SAM 914.02

Page 1 of 9

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 914

Supplemental Assay Method for the Detection of Fungi in Antibody Products

Date: May 15, 2023

Number: SAM 914.02

Supersedes: SAM 914.01, May 2, 2017

Standard Requirement: 9 CFR 113.450(h)(2)(iii)

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.
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 2 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

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 sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References 

8. Summary of Revisions

Appendices
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 3 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

1. Introduction

This Supplemental Assay Method (SAM) describesthe test procedure used to detect 

fungi contamination in dried and liquid antibody products for oral administration, per title 

9, Code of Federal Regulations (9 CFR), part 113.450(h)(2)(iii). This test procedure uses 

Acidified Potato Dextrose agar (APDA) to detect fungi contamination.

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 Lab Armor® bead bath (set to 50°- 60°C)

2.1.3 Vortex mixer

2.1.4 20°-25°C incubator

2.1.5 Analytical balance

2.1.6 Colony counter

2.2 Reagents/supplies

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

2.2.1 Issatchenkia orientalis (American Type Culture Collection (ATCC)

#6258) or equivalent organism

2.2.2 Acidified Potato Dextrose agar (APDA) (Appendix I), National Centers

for Animal Health (NCAH) Media #50102 or as stated in the Outline of 

Production (OP) from the biologics manufacturer.

2.2.3 Tartaric Acid (TA) (Appendix II), NCAH Media #50103

Note: Tartaric acid is a corrosive. Wear appropriate personal protective 

equipment when handling.

2.2.4 Sterile water 
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 4 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

2.2.5 0.15M Phosphate Buffered Saline with 12% sucrose (PBS w/ 12% 

sucrose)

2.2.6 Sterile pipettes, individually packaged

2.2.7 Sterile petri dishes, 100 x 15-mm

2.2.8 Sterile culture tubes 

2.2.9 Lab coat or sterile sleeves and gloves

2.2.10 Spatulas

2.2.11 Weigh boats

2.2.12 Sterile wide mouth snap cap specimen containers

2.2.13 70% ethanol

2.2.14 4 x 4-inch sterile gauze pads

2.2.15 Pipetting aid

2.2.16 Micropipettors, 100-μL to 1.0-mL

2.2.17 Pipette tips, 100-μL to 1.0-mL

2.2.18 Sterile syringes with needles

2.2.19 Glass serum bottles, 2.0-mL

2.2.20 Rubber seals and metal caps for serum bottles

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel 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.
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 5 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

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 on the BSC at the beginning of the work week and leave on all week.

3.2.3 Turn on the bead bath at least one day before use to allow the temperature 

to equilibrate. 

3.2.4 Monitor temperature of incubators, freezers, coolers, and bead baths 

according to SOPs.

3.3 Preparation of reagents/control procedures

3.3.1 Warm the samples and reference culture to 20°- 25°C (room temperature) 

before rehydrating, if needed, to the volume listed on the vial or in the firm’s OP. 

Thaw frozen products in the BSC immediately before testing.

3.3.2 I. orientalis reference stock culture is prepared according to the

manufacturer’s instructions, suspended in PBS with 12% sucrose, mixed on a 

stir plate, 1.0 mL of culture dispensed into 2.0-mL glass serum vials, loosely 

capped, lyophilized, crimped, labeled, and then stored at -75°± 5°C. For each 

test session, inoculate a petri dish with 10-100 CFU/0.1 mL to serve as a positive 

control.

3.3.3 PBS w/ 12% sucrose: The PBS w/ 12% sucrose is prepared by mixing 440

mL of PBS and 60 g sucrose, dispensing 300 mL into a 500-mL flask, and 

sterilizing at 121°C for 20 minutes (min) or by following the manufacturer’s 

recommendations. Store at 20°- 25°C (room temperature) for 1 year.

3.3.4 Preparation of the APDA medium: On the day of the test, melt the APDA 

in an autoclave for 30 minutes at 100°C. Place the bottles of melted media in the

bead bath. Do not begin testing until the agar has cooled to at least 57°C. The 

temperature of the bead bath should be approximately 57°C when the agar is 

ready. Just prior to using the APDA medium, add 1 mL of TA/100 mL of 

molten APDA medium.

3.3.5 Negative/Technique Control: Inoculate one petri dish with 1 mL of the

water that was used as a diluent in the testing session. If no diluent is used in the

testing session, use sterile water packaged in serum vials as the inoculum. Pour 

15-20 mL of 52°- 57°C molten APDA into each plate, swirl gently to mix and

allow to solidify. Once the agar has hardened, invert the petri dish. Incubate this 

control plate with the serial test plates.
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 6 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

 3.4 Preparation of the sample

3.4.1 At least ten containers of final product are required for fungi purity

testing.

3.4.2 Samples are liquid or dried antibody products. When needed, sterile 

purified water in volumes specified on the product label or the diluent specified 

in the firm’s OP is used for rehydrating samples.

3.4.3 Weigh samples of product on an analytical balance, if needed. Rehydrate 

samples of the product according to label or instructions from the firm's OP. 

Dissolve samples by stirring on a stir plate or shake with a vortex mixer. If 

sample does not readily go into solution after manipulating, allow the diluted 

sample to sit in a 30°- 35°C incubator for 1 hour (hr) ± 30 minutes (min). 

