samples with the firm’s OP method is less than the required minimum, the serial or 

subserial is UNSAT.

5.3 If on the RT, the average using the firm’s OP method count is equal to or exceeds

the required minimum, the serial or subserial is SAT.

5.4 If on the initial test the reference culture or positive control culture is not within 

the titer range determined in Section 3.3.2, but the serial being tested has a SAT result, 

the serial or subserial is a no test (NT) for bacterial count without additional testing, and 

the product is released on the results of the firm’s tests. If the reference culture is not 

within its titer range and the serial being tested is below its minimum release titer, the 

serial is retested using double the amount of new vaccine samples. If on the initial test 

there is growth on the negative control plates, the serial or subserial is a NT for bacterial 

count without additional testing. 

6. Record and Report of Test Results

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

7. References 

Title 9, Code of Federal Regulations, part 113.70, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions 

Version .06 

• The coversheet has been updated.

Version .05 

• The Bacteriology Section Leader, the CVB-PEL Director, and page 1 were updated.

• 1-5: These Sections have been updated and rewritten for clarification. 

• Appendices: Media storage conditions have been updated. 

Version .04
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• Sections 1-6 have been updated to reflect current practices.

• Appendices: Media storage conditions have been updated. 

Version .03

• The Contact information has been updated. 

• 6: This section has been rewritten for clarification.

• Appendices: Media storage conditions have been added.

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: 

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

for the test.

• 4.9: The calculation for CFU/dose has been added. 
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Appendices

Appendix I

Tryptose Broth – National Centers for Animal Health (NCAH) Media #10404

Tryptose broth 26.0 g

H2O 1000.0 mL

Autoclave 20 minutes at 121°C. Media is stored at 2o- 5oC for up to 6 months. 

Appendix II

Tryptose Agar with 5% Bovine Blood (defibrinated) – NCAH Media #10218

Tryptose agar 41.0 g

QH2O 950.0 mL

Autoclave 25 minutes at 121°C. Cool in waterbath at 56°C and add 50.0 mL

defibrinated bovine blood. Media is stored at 2o

- 5oC for up to 6 months.