Center for Veterinary Biologics SAM 631.06

Testing Protocol Page 1 of 12

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 631

Supplemental Assay Method for Potency Testing of Salmonella typhimurium

Bacterins 

Date: June 17, 2022

Number: SAM 631.06 

Supersedes: SAM 631.05, May 24, 2016 

Standard Requirement: 9 CFR 113.120 

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 Potency Testing of Salmonella typhimurium Bacterins

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 Post-inoculation 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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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

1. Introduction

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

products containing Salmonella typhimurium, as prescribed in title 9, Code of Federal

Regulations (9 CFR), part 113.120. Mice are vaccinated twice, 14 days apart, and challenged 

with a standard dose of virulent S. typhimurium 7 to 10 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+, (Spectronics Instruments) 

2.1.2 Sterile inoculating loop

2.1.3 Bunsen burner or Bacti-Cinerator® (if non-sterile wire loop is 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 rings on serum vials

2.1.8 Rotary shaker 

2.1.9 Biological safety cabinet 

2.2 Reagents/supplies

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

2.2.1 S. typhimurium challenge culture, 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 S. typhimurium

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

2.2.3 Appropriately-qualified S. typhimurium reference bacterin, available from 

the biologics manufacturer. This reagent is supplied to the CVB, upon request, 

with a reagent information sheet indicating handling and test information. 

2.2.4 Syringes, 1-mL Tuberculin 

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

2.2.6 Glass serum bottle, 10- to 100-mL 

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

2.2.8 Screw-cap glass tubes, 13 x 100-mm and 15 x 125-mm

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

2.2.10 Micropipette tips, up to 1000-μL capacity

2.2.11 Glass rod, bent into L-shape 

2.2.12 Tryptose agar plates or bovine blood agar plates 

2.2.13 Tryptose broth 

2.2.14 Phosphate-buffered saline (PBS)

2.2.15 Brain-heart infusion broth 

2.2.16 Screw cap flask, 1-L 

2.3 Animals

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

some colonies of mice may be relatively resistant to salmonellosis and therefore 

less suitable for this assay.

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

3 dilutions/serial). 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. 

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

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

technique, the handling of live bacterial cultures, and the handling of mice. 

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 

S. typhimurium, necropsy 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 bacteriological 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 S. typhimurium reference bacterin, provided by the biologics 

manufacturer. Refer to current reagent information sheet for details. 

3.4.2 S. typhimurium challenge culture, available from the CVB. Refer to 

current reagent data sheet for details. 

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

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. 1 L 

Adjust pH to 7.2 ± 0.1. Autoclave 20 minutes at ≥ 121°C. Store at 20°- 25°C for 

no longer than 6 months. 

3.4.4 Tryptose broth – NCAH Media #10404 

Tryptose broth powder (BBL or equivalent) 26 g 

Deionized water q.s. 1000 mL 

Autoclave 15 minutes at ≥ 121°C. Cool before using. Store at 20°- 25°C no longer 

than 6 months. 

3.4.5 Tryptose agar – NCAH Media #10093 

Tryptose agar powder (BBL or equivalent) 41 g 

Deionized water q.s. 1000 mL 

Autoclave 25 minutes at ≥ 121°C. Cool in a 56°- 60°C water bath. Pour into 

sterile petri dishes. Allow to cool to 20°- 25°C. Store at 2°- 7°C for no longer than 

6 months. 

3.4.6 Brain-heart infusion broth – NCAH Media #10009 

Brain-heart infusion (BBL or equivalent) 37 g 

Deionized water q.s. 1000 mL 

Autoclave 20 minutes at ≥ 121° C. Store at 20°- 25°C for no longer than 6 

months. 

3.4.7 Bovine blood agar – NCAH Media #10006 

Blood agar base powder 40 g 

Deionized water 950 mL 

Autoclave for 20 minutes at ≥ 121° C. Cool to 45°- 47°C. 

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

Add: 

Defibrinated blood 50 mL

Pour into sterile petri dishes. Allow to 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 tenfold dilutions. 

Typically, test the bacterins at 1:10, 1:100, and 1:1000 dilutions. Refer to the 

current reagent information sheet for any starting dilutions of the reference 

bacterin. It is permissible to make tenfold 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 tenfold 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 tenfold dilutions of the reference bacterin as specified on the 

reagent information sheet. Make identical tenfold dilutions of the test bacterin(s) 

in the diluent approved in the specific Outline of Production for that product. 

(Some oil-adjuvanted products require oil-based diluents.) Place each dilution in a 

separate sterile injection vial. Prepare dilutions immediately prior to use; do not 

store in diluted form. 

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 intraperitoneally with the dose supplied on the reagent information 

sheet. Inject test bacterins intraperitoneally at a dose volume that corresponds to 

1/20 of the lowest dose recommended on the product label or Section VI of the 

current 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 

S. typhimurium and included in data calculations.

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

vaccination.

