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SAM 803.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 803

Supplemental Assay Method for Examination of Trypsin Solutions for 

Porcine Parvovirus Contamination

Date: November 28, 2014

Number: SAM 803.03

Supersedes: SAM 803.02, February 11, 2011

Standard Requirement: 9 CFR 113.53

Contact : Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals:

/s/Geetha B. Srinivas Date: 27Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Jan15

Rebecca L.W. Hyde, Section Leader

Quality Management

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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Supplemental Assay Method for Examination of Trypsin Solutions for Porcine Parvovirus Contamination

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Personnel qualifications/training

4. Performance of the Test

5. Report of Test Results

6. References

7. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test method that uses a cell 

culture system and uses different methods, e.g., immunofluorescence, hemagglutination, and 

aniline dye staining, to determine whether trypsin contains endogenous porcine parvovirus 

(PPV).

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 A supply (8 or more) of sterile plastic or glass tissue culture flasks of at 

least 75-cm

2

surface area each

2.1.2 At least 2 racks of sterile Leighton tubes with coverslips or 2 flats of Tech 

slides

2.2 Reagents/supplies

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

2.2.1 Porcine parvovirus susceptible cells, either primary embryonic or a swine 

cell line, which will grow out in 4 to 5 days

2.2.2 A certified contamination free bovine calf serum

2.2.3 A medium that has been found of sufficient nutriative value to grow a 

monolayer of primary swine kidney cells in 4 to 6 days. Suggested media are 

CMRL (Grand Island Biological Company) or Alpha MEM (Flow Laboratories), 

both supplemented with 1% sodium pyruvate and containing 50 mcg Gentimycin 

per mL.

2.2.4 Porcine parvovirus fluorescent antibody conjugate

2.2.5 An aniline dye stain that will demonstrate intranuclear inclusion bodies 

(e.g., May Gruenwald-Giemsa or Shorr’s)

2.2.6 Guinea pig red blood cells
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3. 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.

4. Performance of the Test

4.1 All operations involving trypsin, until it is inoculated, are conducted at ice bath 

temperature.

4.2 Five grams of trypsin are put into solution in a sufficient volume to fill the head of 

a high speed centrifuge. For example, 5 gms in 162.0 mL (a 3.09% solution) will fill the 

50 Ti head of a Spinco L-2 Sterile rabbit serum at 2% level may be used to mark the 

pellet area and provide a cushion.

4.3 The filtered solution is then filled into the centrifuge tubes and centrifuged at 

80,000 x g for 1 hour. The supernatant material is decanted and discarded. The area 

where the pellet would be normally found in each tube is scraped with a small sterile 

metal spatula into 0.2 to 0.3 mL of sterile distilled water. The material is aspirated with a 

sterile 10-mL syringe with an attached 6-inch metal cannula and all material is pooled.

4.4 The reconstituted material is divided into 2 equal aliquots and inoculated into 2 

flasks containing freshly seeded swine cells. The containers are then incubated until 

confluent monolayers form (4 to 7 days). Two cell control flasks are set up in parallel.

4.5 After the cells are confluent, the cells in both the inoculated and the control flasks 

are scraped off with a rubber policeman or removed by pre-tested clean trypsin and the 

material pooled as to test material or control. Further dispersion of the cells is made by 

forcing them through a syringe and 20-gauge needle. When it is observed that the cells 

are well broken apart, they are centrifuged at slow speed and reconstituted in fresh media 

to original volume and 2 fresh flasks reseeded as well as 10-12 Leighton tubes or 5-6.

Tech slides for both inoculated and controls Incubation is as before.

4.6 The 3rd day postseeding and every day thereafter, 2 test coverslips or 1 test Tech 

slide and 2 control coverslips or 1 Tech slide are stained with anti-porcine parvovirus 

fluorescent antibody conjugates. This is continued until the flasks are ready to subculture.

If typical nuclear staining is observed, the fluid from the inoculated flasks is saved when 

the subculture is made, and PPV may be further verified by a hemagglutination test.
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5. Report of Test Results

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

6. References

Title 9, Code of Federal Regulations, section 113.51, U.S. Government Printing Office, 

Washington, DC.

7. Summary of Revisions

Version .03

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .02

 The Contact information has been updated