Center for Veterinary Biologics SAM604.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 604

Supplemental Assay Method for the Evaluation of Koch’s Old Tuberculin

Date: June 17, 2022 

Number: SAM 604.05 

Supersedes: SAM 604.04, February 8, 2016 

Standard Requirement: 9 CFR 113.406 

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 the Evaluation of Koch’s Old Tuberculin

Table of Contents

1. Introduction

2. Materials

2.1 Reagents/supplies

2.2 Animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Selection and handling of test animals

3.3 Preparation of reagents 

3.4 Preparation of supplies

3.5 Test animal sensitization

4. Performance of the Potency Test

 

4.1 Preparation of guinea pigs for the potency assay 

4.2 Preparation of tuberculin dilutions

4.3 Intradermal injection of test animals

5. Interpretation of the Test Results

5.1 Recording of test results

5.2 Criteria for a valid test

6. Reporting of Test Results

 

7. References

8. Summary of Revisions 

Appendix 

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Supplemental Assay Method for the Evaluation of Koch’s Old Tuberculin

1. Introduction

This is a Supplemental Assay Method (SAM) for the evaluation of production lots of Koch’s old 

tuberculin in accordance with title 9, Code of Federal Regulations (9 CFR), part 113.406. 

2. Materials

2.1 Reagents/supplies

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

2.1.1 Mycobacterium tuberculosis reference tuberculin, current lot. This 

reference is obtained from the United States Department of Agriculture (USDA), 

Animal and Plant Health Inspection Service (APHIS), Veterinary Services (VS), 

Center for Veterinary Biologics (CVB).

2.1.2 M. tuberculosis sensitizing agent, current lot. This reagent is available 

from the CVB.

2.1.3 Saline solution, 0.85% 

2.1.4 Validated digital calipers or metric ruler made of clear plastic

2.1.5 Needles, 20-gauge x 1-inch and 26-gauge x 3/8-inch

2.1.6 Disposable syringes, 1-mL and 3-mL 

2.1.7 Pipettes, 1-mL, 2-mL, 5-mL and 25-mL 

2.1.8 Glass serum bottles, 10-mL and 125-mL 

2.1.9 Rubber seals and metal caps for serum bottles 

2.1.10 Crimper for aluminum seals

2.1.11 Depilatory cream

2.1.12 Animal clippers, equipped with a sharpened #40 or #50 blades, or 

equivalent 

2.1.13 Ear tags for small animals and ear tag applicator 

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Supplemental Assay Method for the Evaluation of Koch’s Old Tuberculin

2.1.14 Sprayola livestock paint and applicator 

2.1.15 Cage cards

2.2 Animals

Guinea pigs, 500-700 g, white-haired, nonpregnant females. Ten guinea pigs are required 

for each lot to be tested. Two additional guinea pigs are required as nonsensitized 

controls. All guinea pigs used for a test must be from the same source and housed and fed 

in the same manner.

3. Preparation for the Test

3.1 Personnel qualifications/training

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

chemicals, equipment, and glassware and have specific training and experience in the 

safe handling of laboratory animals. 

3.2 Selection and handling of test animals

3.2.1 Select guinea pigs that are healthy, free of external parasites, and have an 

unblemished hair coat. 

3.2.2 Examine guinea pigs the day they are received, and house according to 

standard operating procedures. 

3.2.3 When the test is concluded, instruct the animal caretakers to euthanize the 

guinea pigs, unless they can be used as second use animals for additional testing 

or blood collection. 

3.3 Preparation of reagents

3.3.1 Saline solution, 0.85% (National Centers for Animal Health (NCAH) 

Media #30201) 

Sodium chloride 8.5 g 

Water q.s. to 1.0 L 

Autoclave at ≥121°C for 15 minutes. Store at 20°- 25°C for no longer than 

six months. 

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3.4 Preparation of supplies

3.4.1 Sterilize all glassware before use.

3.4.2 Use only sterile supplies (syringes, needles, rubber seals, metal caps, etc.).

3.5 Test animal sensitization

3.5.1 Allow the Sensitinogen to warm up to room temperature before use. 

3.5.2 Sensitize 10 guinea pigs per lot of tuberculin to be evaluated. Wait 30 to 

120 days before performing the potency portion of the assay. 

