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SAM 910.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 910

Supplemental Assay Method for Detection of Mycoplasma Contamination

Date: February 21, 2023

Number: SAM 910.05

Supersedes: SAM 910.04, February 16, 2017

Standard Requirement: 9 CFR Part 113.28

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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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 of reagents/control procedures

3.4 Preparation of the samples

4. Performance of the Test

5. Interpretation of the Test Results

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 procedures used to detect 

Mycoplasma contamination in live viral products, master cell stocks, and master seed viruses, as 

prescribed in title 9, Code of Federal Regulations (9 CFR), section 113.28. If Mycoplasma

contamination is present, colonies will form on the agar as seen under a stereoscope.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Laminar-flow Class II biosafety cabinet (BSC)

2.1.2 Dissecting stereoscope (35 – 100X magnification) 

2.1.3 33°- 37°C incubator (humidified 4-6% CO2) for agar plate incubation

2.1.4 33°- 37°C incubator for broth incubation and anaerobic jar

2.1.5 Anaerobic jar system (Oxoid AnaeroJar 3.5 L system and components)

2.1.6 Vacuum pump (Gast Manufacturing Corporation)

2.1.7 Schrader valve chuck fitted to a 24-inch length of rubber hose 

2.1.8 Compressed gas regulator

2.1.9 Vortex mixer

2.2 Reagents/supplies

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

2.2.1 Mycoplasma broth (Appendix I), National Centers for Animal Health 

(NCAH) Media #10162

2.2.2 Mycoplasma agar (Appendix II), NCAH Media #10167 

2.2.3 DPN/L cysteine (Appendix III), NCAH Media #30039

2.2.4 Mycoplasma hyorhinis (ATCC #17981) 
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2.2.5 Acholeplasma laidlawii (ATCC #23206)

2.2.6 Compressed anaerobic gas mixture (5% CO2 in 95% N2)

2.2.7 70% ethanol

2.2.8 1% Virkon disinfectant solution

2.2.9 Sterile water

2.2.10 Lab coat and gloves

2.2.11 Sterile gauze pads, 4 x 4-inch

2.2.12 Sterile syringes and needles

2.2.13 Sterile serological pipettes, various sizes

2.2.14 Pipet-aid

3. Preparation for the Test

3.1 Personnel qualifications/training

The 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. The personnel must also have knowledge of safe operating procedures,

policies, and guidelines; 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 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn the BSC on 30 minutes prior to the test session.

3.2.3 Monitor temperature of incubators, freezers, and coolers according to 

SOPs.
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3.3 Preparation of reagents/control procedures

Use M. hyorhinis and A. laidlawii as the indicator organisms to determine the growthpromoting qualities of the Mycoplasma broth and agar as required in the 9 CFR 

113.28(d)(4). Use M. hyorhinis as the positive control for the test procedure.

Note: Preferably, handle the control cultures after preparing the test samples and negative 

controls to prevent contamination of the samples being tested. If this is not possible, 

thoroughly disinfect the BSC and supplies before commencing to Section 4.

3.3.1 Thaw the frozen vials of M. hyorhinis and A. laidlawii stock cultures in 

the BSC. Rehydrate lyophilized stock cultures with Mycoplasma broth according 

to the reagent data sheet. Mix the stock cultures thoroughly by vortexing 

immediately prior to use.

3.3.2 Prepare working dilutions of the indicator organism stock cultures 

according to the reagent data sheet specifications. Mix the dilutions thoroughly by 

vortexing. Prepare a sufficient volume of each working dilution for each indicator 

organism (i.e., 9-20 mL volumes of each of the working dilutions).

3.3.3 Test the broth for growth-promoting qualities.

3.3.3.1 Inoculate each of 10 tubes containing 9 mL of Mycoplasma broth 

with 1 mL of the dilution that contains 10 CFU/mL of A. laidlawii.

3.3.3.2 Inoculate each of 10 tubes containing 9 mL of Mycoplasma broth 

with 1 mL of the dilution that contains 100 CFU/mL of A. laidlawii.