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

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 3 tubes containing 10 mL of brain-heart infusion broth with 

500 μL of reconstituted culture. 

4.2.3 Incubate the inoculated tubes at 35°- 37°C for 16 to 20 hours. 

4.2.4 Perform a Gram stain on the overnight culture, using standard methods. If 

the bacteria in the Gram stain are short, Gram negative rods (evidence of pure 

culture), proceed to the next step. If the challenge appears contaminated, discard 

affected tubes.

4.2.5 Adjust the density of the overnight culture to 57-61% T at 620 nm using a 

Spectronic 20D+ spectrophotometer. Dilute the culture, as needed, with tryptose 

broth. Place the culture in a 13 x 100-mm screw-cap tube for spectrophotometric 

determination. Use sterile tryptose broth in a 13 x 100-mm tube as a blank for the 

spectrophotometer. 

4.2.6 Prepare a 10-1 dilution of the standardized culture in tryptose broth. This 

inoculum is 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 of the standardized culture for LD50

determination (10-3 to 10-5) and post-inoculation plate count (10-5 to 10-7). 

Dispense an aliquot of each LD50 dilution in a separate serum vial and seal. 

Alternatively, save an aliquot of the challenge inoculum or 10-5 dilution to prepare 

the additional plate count dilutions later (see Section 4.4.1).

4.2.8 Place the vial(s) of challenge inoculum and additional dilution tubes on 

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

for post-inoculation plate count. 

4.3 Timing and administration of challenge

4.3.1 Challenge all vaccinates 7 to 10 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 0.25 mL of challenge inoculum 

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

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

4.3.4 Inoculate separate groups of 10 non-vaccinated control mice 

intraperitoneally with 0.25 mL of each of the LD50 dilutions. 

4.4 Postinoculation plate count in a biological safety cabinet

4.4.1 After mice are challenged, prepare 10-5 to 10-7 tenfold dilutions using 

tryptose broth diluent (if not previously prepared in Section 4.2.7).

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

an agar plate. Plate each dilution in triplicate using 0.1 mL on bovine blood agar 

or tryptose 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. 

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

4.4.4 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

4.4.5 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.

4.5 Observation of mice after challenge

4.5.1 Observe the mice up to twice daily for 14 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 salmonellosis, necropsy 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.

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

5. Interpretation of the Test Results

Interpret the test as prescribed in 9 CFR 113.120. 

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 At least 2 dilutions of the reference shall protect > 0% and < 100% of the mice for 

a valid test. The lowest dilution of the reference shall protect > 50% of the mice and the 

highest dilution of the reference shall protect < 50% of the mice. 

5.4 If the PD50 of the reference cannot be calculated because the lowest dilution tested 

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

1. If the serial is not retested, it is unsatisfactory.

2. If the protection provided by the lowest dilution of the standard exceeds that 

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

is unsatisfactory without additional testing.

3. If the total number of mice protected by the reference (sum of survivors in all 

dilution groups) exceeds the total number protected by the test serial by 8 mice or 

more, the test serial is unsatisfactory without additional testing.

5.5 If the PD50 of the test serial in a valid test cannot be calculated because the highest 

dilution protected more than 50% of the mice, the serial is satisfactory without further 

testing.

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

relative potency (RP) for each serial.

5.7 If the RP of the test serial(s) is ≥ 0.3, the serial is satisfactory. 

5.8 If the RP of the test serial(s) is < 0.3, the test serial is unsatisfactory. 

5.9 A test serial with an RP < 0.3 may be retested by conducting 2 independent 

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

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

the following manner: 

1. Average the RP values of the retests. 

2. If the average RP of the retests is < 0.3, the serial is unsatisfactory. 

3. If the average RP of the retests is ≥ 0.3 AND the RP obtained in the original 

test is < 1/3 than the average (RP) of the retests, the test bacterin is satisfactory. 

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

4. If the average of the retests is ≥ 0.3 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 ≥ 0.3, the test 

bacterin is satisfactory. If the new average RP is < 0.3, 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 113.120, 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. 

8. Summary of Revisions 

Version .06 

• Updated coversheet and contact information. 

Version .05 

• The Section Leader and Director information has been updated. 

Version .04

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Supplemental Assay Method for Potency Testing of Salmonella typhimurium Bacterins

• The CVB no longer provides the qualified reference bacterin. The SAM was updated 

to reflect this change.

Version .03

• The Contact information has been updated. 

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

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. 

• 2.2.8 Screw-cap tubes, 15 x 125-mm, have been added. 

• 2.2.12 Bovine blood agar has been added as an additional media option for the plate 

counts. 

• 2.2.16 A 1-L flask has been added

• 2.3.2 The number of mice currently used for the test has been updated. 

• 3.4.7 The recipe for 5% bovine blood agar plates has been added.

• The use of a biological safety cabinet has been added throughout the document. 

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

document. 

• The currently used spectrophotometer parameters have been updated. 

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

• The contact has been changed to Janet Wilson. 

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