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SAM 634.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 634

Supplemental Assay Method for Potency Testing of Pasteurella multocida

Bacterins of Bovine Origin

Date: December 18, 2017

Number: SAM 634.05

Supersedes: SAM 634.04, April 7, 2014

Standard Requirement: 9 CFR, Part 113.121

Contact: Janet M. Wilson, (515) 337-7245

Approvals: /s/Larry R. Ludemann Date: 19Dec17

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 27Dec17

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 27Dec17 

 Linda S. Snavely Date

 Quality Management Program Assistant
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Selection and handling of test mice

3.3 Preparation of supplies/equipment

3.4 Preparation of reagents

4. Performance of the Test

4.1 Vaccination of test animals

4.2 Preparation of challenge in a biological safety cabinet

4.3 Timing and administration of challenge

4.4 Postinoculation plate count in a biological safety cabinet

4.5 Observation of mice after challenge

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes procedures for potency testing biological 

products containing Pasteurella multocida of bovine origin, as prescribed in title 9, Code of 

Federal Regulations (9 CFR), part 113.121. Mice are vaccinated twice, 14 days apart, and 

challenged with a standard dose of virulent P. multocida 10 to 12 days after the second 

vaccination. 

2. Materials

2.1 Equipment/instrumentation 

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

below.

2.1.1 Spectrophotometer, Spectronic 20D+ (Spectronic Instruments)

2.1.2 Sterile Inoculating loop

2.1.3 Bunsen burner or Bacti-Cinerator® (if non-sterile wire loop used)

2.1.4 Incubator, 35°- 37°C

2.1.5 Micropipettors, 20- to 1000-μL

2.1.6 Test tube mixer, vortex-type

2.1.7 Crimper for aluminum caps on serum vials

2.1.8 Biological safety cabinet

2.2 Reagents/supplies

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

2.2.1 P. multocida challenge culture, Strain P-1062 Type A, available from the 

Center for Veterinary Biologics (CVB). Refer to the current reagent data sheet for 

additional information.

2.2.2 Test bacterin(s) containing P. multocida
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2.2.3 P. multocida reference bacterin, available from the CVB. Refer to the 

current reagent data sheet for additional information.

2.2.4 Syringes, 1-mL

2.2.5 Needles, 26-gauge, 3/8-inch

2.2.6 Glass-serum bottle, 20- to 100-mL

2.2.7 Rubber stopper, 13 x 20-mm, and aluminum cap for serum bottle

2.2.8 Glass screw-cap tubes, 13 x 100-mm

2.2.9 Pipettes, 5-mL, 10-mL, 25-mL

2.2.10 Micropipette tips, up to 1000-μL capacity

2.2.11 Bovine blood agar plates

2.2.12 Tryptose broth 

2.2.13 Phosphate-buffered saline (PBS)

2.2.14 Sterile cotton swabs

2.3 Animals

2.3.1 Mice, 16-22 g. Although the 9 CFR does not specify a specific mouse 

type, the CVB uses CF-1 mice. 

2.3.2 Sixty mice are required for each bacterin to be tested (20 mice/dilution;

3 dilutions/bacterin). Sixty additional mice are required for the reference bacterin. 

Thirty mice are required to determine the LD50 of the challenge inoculum. All 

mice must be from the same source colony and of similar weight and/or age.

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware; and must have specific training and experience in 

sterile technique, the handling of live bacterial cultures, and the handling of mice.
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3.2 Selection and handling of test mice

3.2.1 Mice of either sex may be used, but females are recommended.

3.2.2 All mice must be housed and fed in a similar manner.

3.2.3 Identify each cage of mice by treatment group.

3.2.4 If any mice die after vaccination, but prior to challenge with live 

P. multocida, perform a necropsy on these mice to determine cause of death if the 

cause of death is not outwardly apparent. If the cause of death is unrelated to 

vaccination, file the necropsy report with the test records, and no additional action 

is needed. If death is attributable to the test bacterin, report the death immediately 

to the CVB-Inspection and Compliance, which may request further safety testing 

of the bacterin.