3.5.3 Administer 0.5 mL of M. tuberculosis sensitizing agent intramuscularly to 

each guinea pig. Split the dose, administering 0.25 mL into each rear leg. Use 

3-mL syringes fitted with 20-gauge x 1-inch needles. Identify the sensitized 

guinea pigs by cage cards or equivalent. 

3.5.4 Retain 2 guinea pigs as nonsensitized controls. 

4. Performance of the Potency Test

4.1 Preparation of guinea pigs for the potency assay

Clip the abdomen of each guinea pig with animal clippers. Generously apply a depilatory 

cream to the clipped abdomen. Wait at least 3 minutes. Wash off the depilatory cream 

with warm water within 10 minutes of application and dry the abdomen with a soft towel. 

Allow the guinea pigs to rest for a minimum of 4 hours before administering the 

tuberculin injections. 

4.2 Preparation of tuberculin dilutions

4.2.1 Preparation of control dilutions 

1. Vortex the bottle several times to mix. Remove 0.5 mL of reference 

tuberculin from the bottle using a 1-mL pipette. Add tuberculin to a 10-

mL serum bottle containing 1.5 mL saline, to create a 1:4 dilution. Cap the 

bottle, label, and mix well by vortexing. 

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2. Add 1 mL of the 1:4 dilution of tuberculin, measured in a 1-mL pipette, 

to a 10-mL serum bottle containing 1.5 mL of saline to create a 1:10 

dilution. Cap the bottle, label, and mix well by vortexing. 

3. Repeat Steps 1 and 2 with the lot of tuberculin to be tested. 

4.2.2 Preparation of assay dilutions

Note: Prepare dilutions of the reference tuberculin and the lot to be tested in 

exactly the same manner.

1. Dispense 99 mL of saline into a 125-mL serum bottle. Add 1.0 mL of 

reference tuberculin, measured in a 1-mL pipette. This is a 1:100 dilution. 

Cap and label the bottle. Mix well by vortexing. 

2. Label 2, 10-mL serum bottles 1:200 and 1:400, respectively. Use a 

5-mL pipette to add 5.0 mL of saline to each of the bottles labeled 1:200 

and 1:400. 

3. Use a 5-mL pipette to add 5.0 mL of the 1:100 dilution prepared in 

Step 1 into the bottle labeled 1:200. Close the bottle with a rubber stopper. 

Mix well by vortexing. 

4. Use a 5-mL pipette to transfer 5.0 mL of the 1:200 dilution into the 

bottle labeled 1:400. Close the bottle with a rubber stopper. Mix well by 

vortexing. 

5. Clamp aluminum seals over the rubber stoppers on the 3 serum bottles 

containing the diluted tuberculin. 

6. Repeat Steps 1-5 with each lot of tuberculin to be tested. 

4.3 Intradermal injection of test animals

4.3.1 Identify 6 injection sites on the abdomen of each sensitized guinea pig, 

with 3 sites on each side, evenly spaced, equidistant from the midline. (See 

sample worksheet in the Appendix.) Do not mark the sites on the abdomen with 

ink. 

4.3.2 Each sensitized guinea pig will receive a total of 6 injections, 1 at each 

previously identified site as recorded on the worksheet. Each preparation (1:100, 

1:200, or 1:400 dilution of the reference tuberculin; or 1:100, 1:200, or 1:400 

dilution of the test tuberculin) will be injected into each site. 

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4.3.3 Administer 0.05 mL of each preparation at the sites by intradermal 

injection. Needles must be sharp and it is recommended that they are replaced 

after insertion through a rubber stopper or after injecting 10 guinea pigs. Use 

1-mL tuberculin syringes fitted with 26-gauge x 3/8-inch needles. 

4.3.4 Identify 4 injection sites on each nonsensitized (control) guinea pig, 

equidistant, 2 on each side of the midline. (See sample worksheet in the 

Appendix.) Do not mark the sites on the abdomen with ink. 

4.3.5 Each nonsensitized (control) guinea pig will receive a total of 4 injections, 

1 at each previously identified site. Each preparation (1:4 or 1:10 dilution of the 

reference tuberculin; or 1:4 or 1:10 dilution of the test tuberculin) will be injected 

into each site. 

