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SAM 214.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 214

Supplemental Assay Method for Immunogenicity and Spore Count Testing of 

Anthrax Spore Vaccine-Nonencapsulated

Date: March 25, 2014

Number: SAM 214.05

Supersedes: SAM 214.04, November 16, 2010

Standard Requirement: 9 CFR 113.66

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

Approvals: /s/Larry R. Ludemann Date: 05May14

Larry R. Ludemann, Section Leader

Bacteriology

/s/Byron E. Rippke Date: 07May14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09May14

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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Table of Contents

1. Introduction

Part I. Immunogenicity Test

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Test animals

3. Preparation for the Immunogenicity Test

3.1 Personnel qualifications/training

3.2 Preparation of supplies

3.3 Handling of test animals

3.4 Preparation of reagents

4. Performance of the Immunogenicity Test

4.1 Vaccination of test animals

4.2 Timing of challenge

4.3 Preparation of challenge dilutions

4.4 Inoculation of guinea pigs

4.5 Observation of guinea pigs following challenge

5. Interpretation of the Immunogenicity Test Results

6. Reporting of the Immunogenicity Test Results

Part II. ASV Spore Count Test

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Spore Count Test

3.1 Personnel qualifications/training

3.2 Preparation of supplies

3.3 Preparation of reagents
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4. Performance of the Spore Count Test

5. Calculation of Spore Counts

6. Interpretation of the Spore Count Test Results

7. Reporting of the Spore Count Test Results

8. References

9. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes methods for immunogenicity and spore 

count testing of Anthrax Spore Vaccine-Nonencapsulated (ASV-N) as prescribed in title 9, Code 

of Federal Regulations (9 CFR), section 113.66. Part I describes the guinea pig vaccinationchallenge test used to determine immunogenicity of ASV-N Master Seed. Part II describes the 

spore count procedure for release testing of final container samples of ASV-N. 

Part I. Immunogenicity Test

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Class II biological safety cabinet

2.1.2 Autoclave

2.1.3 Pipette filler

2.2 Reagents/supplies

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

unless specified as required for the test.

2.2.1 Bacillus anthracis challenge culture, IRP 137 (This culture must be 

obtained from the United States Department of Agriculture, Animal and Plant 

Health Inspection Service, Veterinary Services, Center for Veterinary Biologics

[CVB], Ames, IA 50010.)

2.2.2 Glycerol diluent

2.2.3 Disposable syringes, 3-mL and 5-mL

2.2.4 Needles, 18-gauge x 1 1/2-inch and 23-gauge x 1-inch

2.2.5 Glass pipettes, 2-mL and 5-mL

2.2.6 Glass volumetric pipettes, 25-mL or 100-mL
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2.2.7 Glass screw-top Erlenmeyer flask, 500-mL, with cap

2.2.8 Glass screw-top tubes, 16 x 125-mm, with caps

2.2.9 Glass screw-top dilution bottles, 125-mL or 160-mL

2.2.10 Glass serum bottles, 100-mL

2.2.11 Rubber seals and metal caps for serum bottles

2.2.12 Disposable clothing

2.2.13 Occupational Safety and Health Administration (OSHA)-approved dust 

and mist respirator

2.3 Test animals

Guinea pigs, 400-500 g. Forty-two guinea pigs (30 vaccinates and 12 controls) from the 

same source are required for each lot of Master Seed to be tested. (Guinea pigs of either 

sex or any color may be used.)

3. Preparation for the Immunogenicity Test

3.1 Personnel qualifications/training

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

chemicals, equipment, and glassware. In addition, personnel need specific training and 

experience in the safe handling of live B. anthracis cultures.

3.2 Preparation of supplies

3.2.1 Sterilize all glassware before use.

3.2.2 Make sure all disposable supplies (pipettes, syringes, needles, rubber 

stoppers, etc.) are sterile.

3.3 Handling of test animals

3.3.1 Select guinea pigs that appear healthy, are free of external parasites, and 

have an unblemished hair coat.
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3.3.2 Examine and cage the guinea pigs the day they are received. (Up to 4 

guinea pigs of the same sex may be housed in each cage.)

3.3.3 House the test animals in a room with access limited to authorized

personnel only.

3.3.4 Wear disposable outer clothing and an OSHA-approved dust and mist 

respirator for protection during and after vaccination and challenge. Use the 

disposable clothing and respirator only once and discard them before exiting the 

animal room.

3.3.5 Euthanize and incinerate the guinea pigs when the test is concluded. 

Disinfect the contaminated equipment and cages. Sterilize the bedding before 

disposal.