3.2.5 When the test is concluded, instruct the animal caretakers to euthanize and 

incinerate the mice and to sanitize the contaminated rooms.

3.3 Preparation of supplies/equipment

3.3.1 Use only sterile supplies.

3.3.2 Operate and maintain all equipment according to manufacturers’ 

recommendations and applicable standard operating procedures.

3.4 Preparation of reagents

3.4.1 P. multocida Reference Bacterin. Refer to the current reagent data sheet

for details. 

3.4.2 P. multocida challenge culture, Strain P-1062 Type A. Refer to the current 

reagent data sheet for details.

3.4.3 Phosphate-buffered saline – National Centers for Animal Health (NCAH)

Media #10559

Sodium chloride 8.0 g

Potassium chloride 0.2 g

Sodium phosphate, dibasic 1.15 g

Potassium phosphate, monobasic 0.2 g

Deionized water q.s. to 1000 mL

Adjust pH to 7.2 ± 0.1. Autoclave 20-30 minutes at ≥ 121°C following 

manufacturer’s recommendations. Store at 20°- 25°C for no longer than 1 year.
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3.4.4 Tryptose broth – NCAH Media #10404

Tryptose broth powder 26 g

Deionized water q.s. 1000 mL

Autoclave 15-30 minutes at ≥ 121°C following manufacturer’s recommendations. 

Cool before using. Store at 20°- 25°C for no longer than 1 year.

3.4.5 5% bovine blood agar – NCAH Media #10006

Blood agar base powder 40 g 

Deionized water q.s. to 950 mL

Autoclave 20-30 minutes at ≥ 121°C following manufacturer’s recommendations. 

Cool to 45°- 47°C. 

Add:

Defibrinated bovine blood 50 mL

Pour into sterile petri dishes. Cool to 20°- 25°C. Store at 2°- 7°C for no longer 

than 6 months.

4. Performance of the Test

4.1 Vaccination of test animals

4.1.1 Check the label on each product and Section VI of the current Outline of 

Production to confirm identity and dose volume.

4.1.2 Test each test bacterin and the reference bacterin at 3 fivefold dilutions. 

Typically, test the bacterins undiluted, 1:5 and 1:25. Refer to the current reagent 

data sheet for any starting dilutions of the reference bacterin. It is permissible to 

make fivefold dilutions other than those described as long as the reference and 

test bacterins are tested at the same dilutions. For viscous bacterins, it is advisable 

to start at 1:2 or 1:3 and make fivefold dilutions from this starting point to 

increase injectability of the product at the low dilution.

4.1.3 Thoroughly mix product by inverting end-to-end at least 10 times. Make 

the appropriate fivefold dilutions of the reference bacterin in PBS. Make identical 

fivefold dilutions of the test bacterin(s) in the diluent approved in the specific 

Outline of Production for that product. (Some oil-adjuvanted products require oilbased diluents.) Place each dilution in a separate sterile serum bottle. Prepare 

dilutions immediately prior to use; do not store in diluted form.
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4.1.4 Vaccinate separate groups of 20 mice with each of the test bacterin

dilutions and reference bacterin dilutions. For reference bacterin groups, inject 

each mouse with 0.1 mL intraperitoneally. Inject test bacterins intraperitoneally at 

a dose volume that corresponds to 1/20 of the lowest dose recommended on the 

product label or Outline of Production. This volume must not be < 0.1 mL

Note: It is permissible to vaccinate a few extra mice in each group to 

compensate for any potential deaths that may occur prior to challenge and 

are not related to vaccination. However, if extra mice are vaccinated, all 

surviving at the time of challenge must be challenged with live P. multocida

and included in data calculations.

4.1.5 Revaccinate the mice in a similar manner 14 days after the first 

vaccination.