 

3.4.4 Label 10 plates for each serial with the sample number or name and the 

container number.

 4. Performance of the Test

4.1 Dress in a clean lab coat or sterile sleeves and gloves to perform purity testing.

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

immediately prior to use and between testing each serial.

4.3 Place the necessary testing materials (micropipettors, pipet tips, 4 x 4-inch gauze 

squares, petri dishes, etc.) and the diluted (as stated in the firm’s OP) sample into the 

BSC.

4.4 Mix the sample thoroughly. Dispense 1 mL of inoculum from one sample 

container into a petri dish. 

4.5 Repeat Section 4.4 for the other nine sample containers for this serial.

4.6 Dispense 15-20 mL of 52°- 57°C molten APDA medium into each of the 10

dishes for this serial. Swirl gently to mix and then allow the agar to cool in the BSC.

Once the agar has hardened, invert the petri dishes and place the dishes in a 20°- 25°C 

incubator for 5 days. 

4.7 Repeat Sections 4.3 through 4.6 for the other serials of biologic to be tested.

4.8 Once all the test serials have been put on test, prepare the negative/technique

controls for the testing session (see Section 3.3.5). 
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 7 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

4.9 Once the purity portion of the test has been completed, prepare the positive 

control organism in an area that is separate and apart from the clean area where the purity 

test was conducted (see Section 3.3.2). 

4.9.1 Inoculate a petri dish with 10-100 CFU/0.1 mL of I. orientalis. 

4.9.2 Dispense 15-20 mL of 52°- 57°C molten APDA medium into the plate. 

Swirl gently to mix and then allow the agar to cool in the BSC. Once the agar has 

hardened, invert the petri dish.

4.10 All plates are incubated at 20°- 25°C for 5 days.

5. Interpretation of the Test Results

5.1 Criteria for a valid test:

5.1.1 There must be no growth in the negative/technique control and media.

control plates.

5.1.2 The positive control plate is examined for fungi characteristic growth and 

must contain a range of 10-100 CFU/0.1 mL. Characteristic growths are identified 

as creamy to white colony growth for yeasts and filamentous colony growth of 

various colors (like yellow, orange, red, etc.) for molds.

5.1.3 If these criteria are not met, the test is considered invalid or a no test (NT). 

Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory (UNSAT) sterility result for that product.

5.2 After incubation, all plates are examined macroscopically for typical fungi 

growth. If fungi characteristic colony growth (see Section 5.1.2) appears on the positive 

control plate and does not appear on any negative control plate, the test is valid. 

5.3 If no fungi colonies are detected on any of the sample agar plates, the serial is 

determined to be negative for fungi contamination and is satisfactory (SAT). If fungi

characteristic colony growth is observed on any of the sample agar plates, the test is 

considered inconclusive (INC), and one retest (RT; see Section 5.5) to rule out faulty 

technique, may be conducted. If a RT is not conducted within 21 days, the serial is 

UNSAT by the first test.

5.4 If growth is observed on the negative or media control plate(s), or no 

characteristic growth is observed on the positive control plate, the test is a NT and may

be repeated.
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 8 of 9 

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

5.5 If a RT is ordered, double the number of samples used in the first test are 

required. If fungi characteristic colony growth is observed on any of the RT plates, the 

serial is UNSAT. 

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs. 

7. References

Title 9, Code of Federal Regulations, part 113.450(h)(2)(iii), U.S. Government Printing 

Office, Washington, DC. 

Standard methods for the examination of dairy products. 1992, 16th Edition, Robert T. 

Marshall editor. American Public Health Association, Washington, DC. 

8. Summary of Revisions 

Version .02 

 The coversheet has been updated and document reformatted.
Center for Veterinary Biologics SAM 914.02

Testing Protocol Page 9 of 9

Supplemental Assay Method for the Detection of Fungi in Antibody Products 

Appendices

Appendix I

Acidified Potato Dextrose Agar (APDA) – National Centers for Animal Health (NCAH)

 Media #50102

 Potato Dextrose Agar 39.0 g

QH20 1000.0 mL

Tartaric acid 10% Solution 10.0 mL

(NCAH Media #50103)

For plates: Combine agar and water. Mix well. Autoclave 15 minutes at 121°C. Cool in a 

52°- 57°C water bath for a minimum of 1 hour. Aseptically add tartaric acid solution and 

mix well. Dispense. Store at 2°- 5°C for up to 6 months.

For bottles: Combine agar and water. Mix well. Bring to a boil and dispense. Autoclave 

15 minutes at 121°C. Prepare 100 mL of tartaric acid solution and deliver separately with 

bottles. Store at 2°- 5°C for up to 6 months.

Appendix II

Tartaric Acid (TA) – NCAH Media #50103

 Tartaric Acid 10.0 g

QH20 100.0 mL

Note: Tartaric acid is a corrosive. Wear appropriate personal protective equipment 

when handling.

In a low flow hood, combine acid and water. Mix well. Filter sterilize. Aseptically 

dispense. Label with a corrosive sticker. Store at 20°- 25°C (room temperature) for up to

a year.