3.3.3.3 Incubate the cultures for 10 days at 33°- 37°C.

3.3.3.4 On day 10 of the test, read the test vessels as positive for growth if 

a dark precipitate is seen on the bottom of the vessel. Record growth as the 

number of positive per 10 vessels for each dilution.

3.3.4 Test the agar for growth-promoting qualities and verify the concentration 

of the M. hyorhinis working dilutions.

3.3.4.1 Inoculate four agar plates with 0.1 mL of the dilution of M.

hyorhinis that contains 10 CFU/mL.

3.3.4.2 Inoculate four agar plates with 0.1 mL of the dilution of M. 

hyorhinis that contains 100 CFU/mL.

3.3.4.3 Tilt the plates in a circular motion to allow the inoculum to 

flow over the surface. Make a short continuous “Z” streak across the 
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agar surface with a pipette. Incubate two plates of each dilution 

under anaerobic conditions (Appendix IV) and the other two 

plates in a humidified 4-6% CO2 incubator. 

3.3.4.4 Examine the agar plates after 10-14 days of incubation.

3.4 Preparation of the samples

3.4.1 Obtain sufficient Mycoplasma broth and agar as needed for the test.

3.4.2 Refer to the Outline of Production (OP) for specific testing instructions for 

each sample. 

3.4.3 Number the media test vessels and agar plates to coincide with the 

samples and controls to be tested.

4. Performance of the Test

4.1 Dress for the Mycoplasma test by wearing a lab coat and gloves.

4.2 Wipe down the interior surfaces of the BSC used for testing with 70% ethanol

prior to placing supplies, equipment, samples, control cultures, and media in the BSC.

4.3 Aseptically add 2 mL of DPN/L cysteine to each flask of Mycoplasma broth that 

will be used for testing.

4.4 Thaw frozen products immediately before testing. Immediately prior to opening a 

sample container, disinfect the surface of sample vials and ampules with a 4 x 4-inch 

gauze pad soaked in 70% ethanol in the BSC. Rehydrate lyophilized products according 

to the OP with Mycoplasma broth. Dilute poultry vaccines that are to be administered by 

drinking water with 30 mL Mycoplasma broth per 1,000 doses, or as indicated in the OP.

4.5 Mix the sample thoroughly and then place 1 mL of the product into a flask 

containing 100 mL of supplemented Mycoplasma broth. Close the flask and mix the 

culture thoroughly by swirling.

4.6 Place 0.1 mL of product on each of two agar plates. Tilt each plate in a circular 

motion to allow the inoculum to flow over the surface. Make a short continuous “Z” 

streak across the agar surface with a pipette. Leave the plates in the closed, upright 

position in the BSC until the inoculum has been absorbed.

4.7 Repeat the procedures in Sections 4.4 through 4.6 for the remaining samples to 

be tested.
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4.8 Prepare the control vessels for the test session.

4.8.1. For the negative control, incubate one flask containing 100 mL of

uninoculated Mycoplasma broth supplemented with DPN/L cysteine. To confirm 

purity of the supplemented broth at the initiation of the test, place 0.1 mL of 

uninoculated supplemented broth on each of two agar plates. Tilt each plate in a 

circular motion to allow the inoculum to flow over the surface. Make a short 

continuous “Z” streak across the agar surface with a pipette. Leave the plates in 

the closed, upright position in the BSC until the inoculum has been absorbed.

4.8.2. For the positive control, inoculate one flask containing 100 mL of 

Mycoplasma broth supplemented with DPN/L cysteine with 1 mL of the M. 

hyorhinis dilution that contains 100 CFU/mL.

4. 9 Incubate the test mediums.

4.9.1. Incubate all culture flasks from the testing session in a 33o- 37oC incubator 

for 14 days.

4.9.2. Incubate half of the agar plates inverted and covered in a humidified 4-6% 

CO2 incubator for 10-14 days.

4.9.3. Incubate the other half of the agar plates inverted in an anaerobic jar, 

prepared as described in Appendix IV, and placed in a 33°- 37°C incubator for 

10-14 days.

4.10 Subculture the broth cultures on post-inoculation days 3, 7, 10, and 14.

4.10.1. Handle the cultures in the following order to prevent cross-contamination: 

negative control, test samples, and positive control. 