4.3.6 Administer 0.05 mL of each preparation by intradermal injection at the 

sites determined on the sample worksheet in the Appendix. Use 1-mL tuberculin 

syringes fitted with 26-gauge x 3/8-inch needles. 

4.3.7 Guinea pigs are identified by small animal ear tags. Alternatively, mark 

guinea pigs with Sprayola to differentiate them. Animals may be marked with 

different colors on the head, back, rear, or foot for identification.

5. Interpretation of the Test Results

5.1 Recording of test results

5.1.1 Measure the test reactions at 24 hours following injection.

5.1.2 Measure the greater and lesser diameters of erythema and/or swelling to 

the closest mm at each injection site. Gently palpate the lesion to determine the 

margin of the swelling, which may or may not extend beyond the margin of 

erythema. Record the results. 

5.1.3 Calculate the area of erythema and/or swelling (in mm2

) by multiplying 

the greater and lesser diameter measurements.

5.1.4 For each dilution of tuberculin tested, add together the reaction areas for 

that dilution from each of the 10 guinea pigs. Record the sum reaction for each 

dilution. 

5.1.5 Add together the sums obtained for each dilution of the reference 

tuberculin to generate the grand total for the reference tuberculin. Similarly 

calculate a grand total for the test serial. Record these results.

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5.1.6 Divide the grand total for the test serial by the grand total for the reference 

tuberculin. Multiply by 100 to express the test serial reaction as a percentage of 

the reference tuberculin reaction. Record all calculations.

5.2 Criteria for a valid test

5.2.1 The control guinea pigs must have no signs of reaction at 24 hours. 

5.2.2 For a satisfactory serial, the reaction of the test serial must be 25% of the 

reaction of the reference tuberculin, i.e., the percentage calculated in Section 

5.1.6 must fall between 75% and 125%. 

6. Reporting 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 113.406, U.S. Government Printing 

Office, Washington, DC. 

7.2 History of reagents: The M. tuberculosis sensitizing agent is a moist, heat-killed 

preparation of M. tuberculosis strains Pn, C, and DT. These strains were originally 

obtained from Cooper Animal Health. M. tuberculosis reference tuberculin was obtained 

from the USDA, APHIS, VS, NVSL, Ames, IA 50010. 

8. Summary of Revisions 

Version .05

• Updated coversheet and contact information. 

Version .04

• Updated Section Leader and Director. 

• Alternative guinea pig identification method added. 

Version .03

• The Contact information has been updated. 

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• 2.1.3: The formula for sterile saline (NCAH Media# 30201) does not require a pH.

• 2.1.7: 2-mL pipettes have been added to the list of reagents/supplies as they are also 

used to prepare dilutions. 

• 2.1.12: #50 clipper blades have been added to the list of reagents/supplies as they are 

also used to remove hair from the abdomen of the guinea pigs. 

• 3.3.1: Adjusting the pH level has been removed because Saline solution, 0.85% 

(NCAH Media #30201) does not list a pH in the formula. 

• 3.5.1: The Sensitinogen is warmed up to room temperature before use to aid in 

mixing of the reagent. 

• 4.2: Syringes are no longer used to prepare dilutions and have been replaced by 

pipettes.

• 4.3.3: The entire needle length is no longer being used for each injection as inserting 

the needle just past the beveled edge is the correct depth into the dermis.

• All references to the National Veterinary Services Laboratories (NVSL) have been 

changed to the National Centers for Animal Health (NCAH) throughout the 

document. 

Version .02 

• The Contact has been changed from Charles Egemo to Janet Wilson.

• 2.1.4: The phrase “Validated digital calipers” has been added,

• 2.1.7: One-mL pipettes have been added to the list of equipment.

• 2.1.12: The phrase “equipped with a sharpened #40 blade, or equivalent” has been 

added.

• 2.1: “Ear tags for small animals” and “Cage cards” have been added to the list of 

reagents and supplies.

• 3.2: This section has been revised to reflect current practices.

• 3.3.1: The phrase “Store at 20°- 25°C for no longer than six months” has been added.

• 4.1/4.2/4.3: These sections have been revised to reflect current practices.

• 5.1: This section has been revised to reflect current practices.

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Appendix

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