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SAM 600.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 600

Supplemental Assay Method for Testing for Purity, Potency and Dissociation 

of Brucella abortus Vaccine, Strain 19

Date: January 3, 2022

Number: SAM 600.03

Supersedes: SAM 600.02, December 1, 2015

Standard Requirement: 9 CFR 113.65

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

Approvals: /s/Melisse M. Schilling Date: 01/20/2022

Melisse M. Schilling, Section Leader

Bacteriology 

/s/Paul J. Hauer Date: 01/20/2022

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.
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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 ofsamples/reagents/control procedures

4. Performance of the Test

5. Interpretation of the Test Results

5.1 Purity testing

5.2 Potency testing

5.3 Dissociation testing

6. Report of Test Results

7. References

8. Summary of Revisions 

Appendices
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1. Introduction

This Supplemental Assay Method (SAM) describes the test procedure used to determine the 

purity, dissociation and potency (viability) of Brucella abortus Vaccine (Strain 19), as prescribed 

in the Code of Federal Regulations, Title 9 (9 CFR), Part 113.65. Purity is determined by 

inoculating dextrose Andrades broth and thioglycollate broth; dissociation by inoculating potato 

agar plates; and potency by inoculating tryptose agar plates after diluting in 1% peptone water.

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 Inoculum spreader

2.1.4 Disposable syringes and needles - appropriate sizes

2.1.5 Sterile disposable pipettes - appropriate sizes

2.1.6 Small disposable bottles

2.1.7 Pipetting aid

2.1.8 35°- 37°C incubator

2.1.9 Class II Biosafety cabinet

2.1.10 Lab coat or sterile sleevesgloves, and protective eyewear

2.1.11 Sterile gauze pads, 4 x 4-inch

2.1.12 Test tube rack

2.1.13 SharpeeTM container
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2.2 Reagents/supplies

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

2.2.1 Dextrose Andrades broth (Appendix I) - National Centers for Animal 

Health (NCAH) Media #10141

2.2.2 Tryptose agar (Appendix II) - NCAH Media #10093

2.2.3 Thioglycollate broth (Appendix III) - NCAH Media #10135

2.2.4 Potato agar (Appendix IV) - NCAH Media #10452

2.2.5 1% peptone saline solution (Appendix V) - NCAH Media #10138

2.2.6 Crystal violet solution (Appendix VI) - NCAH Media #30270

2.2.7 B. abortus, Strain 19 reference culture - National Veterinary Services 

Laboratories (NVSL) Diagnostic Bacteriology Laboratory (DBL) reagent code

#15

2.2.8 Chemical disinfectant

2.2.9 70% ethyl alcohol

2.2.10 Sterile water in serum vials

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel performing the test 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 Turn the laminar-flow hood on 30 minutes before use and turn off after 

use.
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3.2.2 Monitor the incubator, freezers and coolers daily for temperature.

3.3 Preparation of samples/reagents/control procedures

3.3.1 Allow the samples and reference cultures to warm to room temperature 

before rehydrating to the appropriate volume.

3.3.2 Negative and Positive Controls: Incubate 1 each of uninoculated tryptose 

and potato agar plates and 1 each of uninoculated dextrose Andrades and 

thioglycollate broth tubes with the test sample plates and tubes as negative 

controls. Dilute the B. abortus reference culture (positive control) and plate the 

same as the test samples.

3.3.3 Store plates used for making counts at refrigerator temperature. Allow the 

plates to be used for counts to dry under a biosafety cabinet before use. At the 

time of use, plates are no more than 30 days old.

4. Performance of the Test

4.1 Number all tubes, bottles, and plates.

4.2 Disinfect the top of the vials with 70% ethyl alcohol-soaked gauze pads. 

Reconstitute the desiccated products with enclosed sterile diluent, using the appropriate 

size syringe and needle. Mix until the vaccine cake is well dissolved.

4.3 Using a 10-mL syringe, remove approximately 10 mL of the vaccine from the vial 

of product. Place 0.2 mL each in 1 tube of dextrose Andrades broth and 1 tube 

thioglycollate broth. Place 0.1 mL onto a plate of potato agar. Transfer the rest of the 

product in the syringe to a sterile test tube.

4.4 With an inoculating loop, streak the 0.1 mL inoculum on the potato agar plate for 

bacterial isolation of individual colonies.

4.5 Mix the vaccine in the sterile test tube with a vortex mixer. Transfer 1.0 mL of the 

vaccine from the tube to a dilution bottle containing 99 mL of 1% peptone water using a 

sterile pipette. Mix the dilution bottle well by inversion.

4.6 Transfer 1.0 mL from the first 1% peptone water dilution bottle (10-2) to a second 

bottle containing 99 mL of 1% peptone water (10-4

). Mix the dilution bottle well by 

inversion.
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4.7 Transfer 1.0 mL from the second (10-4) 1% peptone water dilution bottle to a third 

dilution bottle containing 99 mL of 1% peptone water (10-6). Mix the dilution bottle well 

by inversion.

4.8 Transfer 10.0 mL from the third (10-6) 1% peptone water dilution bottle to a 

fourth dilution bottle containing 40 mL of 1% peptone water (10-7). Mix the dilution 

bottle well by inversion.

4.9 Plate 0.1 mL of the fourth dilution bottle onto each of 4 tryptose agar plates using 

a pipette.

4.10 Spread the inoculum over the surface of the plates using a sterile inoculum 

spreader.