4.10.2. Swirl each flask of broth immediately prior to dispensing to thoroughly 

mix the culture.

4.10.3. Place 0.1 mL of culture on each of two agar plates. Tilt each plate in a 

circular motion to allow the inoculum to flow over the surface. Make a short 

continuous “Z” streak across the agar surface with a pipette. Leave the plates in 

the closed, upright position in the BSC until the inoculum has been absorbed.

4.10.4. Incubate the mediums as indicated in Section 4.9.

4.11 At 10-14 days postinoculation, examine the agar plates for Mycoplasma colonies 

using a stereoscope at 35 – 100X magnification.
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4.12 After each test session, disinfect the interior of the BSC and counter tops with

70% ethanol. Discard the biological samples and any extra media according to SOPs. 

5. Interpretation of the Test Results

5.1 If Mycoplasma colony growth appears on any of the positive control plates and 

does not appear on any of the negative control plates, the test is valid. 

5.2 If no Mycoplasma colonies are detected on any of the sample agar plates, the

sample is determined to be negative for Mycoplasma contamination and is satisfactory 

(SAT).

5.3 If Mycoplasma colony growth is found on any of the sample agar plates, the 

sample is positive for Mycoplasma contamination and is unsatisfactory (UNSAT). 

5.4 If mold or bacteria is detected on the agar plates at any time during the test and 

the sample cannot be ruled out as the source of the contamination, a sterility test will be 

performed according to the appropriate SAM. This will add a 3-week delay before the test 

results are reported for the serial. If the sterility test indicates that the serial is contaminated, 

the Center for Veterinary Biologics (CVB) will report a No Test (NT) for the Mycoplasma

test and an UNSAT for the sterility test. The final serial disposition will be reported as 

UNSAT. 

6. Report of Test Results

Report the test results as described by SOPs. 

7. References

Title 9, Code of Federal Regulations, section 113.28, U.S. Government Printing Office, 

Washington, DC. 

8. Summary of Revisions 

Version .05 

• The coversheet has been updated.

Version .04 
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• The Bacteriology Section Leader, the CVB-PEL Director, and page 1 were updated.

• Updated Section 2. 

• Deleted approximately from the entire document. 

• Sections 3.3 and 5.4: Rewritten for clarification.

• Appendix I: Updated media storage limit.

Version .03

• Updated and reorganized Sections 2-5 to reflect current practices and procedures. 

• Appendix I and Appendix III: Updated storage limits and preparation protocols of 

media.

• Added Appendix IV to describe the use of an anaerobic jar to produce anaerobic 

conditions for Mycoplasma spp. growth. 

Version .02 

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. One significant change was made that might impact 

the designation of the serial but not the Mycoplasma test; the following changes were made to 

the document: 

• The Contact information has been updated. 

• 2.1: The Bunsen burner has been removed from the list of equipment that is needed 

for the test. The disinfectant has been removed from the list of equipment that is 

needed for the test. Clarification on the type of incubators needed has been added. 

• 2.2.5/2.2.6/2.2.7: The tubes, flasks, and petri dishes have been separated out into 

individual items and information on the sizes used has been added. 

• 3.3.2: The concentration of the inoculum used for testing has been clarified and the 

growth promotion testing from sample preparation has been separated into another 

statement. 

• 4.8: Preparation of the inoculum has been rewritten for clarity. 

• 4.13: The references have been clarified. 
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• 5.1: The test conclusions have been separated into individual items and clarified.

• 5.4: Follow-up testing (conducted in accordance with CVB Notice 09-02) has been 

added. This change will not impact the outcome of the Mycoplasma test but the 

follow-up sterility test could impact the overall disposition of the serial on test.

• Appendices: Media storage parameters have been added.
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Appendices

Appendix I

Mycoplasma (MG) Broth – National Centers for Animal Health (NCAH) Media #10162

Heart Infusion Broth 25 g

Proteose Peptone #3 10 g

Yeast Extract 5 mL

1% Thallium Acetate 25 mL

1% Tetrazolium Chloride 5.5 mL

Penicillin (100,000 U/mL) 5 mL

Horse Serum (Heat inactivate.) 100 mL

QH2O 970 mL

Adjust pH to 7.9 with 10% NaOH. 

