Center for Veterinary Biologics SAM 636.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 636

Supplemental Assay Method for the Evaluation of Tuberculin Purified 

Protein Derivative (PPD)

Date: June 17, 2022

Number: SAM 636.05 

Supersedes: SAM 636.04, May 27, 2016 

Standard Requirement: 9 CFR 113.409 

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 Tuberculin Purified Protein Derivative (PPD)

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 Tuberculin Purified Protein Derivative (PPD)

1. Introduction

This is a Supplemental Assay Method (SAM) for the evaluation of production lots of tuberculin 

purified protein derivative (PPD) in accordance with title 9, Code of Federal Regulations, 

(9 CFR), part 113.409. 

2. Materials

2.1 Reagents/supplies

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

2.1.1 Reference PPD tuberculin, current lot. This reference is obtained from the 

United States Department of Agriculture, Animal and Plant Health Inspection 

Service, Veterinary Services, National Centers for Animal Health (NCAH). 

2.1.2 Mycobacterium bovis sensitizing agent, current lot. This reagent is 

available from the NCAH. 

2.1.3 M. avium sensitizing agent, current lot. This reagent is available from the 

NCAH. 

2.1.4 Phosphate buffer, PPB II 

2.1.5 Validated digital calipers or metric ruler made of clear plastic

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

2.1.7 Disposable luer-locking syringes, 1-mL and 3-mL 

2.1.8 Pipettes, 1-mL, 2-mL, 5-mL, 10-mL and 25-mL 

2.1.9 Glass serum bottles, 20-mL and 30-mL 

2.1.10 Rubber seals and metal caps for serum bottles 

2.1.11 Crimper for aluminum seals

2.1.12 Depilatory cream, Nair®

2.1.13 Animal clippers, equipped with a sharpened #40 or #50 blade 

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Supplemental Assay Method for the Evaluation of Tuberculin Purified Protein Derivative (PPD)

2.1.14 Ear tags for small animals

2.1.15 Ear tag applicator 

2.1.16 Sprayola animal paint and applicator 

2.1.17 Cage cards

2.2 Animals

Guinea pigs, 500-700 g, white-haired, nonpregnant females. Twenty-three guinea pigs are 

required for each lot to be tested. For each group of unknowns to be tested 

simultaneously, 20 guinea pigs are required for the reference PPD. 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. They must have experience in the performance of 

this assay.

3.2 Selection and handling of test animals

3.2.1 Select guinea pigs that are healthy and 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 the 

current 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. 

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Supplemental Assay Method for the Evaluation of Tuberculin Purified Protein Derivative (PPD)

3.3 Preparation of reagents

Phenolized Phosphate Buffer Number 2 (PPB II) – NCAH Media #10528 

Na2HPO4 1.89 g 

KH2PO4 0.36 g 

NaCl 8 g 

Distilled water 900 mL 

Adjust pH to 7.2 ± 0.1. Autoclave at ≥121°C for 20 minutes. When cool, add: 

5% phenol (in water) 100 mL 

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 Sensitize 20 guinea pigs per lot of PPD to be evaluated. Sensitize 10 of the 

animals with M. bovis sensitinogen, and sensitize the remaining 10 animals with 

M. avium sensitinogen. For each group of PPD lots that will be tested 

simultaneously, similarly sensitize 20 additional guinea pigs to be used for testing 

the M. bovis reference PPD. Wait 35 days before performing the potency portion 

of the assay.

3.5.2 Administer 0.5 mL of the respective sensitinogen 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. 

3.5.3 Retain 3 guinea pigs as nonsensitized controls for each unknown lot of 

PPD to be tested. Unsensitized controls are not necessary for the reference PPD.

4. Performance of the Potency Test

4.1 Preparation of guinea pigs for the potency assay

Clip the entire 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 

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towel. Allow the guinea pigs to rest for at least 4 hours before administering the 

tuberculin injections. 

4.2 Preparation of tuberculin dilutions

Preparation of assay dilutions:

4.2.1 Determine the protein concentration for the serial using the current version 

of SAM 513. The concentration must be 1 mg/mL, so a predilution may need to 

be made before preparing the following dilutions. Make 4 dilutions of the test lot 

of PPD, using sterile PPB II as the diluent. Dilute to achieve final concentrations 

of 0.6, 1.2, 2.4, and 4.8 μg protein per 0.1-mL dose, respectively as indicated 

below. Place each dilution in a serum bottle. Cap and label the bottles. 

Final concentration (μg 

protein per 0.1-mL dose)

 Contents of serum vial

 4.8 1.2 mL of 1 mg/mL aliquot + 23.8 mL PPB II

 2.4 10 mL of 4.8 μg protein per 0.1-mL dose

dilution + 10 mL PPB II

 1.2 5 mL of 4.8 μg protein per 0.1-mL dose

dilution + 15 mL PPB II

 0.6 2.5 mL of 4.8 μg protein per 0.1-mL dose

Dilution + 17.5 mL PPB II

4.2.2 Repeat Step 1 with each additional serial of PPD to be tested and with the 

M. bovis reference PPD. Determine the protein concentration for the M. bovis

reference. The concentration must be 1 mg/mL, so a predilution may need to be 

made before preparing the 4 dilutions listed above. 

4.3 Intradermal injection of test animals

4.3.1 Identify 4 injection sites on the abdomen of each guinea pig, with 2 sites 

on each side, equidistant from the midline (see the Appendix). Sites must be 

spaced sufficiently far apart to avoid overlapping of subsequent skin reactions. 

Do not mark the sites on the abdomen with ink. Randomly assign and record 

which dilution (0.6, 1.2, 2.4, or 4.8 μg protein per 0.1-mL dose) will be injected 

into each site. 