4.1.6 Retain 30 non-vaccinated mice to determine LD50 of the challenge.

4.2 Preparation of challenge in a biological safety cabinet

4.2.1 Reconstitute a vial of challenge in 1 mL tryptose broth. 

4.2.2 Inoculate 2 blood agar plates with a loopful of reconstituted culture and 

streak for isolation.

4.2.3 Incubate the inoculated blood agar plates at 35°- 37°C for 16 to 18 hours.

4.2.4 Use plates that have pure growth by visual inspection to prepare the 

challenge inoculum.

4.2.5 Scrape several bacterial colonies from the surface of the blood agar plates 

using a sterile cotton swab and suspend in tryptose broth in a 13 x 100-mm tube. 

Add bacterial growth until the suspension measures 76-80% T at 630 nm using a 

Spectronic 20D+ spectrophotometer or equivalent. Use sterile tryptose broth in a 

13 x 100-mm tube as a blank for the spectrophotometer. 

4.2.6 Prepare a 10-5dilution of the standardized culture in tryptose broth. This is 

the inoculum used to challenge the mice. Dispense challenge liquid into a serum 

vial and seal with a rubber stopper and aluminum ring. 

4.2.7 Prepare additional tenfold dilutions for challenge LD50 determination 

(10-7to 10-9) and postinoculation plate counts (10-5to 10-7). Dispense an aliquot of 

each LD50 dilution in a separate serum vial and seal.
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4.2.8 Place the vials of challenge inoculum and additional dilution tubes on ice. 

Keep on ice through challenge procedure and until culture is added to plates for

postinoculation plate count.

4.3 Timing and administration of challenge

4.3.1 Challenge all vaccinates 10 to 12 days after the second vaccination.

4.3.2 Challenge non-vaccinated LD50 controls at the same time as the

vaccinates.

4.3.3 Inoculate each vaccinated mouse with 0.2 mL of challenge inoculum 

intraperitoneally, using a 1-mL syringe and 26-gauge, 3/8-inch needle.

4.3.4 Inoculate separate groups of 10 non-vaccinated control mice 

intraperitoneally with 0.2 mL of each of the LD50 dilutions.

4.4 Postinoculation plate count in a biological safety cabinet

4.4.1 All bacterial suspensions must be mixed well prior to placing an aliquot on 

an agar plate. Plate each dilution (10-5to 10-7) in triplicate using 0.1 mL on bovine 

blood agar. Inoculum must be spread evenly on the surface of the agar plates and 

not allowed to pool around the edges. Complete all plate inoculations within 1 

hour of challenge.

4.4.2 Incubate the plates aerobically at 35°- 37°C for 18 to 30 hours.

4.4.3 Using the dilution yielding 30-300 colonies per plate, calculate the colony 

forming units (CFU)/challenge dose according to the following formula:

Colony count 

sum

x

1

x

1

x

Challenge 

dilution

x

Challenge 

vol. (mL)



CFU

Number of 

plates

Dilution factor 

plated

Plated volume 

(mL)

1 Dose Dose

4.4.4 Record the plate count (CFU/dose) of the challenge on the test result form. 

This information is for informational purposes to track trends and to troubleshoot 

problem tests. The 9 CFR does not specify a minimum or maximum CFU/dose for 

this test.
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4.5 Observation of mice after challenge

4.5.1 Observe the mice up to twice daily for 10 days after challenge. Record 

deaths and euthanize any moribund mice as recommended by the Institutional 

Animal Care and Use Committee.

4.5.2 If deaths occurring after challenge are suspected to be due to causes other 

than pasteurellosis, perform a necropsy on such mice to determine the cause of 

death. If cause of death is unrelated to vaccination and/or challenge, do not 

include the deaths in the total deaths for the test.

5. Interpretation of the Test Results

Interpret the test as prescribed in 9 CFR 113.121.