4.11 Repeat Sections 4.3 through 4.10 with the second vial of the serial being tested.

4.12 Repeat Sections 4.3 through 4.10 with 1 reference or positive control vial of

B. abortus.

4.13 Invert all the agar plates (tryptose and potato agar) and incubate them and the 

dextrose Andrades and thioglycollate broths at 35°- 37°C for 4 days.

4.14 Negative (media) controls of both broths and agars are incubated along with the 

test.

4.15 Flood the plates of potato agar with crystal violet stain (see Appendix VI) for 20 

seconds after incubation. Remove stain from plates by using a pipette with a pipetting 

device and place the stain in a bottle containing disinfectant. Observe the plates with a 

colony counter or dissecting microscope.

4.16 After incubation, average the colony forming units (CFU) for the 4 tryptose agar 

plates (see Section 4.9) and divide by 10, which will give billion (B) CFU per dose 

(CFU/D) per vial. Determine the mean CFU/D for the number of vialstested.

5. Interpretation of the Test Results

5.1 Purity testing

5.1.1 A serial is considered satisfactory (SAT) for purity when, observed 

macroscopically, there is no abnormal growth or color change of the indicator in 

the dextrose Andrades broth and no abnormal growth characteristics in the 

thioglycollate broth, once compared to the reference control tubes.
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5.1.2 If abnormal growth is observed macroscopically in any tube, the serial or 

subserial may be retested using 4 new vaccine samples, provided that if the retest 

(RT) is not done, the serial or subserial is unsatisfactory (UNSAT) for purity. 

Gram stains are prepared from any abnormal growth for microscopic examination.

5.2 Potency testing

5.2.1 A serial is considered SAT for potency when the average count fromvials 

is between 3 and 10 B CFU/D of live B. abortus organisms.

5.2.2 If on the initial test the CFU/D is above 10 B CFU/D, the serial or subserial 

may be retested using 4 new vaccine samples, provided that if the retest is not done, 

the serial or subserial is UNSAT. If on the RT, the average count of

the 4 vaccine samples is above the required range, RT the serial or subserial every 

3 weeks until the average count drops below 10 B CFU/D.

5.2.3 If on the initial test the CFU/D is below 3 B CFU/D, the serial or subserial 

is RT with 4 vials. If the CFU/D of the retested vials is between 3 and 10 B 

CFU/D, the serial is SAT. If the CFU/D of the retest is below 3 B CFU/D, the 

serial is UNSAT.

5.3 Dissociation testing

5.3.1 A serial is considered SAT for dissociation when colonies on potato agar 

do not take the stain and remain white, an indication of smooth colony type.

5.3.2 A serial is considered UNSAT for dissociation if there are > 5% rough 

colony type or > 15% undesirable colony types which have stained red or purple.

6. Report of Test Results

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

7. References

Code of Federal Regulations, Title 9, Part 113.25, U.S. Government Printing Office, Washington, 

DC.
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8. Summary of Revisions

Version .03

• The Bacteriology Section Leader has been updated.

• Minor edits throughout document.

Version .02

• Reviewed 01Dec15, but no revisions needed.

Version .01

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:

• The document number has been updated from STSAM0600 to SAM 600.

• The Contact phone number has been updated.

• 2.1: The Bunsen burner has been removed.

• 3.1: Personnel qualifications have been clarified.

• 3.2.2/3.2.3: The references to internal documents have been removed.

• Appendix: Additional storage conditions for media have been added.
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Appendices

Appendix I

NCAH Media #10141

Dextrose Andrades broth

Beef infusion (double strength) 500.0 mL

QH2O 500.0 mL

Bacto peptone 10.0 g

Sodium chloride 5.0 g

Mix and adjust pH to 7.4 with 10% sodium hydroxide (NaOH). Autoclave 30 

minutes in flask at 121°C. Filter add:

1% dextrose 10.0 g

Regular Andrades indicator (0.5% stock solution) 10.0 mL

Read just pH to 7.4 with 10% NaOH. Autoclave 12 minutes at 121°C. Store at 

20°- 25°C for up to 30 days.

Appendix II

NCAH Media #10093

Tryptose agar

Tryptose agar 41.0 g

H2O 1000.0 mL

Autoclave at 121°C for 25 minutes. Cool in waterbath at 56°C. Store at 2°- 5°C 

for up to 30 days.

Appendix III

NCAH Media #10135

Fluid thioglycollate medium

Fluid thioglycollate medium 29.5 g 

QH2O 1000.0 mL

Mix and heat to boiling. Autoclave 20 minutes at 121°C. Store at 20°- 25°C for 

up to 30 days.
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 Appendix IV

NCAH Media #10452

Potato agar (powder)

Potato infusion agar 49.0 g

Glycerol 20.0 mL

QH2O 1000.0 mL

Stir and autoclave 20 minutes. Store at 2°- 5°C for up to 30 days.

 Appendix V

NCAH Media #10138

Peptone solution 1% + 0.5% NaCl

Bacto peptone 10.0 g

NaCl 5.0 g

QH2O 1000.0 mL

Autoclave for 20 minutes at 121°C. Store at 20°- 25°C for up to 30 days.

 Appendix VI

NCAH Media #30270

Crystal violet ammonium oxalate (stock sol) DBL

Sol 1:

Crystal violet 2.0 g

Absolute ethanol 20.0 mL

Mix until 

dissolved.

Sol 2:

Ammonium oxalate 0.8 g

QH2O 80.0 mL

Mix until dissolved, then mix Sol 1 and Sol 2 together to form a stock sol 

(should be stored in amber bottle at 20°- 25°C for up to 30 days). The stock sol 

is diluted 1:40 in distilled water just before use.