Filter through 0.2-μm sterilized mini capsule filter and dispense 100 mL volumes into 

sterilized glass 125-mL flasks, 9 mL volumes in sterile 16 x 125-mm screw-capped tubes, 

and 30 mL volumes in sterile 50-mL screw-capped tubes.

Media must be prepared in a sterile room. Media may be stored up to 3 months at 

20°- 25°C.

Appendix II

Mycoplasma (MG) Agar – NCAH Media #10167 

Heart Infusion Agar 25 g

Heart Infusion Broth 10 g

Proteose Peptone #3 10 g

1% Thallium Acetate 25 mL

QH2O 995 mL

Heat to boiling. Cool and adjust pH to 7.9 with 10% NaOH. Autoclave 20 minutes.

Cool to 56oC and add:

Horse Serum (HI) 126 mL

Yeast Extract (Sterile) 5 mL

0.5% Penicillin 5.2 mL

1% DPN-Cysteine 21 mL

TOTAL (1152 mL)

Dispense 12 mL in sterile 15 x 60-mm petri dishes. Media must be prepared in a sterile 

room. Media may be stored up to a month at 20°- 25°C.
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Appendix III

DPN/L-Cysteine – NCAH Media #30039

Nicotinamide-adenine-dinucleotide (DPN, NAD) 5 g 

Q.S. H2O to 500 mL

L-Cysteine 5 g

Q.S. H2O to 500 mL

Mix each chemical separately until dissolved. Pour solutions together and let mix. Filter 

and dispense 10 mL volumes in sterile 16 x 150-mm screw-capped tubes. Media may be 

stored up to a year at -20°C.

Appendix IV

An anaerobic jar is used to maintain anaerobic conditions for a variety of fastidious

Mycoplasma species. The following procedures are used for the Oxoid AnaeroJar 3.5 L 

System:

1. Remove the clamp, lid with O-ring seal, and the plate rack from the anaerobic 

jar. Invert the agar plates and stack them in the plate rack. Be sure to use an even 

number of plates to keep them from tipping over inside the jar. An uninoculated 

agar plate may be used as an extra plate.

2. Place a small stack of 4 x 4 gauze pads moistened with water on the bottom of 

the jar to provide humidity during incubation. Place the plate rack with stacked

plates back into the anaerobic jar. Place the lid with O-ring seal onto the 

anaerobic jar and attach the clamp. Turn the knob on the clamp until it is handtight.

3. Attach the rubber hose to the vacuum port of the pump. Attach the end of the 

hose fitted with the Schrader valve chuck to the Schrader valve labeled “vacuum” 

on the lid of the anaerobic jar. The gage on the lid measures both vacuum and 

pressure. Vacuum is measured in inches of mercury (inHg) and are indicated by 

red numbers. Pressure is measured in pounds per square inch (psi) and is indicated 

by black numbers. The gauge should read zero (0).

4. Start the vacuum pump and allow the vacuum in the jar to reach 20 to 25 

inHg. Detach the Schrader valve from the jar lid and then turn off the vacuum 

pump.

5. Detach the hose from the vacuum pump port and attach it to the N2/CO2 gas 

regulator port. Turn on the N2/CO2 gas regulator valve and allow gas to start 

flowing, and then attach the Schrader valve chuck to the unmarked Schrader valve 

on the lid of the anaerobic jar.
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6. Allow the pressure of the anaerobic jar to return to 0. Detach the Schrader 

valve from the jar lid and then turn off the flow of gas.

7. Repeat Steps 3 through 6 twice.

8. Incubate the anaerobic jar in a 33°- 37°C incubator for 10 to 14 days.

9. After use, disinfect the jar and its components. Liberally spray the inside of 

the anaerobic jar lid with fresh 1% Virkon solution. Allow the disinfectant to soak

for 10 minutes. Rinse the lid with deionized water and air dry.

10. Fill the anaerobic jar with water and deposit two Virkon tablets in the water.

Place the plate rack rubber and O-ring seal in the jar. Allow the disinfectant to 

soak for 30 minutes. Rinse thoroughly with hot water, rinse with deionized water, 

and air dry.