3.4 Preparation of reagents

3.4.1 B. anthracis challenge culture

The challenge culture, B. anthracis IRP 137, is suspended in 50% glycerol. Each 

ampule contains approximately 1.8 mL of culture suspension. Store ampules of 

challenge at -70°± 5°C.

3.4.2 Glycerol diluent, 50%

The 50% glycerol diluent is prepared by mixing equal parts of glycerol and 0.85% 

NaCl solution. Place 300 mL of diluent in a 500-mL flask and sterilize at ≥121°C 

for 25 to 30 minutes following manufacturer’s recommendations. Store at room 

temperature up to 1 year.

4. Performance of the Immunogenicity Test

4.1 Vaccination of test animals

4.1.1 Check the label on each container of Master Seed to be tested for identity. 

4.1.2 Thoroughly mix the Master Seed by striking the container against the 

palm of the hand at least 25 times before filling the syringes. Use 3-mL or 5-mL

syringes, fitted with 23-gauge x 1-inch needles.

4.1.3 Vaccinate 30 guinea pigs with the required dosage of the Master Seed as 

recommended on the label (see 9 CFR 113.66(b)(2)).
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4.1.4 Vaccinate each animal only once.

4.1.5 Observe the guinea pigs for 14 to 15 days. Examine each animal carefully 

for sores, edema, and general physical condition. Record deaths.

4.2 Timing of challenge

4.2.1 Challenge vaccinated guinea pigs 14 to 15 days after vaccination.

4.2.2 Challenge nonvaccinated control guinea pigs at the same time.

4.3 Preparation of challenge dilutions

Caution: The B. anthracis challenge is a live spore suspension. Prepare the 

challenge within a Class II biological safety cabinet. Use a pipette filler. Autoclave 

any contaminated equipment and clothing before disposal. 

4.3.1 Warm an ampule of challenge culture suspension to room temperature.

4.3.2 Mix the contents thoroughly by shaking the ampule vigorously. 

4.3.3 Disinfect the ampule with 70% ethyl alcohol. Carefully break open the 

ampule using a pledget of dry, sterile cotton (or several sterile gauze squares) to 

enclose the break line.

4.3.4 Use a 3-mL syringe fitted with an 18-gauge x 1 1/2-inch needle to 

aseptically transfer the culture suspension to a 16 x 125-mm test tube. Thoroughly 

mix the suspension.

4.3.5 Place 4 mL of glycerol diluent into a 16 x 125-mm test tube using a sterile 

5-mL pipette. Use a sterile 2-mL pipette to add 1 mL of culture suspension to the 

4 mL of glycerol diluent.

Note: The 50% glycerol is viscid and must be pipetted with care to ensure 

delivery of the complete volume.

Mix thoroughly. 

4.3.6 Measure 100 mL of glycerol diluent into a 125-mL or 160-mL glass 

dilution bottle using a 25-mL or 100-mL volumetric pipette. Remove 1 mL of 

glycerol diluent using a 2-mL pipette thereby leaving 99 mL of diluent in the 

dilution bottle. Using a 2-mL pipette, transfer 1 mL of the 1:5 challenge dilution 

to the 99 mL of glycerol diluent. Mix thoroughly. (This is the 1:500 dilution.)
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4.3.7 Measure 100 mL of glycerol diluent into a 100-mL serum vial using a 

25-mL or 100-mL volumetric pipette. Remove 1 mL of glycerol diluent using a 

2-mL pipette thereby leaving 99 mL of diluent in the serum vial. Using a 2-mL

pipette, transfer 1 mL of the 1:500 challenge dilution to the 99 mL of glycerol 

diluent. Cap and seal the serum vial. Mix thoroughly. (This is the 1:50,000 

challenge dilution.) The challenge material is kept at room temperature until it is 

used to inoculate the guinea pigs.

4.4 Inoculation of guinea pigs

4.4.1 Thoroughly mix the 1:50,000 challenge dilution before filling syringes. 

(Use 3-mL syringes fitted with 23-gauge x 1-inch needles.)

4.4.2 Inoculate 0.5 mL of the challenge into the thigh muscle of each guinea 

pig. (This inoculum contains approximately 4500 guinea pig lethal dose 50 (LD50)

of spores.)

4.5 Observation of guinea pigs following challenge

Observe the guinea pigs daily for 10 days. Examine the test animals for sores, edema, and 

general physical condition. Record the observations on the daily record. Record the 

number of deaths.

Note: Moribund animals exhibiting clinical signs consistent with the expected 

disease pathogenesis that are unable to rise or move under their own power may be 

humanely euthanized and considered as deaths as outlined in 9 CFR 117.4.