4.3.2 Inject each sensitized guinea pig at the 4 defined injection sites inserting 

the length of the bevel on a 3/8-inch needle. Inject individual guinea pigs with 

only a single lot of PPD, 1 site per dilution, as prepared in Section 4.2.1. Needles 

must be sharp to perform ideal intradermal injections and it is recommended that 

needles be replaced after insertion through a rubber stopper or after injecting 10 

guinea pigs. 

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Supplemental Assay Method for the Evaluation of Tuberculin Purified Protein Derivative (PPD)

1. Insert ear tags in each guinea pig for identification purposes. 

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. Administer each preparation intradermally using 1-

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

2. For each serial of PPD to be tested, inject 10 guinea pigs sensitized 

with M. bovis and 10 guinea pigs sensitized with M. avium. Similarly 

inject 3 nonsensitized guinea pigs. 

3. Inject the M. bovis reference PPD into each of 10 guinea pigs sensitized 

with M. bovis and 10 guinea pigs sensitized with M. avium. 

5. Interpretation of the Test Results

5.1 Recording of test results

5.1.1 Measure the test reactions in good lighting 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 Determine the total area of erythema and/or swelling for each guinea pig 

by adding the areas of the 4 injection sites. 

5.1.5 Add together the totals from Section 5.1.4 for all guinea pigs with the 

same sensitization and same PPD tuberculin injection. Then divide by the number 

of guinea pigs in that treatment group to determine the average response per 

guinea pig to the given PPD tuberculin for the given type of sensitization. 

5.1.6 Calculate the specificity index of each lot of PPD tuberculin by 

subtracting the average response obtained on M. avium-sensitized guinea pigs 

injected with that lot from the average response of M. bovis-sensitized guinea pigs 

injected with that lot. Record all calculations. 

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Supplemental Assay Method for the Evaluation of Tuberculin Purified Protein Derivative (PPD)

5.2 Criteria for a valid test

5.2.1 The specificity index of the M. bovis reference PPD must be at least 

400 mm2

 for a valid test. 

5.2.2 The test lot of PPD tuberculin is unsatisfactory if erythema and/or 

swelling is noted at the injection site(s) of 1 or more of the 3 unsensitized guinea 

pigs injected with that lot. 

5.2.3 If the test is valid and no reactions are observed in unsensitized guinea 

pigs, each lot of PPD tuberculin is classified according to the following table: 

Specificity Index Classification

440 mm2 or greater Satisfactory

360-440 mm2 Inconclusive

Less than 360 mm2 Unsatisfactory

5.2.4 If a lot of PPD tuberculin is inconclusive, perform a second-stage test. If a 

second-stage test is not performed, the lot is unsatisfactory. 

1. Conduct the second-stage test in a manner identical to the first, except 

omit the unsensitized control guinea pigs. 

2. Combine the results obtained on all guinea pigs in stages 1 and 2. 

Calculate the average response on the 20 guinea pigs sensitized with each 

antigen and calculate the specificity index.

3. If the cumulative specificity index is ≥ 400 mm2

, the lot of PPD 

tuberculin is satisfactory; if it is < 400 mm2, the lot is unsatisfactory. 

6. Reporting of Test Results

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

7. References

Title 9, Code of Federal Regulations, part 113.409, U.S. Government Printing Office, 

Washington, DC. 

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Supplemental Assay Method for the Evaluation of Tuberculin Purified Protein Derivative (PPD)

8. Summary of Revisions 

Version .05

• Updated coversheet and contact information. 

Version .04

• The Section Leader and Director information has been updated. 

• Alternative guinea pig identification method added. 

Version .03

• The Contact information has been updated. 

• 2.1.7: Disposable syringes, 5-mL and 10-mL have been removed to reflect the 

syringe size used for sensitizing guinea pigs. 

• 2.1.8: Pipettes, 2- mL and 5- mL, have been added to the list of reagents/supplies to 

reflect the other volumes measured while preparing the tuberculin dilutions.

• 2.1.13: Animal clippers equipped with a sharpened #50 blade have been added since 

it is also used for better hair removal on the guinea pigs. 

• 4.3.2: 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.

• 2.1.1/2.1.2/2.1.3: The source of M. bovis Reference PPD tuberculin, M. bovis

sensitizing agent, and M. avium sentizing agent has been changed to reflect the 

current supplier. 

• 2.1.5: Validated digital calipers have been added. 

• 2.1.7: Five-mL and 10-mL disposable syringes have been added. 

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• 2.1.8: One-mL and 10-mL pipettes have been added. 

• 2.1.9: Thirty-mL glass serum bottles have been added. 

• 2.1.13: The phrase “with a sharpened” has been added. 

• 2.1.14/2.1.15/2.1.16: Ear tags for small animals, ear tag applicator, and cage cards 

have been added to the list of reagents/supplies. 

• 3.2.2: The phrase “Animal Users’ Manual” has been replaced by “standard operating 

procedures”. 

• 3.2.3: This section has been changed to reflect more current procedures. 

• 3.3.1: The phrases “Phenolized” and “Buffer Number 2 (PPB-2)” have been added. 

The NVSL Media number has been changed to #10528. 

• 3.5.1: The phrase “M. bovis” has been added. 

• 3.5.2: The phrase “Identify the sensitized guinea pigs by cage cards or equivalent” 

has been added.

• 4.1: The phrase “wait at least 10 minutes” has been changed to “wait at least 3 

minutes”. The phrase “within 10 minutes of application” has been added. 

• 4.2/4.3: These sections have been updated to reflect current procedures. 

• 4.3.1 Removed worksheet BBFRM0002 and added “the current” worksheet. 

• 5.1.1: The phrase “in good lighting” has been added and the section has been 

updated to reflect current procedures. 

• 5.2.1: The phrase “M. bovis” has been added. 

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Appendix

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