5.1 Calculate the LD50 (theoretical dose/dilution at which the challenge would be 

lethal to 50% of the control mice) of the challenge inoculum using the Reed-Muench or 

Spearman-Kärber method of estimation. A valid test must have an LD50 between 100 and 

10,000. 

5.2 Calculate the PD50 of the reference bacterin and each test bacterin (theoretical 

dose/dilution at which the bacterin would protect 50% of the mice) using the ReedMuench or Spearman-Kärber method of estimation.

5.3 If the PD50 of the reference bacterin cannot be calculated because the lowest 

dilution protects < 50% of the mice or the highest dilution protects > 50% of the mice, 

the test is invalid. The reference bacterin also must protect > 0% and < 100% of the mice 

at 2 or more dilutions in a valid test. 

5.4 If the PD50 of the test bacterin cannot be calculated because the lowest dilution 

tested protects < 50% of the mice, the bacterin may be retested, provided the following:

5.4.1 If the bacterin is not retested, it is unsatisfactory.

5.4.2 If the protection provided by the lowest dilution of the reference exceeds 

that provided by the lowest dilution of the test bacterin by at least 6 mice, the test 

bacterin is unsatisfactory without additional testing.

5.4.3 If the total number of mice protected by the reference bacterin (sum of 

survivors in all dilution groups) exceeds the total number protected by the test 

bacterin by 8 mice or more, the test bacterin is unsatisfactory without additional 

testing.
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5.5 If the PD50 of the test bacterin in a valid test cannot be calculated because the 

highest dilution protected more than 50% of the mice, the bacterin is satisfactory without 

further testing.

5.6 Divide the PD50 of each test bacterin by the PD50 of the reference to calculate the 

relative potency (RP) for each bacterin.

5.7 If the RP of the test bacterin is ≥ 0.50, the test bacterin is satisfactory.

5.8 If the RP of the test bacterin is < 0.50, the test bacterin is unsatisfactory.

5.9 A test bacterin with an RP < 0.50 may be retested by conducting 2 independent 

replicate tests in a manner identical to the initial test. Calculate the results of the retests in 

the following manner:

5.9.1 Average the RP values of the retests. 

5.9.2 If the average RP of the retests is < 0.50, the bacterin is unsatisfactory. 

5.9.3 If the average RP of the retests is ≥ 0.50 AND the RP obtained in the 

original test is  1/3 the average RP of the retests, the test bacterin is satisfactory. 

Consider the initial test to be the result of test system error.

5.9.4 If the average of the retests is ≥ 0.50 BUT the RP of the original test is 

> 1/3 of the average RP of the retests, calculate a new average RP using the RP 

values obtained in all tests (original plus retests). If the new average RP is ≥ 0.50, 

the test bacterin is satisfactory. If the new average RP is < 0.50, the test bacterin is 

unsatisfactory.

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.121, U.S. Government Printing 

Office, Washington, DC.

7.2 Reed LJ, Muench H, 1938. A simple method of estimating 50% endpoints. Am J 

Hygiene, 27:493-497.
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8. Summary of Revisions

Version .05

 The Director was updated on the cover page.

Version .04

 Bacteriology Section Leader updated.

 Clarified media expiration dates.

Version .03

 The Contact information has been updated.

 2.1.3/4.5.1: These sections have been updated to reflect current practices.

 2.2.14/4.2.5: Sterile cotton swabs 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 A sterile inoculating loop and biological safety cabinet have been added.

 4.1.1 Reference to Section VI of the current Outline of Production has been added as 

an additional source of the needed information.

 4.2.1 The amount of media used to reconstitute has been adjusted.

 4.2.2 The number of plates used has been updated to reflect current practices.

 References to the current reagent data sheet have been added throughout the 

document.

 References to internal documents have been replaced with summarized information.

 The autoclave parameters have been adjusted.
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 The spectrophotometer parameters currently in use have been updated.

 The contact has been changed to Janet Wilson.