5. Interpretation of the Immunogenicity Test Results

5.1 The test is interpreted as prescribed in 9 CFR 113.66.

5.1.1 If at least 10 of the 12 controls die from B. anthracis within the 10-day 

post-challenge observation period, the test is valid. If 9 or fewer of the 12 controls 

die from B. anthracis within the 10-day observation period, the test is invalid and 

may be repeated.

5.1.2 If at least 27 of the 30 vaccinates survive the 10-day observation period, 

the Master Seed is satisfactory.

5.1.3 If fewer than 27 of the 30 vaccinates survive the 10-day observation 

period, the Master Seed is unsatisfactory.
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5.2 Observation of sores, edema, and general physical condition provides 

supplemental information concerning the safety and quality of the Master Seed. 

However, potency test results are evaluated only on survival/death patterns.

6. Reporting of the Immunogenicity Test Results

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

Part II. ASV Spore Count Test

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Class II biological safety cabinet

2.1.2 Autoclave

2.1.3 Water bath, set at 50°C

2.1.4 Incubator, set at 37°C

2.1.5 Colony counter

2.1.6 Pipette filler

2.2 Reagents/supplies

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

2.2.1 Reference vaccine

2.2.2 Glycerol diluent

2.2.3 Tryptose agar

2.2.4 Glass test tubes, 20 x 150-mm, with Morton closures
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2.2.5 Glass screw-top test tubes, 20 x 150-mm, with caps

2.2.6 Glass screw-top test tubes, 16 x 125-mm, with caps

2.2.7 Needles, 18-gauge x 1- or 1 1/2-inch

2.2.8 Glass screw-top dilution bottles, 125-mL or 160-mL, with caps

2.2.9 Glass pipettes, 2-mL, 5-mL, and 10-mL

2.2.10 Petri dishes, 100 x 15-mm

2.2.11 Disposable syringes, 3-mL

2.2.12 Glass volumetric pipettes, 25-mL or 100-mL

3. Preparation for the Spore Count Test

3.1 Personnel qualifications/training

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

chemicals, equipment, and glassware. In addition, personnel need specific training and 

experience in the safe handling of live B. anthracis cultures.

3.2 Preparation of supplies

3.2.1 Sterilize all glassware before use.

3.2.2 Make sure all disposable supplies (pipettes, syringes, needles, rubber 

stoppers, etc.) are sterile.

3.3 Preparation of reagents

Caution: The Anthrax Spore Vaccine-Nonencapsulated is a live spore suspension. 

Prepare the reference and serial vaccine dilutions within a Class II biological safety 

cabinet. Use a pipette filler. Autoclave any contaminated equipment and clothing 

before disposal or reuse. 

3.3.1 Use a reference vaccine to be tested with the test vaccine. (The reference 

vaccine serves as the control for the media and the technique. The reference 

should have been shown to produce consistent spore counts in at least 3 previous 

tests.)
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3.3.2 Prepare the 50% glycerol diluent by mixing equal parts of glycerol and 

0.85% NaCl solution. Place 300 mL of diluent in a 500-mL flask. Autoclave at 

≥121°C for 25 to 30 minutes following manufacturer’s recommendations. Store at 

room temperature up to 1 year.

3.3.3 Prepare tryptose agar according to label directions on the day of the test. 

Dispense tryptose agar in 20 mL amounts in 20 x 150-mm test tubes with Morton 

closures. Autoclave 25 to 30 minutes at ≥121°C following manufacturer’s 

recommendations.

4. Performance of the Spore Count Test

4.1 Adjust water bath to 50° 5°C.

4.2 Prepare tryptose agar as described above in Part II, Section 3.3.3. Cool tryptose 

medium in the water bath approximately 1 hour.

4.3 Vigorously shake each vaccine by hand 100 times before making dilutions.

4.4 Measure 100 mL of glycerol diluent into each of two 125-mL or 160-mL dilution 

bottles using a 25-mL or 100-mL volumetric pipette. Remove 1 mL of glycerol diluent 

from each bottle using a 2-mL pipette thereby leaving 99 mL of glycerol diluent in each 

bottle.

4.5 Remove 2-3 mL of vaccine with a 3-mL syringe and 18-gauge x 1 1/2-inch needle

and transfer to a sterile 16 x 125-mm glass screw-cap test tube. Use a 2-mL pipette to 

transfer 1 mL of vaccine to 99 mL of glycerol diluent in the dilution bottle. This 1:100 

dilution approximates 15,000 spores/mL in a 2-mL vaccine dose and 30,000 spores/mL in 

a 1-mL vaccine dose.

4.6 Vigorously shake the 1:100 dilution by hand 100 times. Make another 

hundredfold dilution by transferring 1 mL of the 1:100 dilution into 99 mL of glycerol 

diluent in the next dilution bottle. This 1:10,000 dilution approximates 150 spores/mL in 

a 2-mL vaccine dose and 300 spores/mL in a 1-mL vaccine dose. This dilution may be 

too numerous to count.

4.7 Vigorously shake the 1:10,000 dilution by hand 100 times. Using 10-mL pipettes, 

make a twofold dilution (5 mL 1:10,000 dilution + 5 mL glycerol diluent) in a 20 x 

150-mm test tube. This 1:20,000 dilution approximates 75 spores/mL in a 2-mL vaccine 

dose and 150 spores/mL in a 1-mL vaccine dose.
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4.8 Vigorously shake the 1:10,000 dilution by hand 100 times. Using a 5-mL and 

10-mL pipette, make a fivefold dilution (2 mL 1:10,000 dilution + 8 mL glycerol diluent) 

in a 20 x 150-mm test tube. This 1:50,000 dilution approximates 30 spores/mL in a 2-mL

vaccine dose and 60 spores/mL in a 1-mL vaccine dose. 

4.9 Vigorously shake the 1:50,000 dilution by hand 100 times. Using a 2-mL and 

10-mL pipette, make a tenfold dilution (1 mL 1:50,000 dilution + 9 mL glycerol diluent)

in a 20 x 150-mm test tube. This 1:500,000 dilution approximates 3 spores/mL in a 2-mL

vaccine dose and 6 spores/mL in a 1-mL vaccine dose.

4.10 Vigorously shake each dilution (1:20,000; 1:50,000; 1:500,000) 25 times before 

the sample is withdrawn. Use a 2-mL or 5-mL glass pipette to add 1 mL of each dilution 

to each of 3 sterile petri dishes. Pour 1 tube of tryptose agar into each plate and swirl 

slowly 10 times in a figure-8 motion to mix. Allow the medium to harden before 

inverting plates. 

4.11 Dispense 1-mL of uninoculated glycerol diluent into 1-3 sterile petri dishes. Pour 

1 tube of tryptose agar into each plate and also 1-3 uninoculated sterile petri dishes. Swirl 

slowly 10 times in a figure-8 motion and allow the medium to harden before inverting 

plates. These plates serve as negative controls.

4.12 Incubate the plates at 35°- 37°C for 24 to 28 hours.

4.13 Count the colonies and record the results on the worksheets.

5. Calculation of Spore Counts

5.1 Total the colony counts of the 3 plates made for each dilution and divide by 3. 

(This is the average number of spores per mL in that dilution.)

5.2 Calculate CFU/dose by the following formula:

 CFU/Dose = (AVG CFU)\(50 mL ) Therefore, for a 1 mL dose: CFU/Dose = (AVG CFU)

 (2x10-5)(1 mL plated)\(50 doses) (DILUTION FACTOR)

6. Interpretation of the Spore Count Test Results

The spore count results are interpreted as described in 9 CFR 113.66. To be eligible for release, 

each serial and each subserial of Anthrax Spore Vaccine-Nonencapsulated shall have a spore 

count sufficiently greater than that of the vaccine used in the immunogenicity test. This ensures 

that when tested within the expiration period, each serial and subserial must have a spore count 

of at least twice that used in the immunogenicity test but not less than 2 million spores per dose.
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7. Reporting of the Spore Count Test Results

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

8. References

8.1 Title 9, Code of Federal Regulations, section 113.66, U.S. Government Printing 

Office, Washington, DC.

8.2 History of reagents: B. anthracis challenge culture (IRP 137) was obtained from 

the Central Veterinary Laboratory, Ministry of Agriculture, Weybridge, Surrey, England. 

It was derived from a strain developed by Max Sterne, from a Pasteur anthrax vaccine 

strain, by continuous passage in guinea pigs until virulence could not be further 

enhanced. It was consistently virulent for guinea pigs but not for rabbits, domestic 

animals, or man.

9. Summary of Revisions

Version .05

 Bacteriology Section Leader updated.

 Minor word changes for clarification of procedures.

Version .04

 The Contact information has been updated.

Version .03

 The document number has been changed from BBSAM0214 to SAM 214.

 The use of 125-mL glass screw-top dilution bottles has been added.

 Part I, 4.3.3: The use of sterile gauze pads has been added.

 Part I, 5.1.2: The wording has been changed for clarity.

 Part I, 5.1.3: The wording has been changed for clarity.
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 Part II, 4.11: The number of petri dishes that can be used has been changed from 1-

2 to 1-3.

 Part II, 5.2: The wording has been changed for clarity

Version .02

This document was revised to clarify the 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:

 Part II 4.11 Negative controls have been added.

 References to pipette sizes were added throughout the text for clarification.