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SAM 409.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 409

Supplemental Assay Method for Titrating the Fractions of Combination 

Avian Encephalomyelitis/Pox Vaccine

Date: November 28, 2014

Number: SAM 409.05

Supersedes: SAM 409.04, March 11, 2014

Standard Requirement: 9 CFR 113.325 and 113.326

Contact: Sandra K. Conrad, (515) 337-7200

Debra R. Narwold

Approvals: /s/Geetha B. Srinivas Date: 15Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 15Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 15Jan15

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 Titrating the Fractions of Combination Avian Encephalomyelitis/Pox 

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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 sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for Titrating the Fractions of Combination Avian Encephalomyelitis/Pox 

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1. Introduction

This Supplemental Assay Method (SAM) describes a procedure for titrating a vaccine containing 

both avian encephalomyelitis (AE) vaccine virus and avian pox vaccine virus. The vaccine is 

composed of separate preparations of each virus that are mixed together with a suitable 

stabilizer.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Centrifuge (Beckman J6-MI, JS-4.2 rotor)

2.1.2 Humidified, rotating egg incubator (Midwest Incubators, Model No. 252)

2.1.3 Vortex mixer (Thermolyne Maxi Mix II, Model No. M37615)

2.1.4 Microliter pipette (Rainin Pipetman, P1000, or equivalent)

2.1.5 Cool-lite tester (Val-A)

2.1.6 Egg candling light on stand (Speed King)

2.1.7 Etcher electric engraver (Vibro-graver Acme Burgess, Inc.)

2.1.8 Glass 50-mL centrifuge tube, sterile (Kimax)

2.2 Reagents/supplies

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

reagents and supplies must be sterile.

2.2.1 Cotton swabs/cotton balls

2.2.2 Serological pipettes (Falcon)

2.2.3 Pipette tips (Rainin Clean-Pak disposable microliter pipette tips RT-200)

2.2.4 Syringe, 1-cc tuberculin, single use (Becton, Dickinson and Company)
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2.2.5 Hypodermic needle, 26-gauge x 3/8-inch (Becton, Dickinson and 

Company)

2.2.6 Hypodermic needle, 22-gauge x 1 1/2-inch (Becton, Dickinson and 

Company)

2.2.7 Glass test tubes, 16 x 125-mm with Morton closures

2.2.8 Duco cement

2.2.9 1,1,2-Trichloro 1,2,2-Trifluoroethane (Freon)

2.2.10 Chick embryos from specific-pathogen-free (SPF) source 

1. Use 5- to 6-day-old embryos for the AE titration.

2. Use 9- to 11-day-old embryos for the pox titration.

2.2.11 Solutions

1. Tryptose Phosphate Broth (TPB)

TPB 29.5 g

q.s. with distilled or deionized water (DW) 1000.0 mL

Sterilize by autoclaving

2. Penicillin/Streptomycin (pen/strep)

penicillin g 500 units/mL

streptomycin 2 mg/mL

q.s. with DW 1000.0 mL

Sterilize by filtration.

3. 70% alcohol

ethyl alcohol 70 mL

q.s. with DW 30 mL
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4. Iodine, 2% in alcohol

iodine 2 g

ethyl alcohol (70%) 100 mL

2.2.12 Sterile DW

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 and training in the operation of 

the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

Operate all equipment/instrumentation according to manufacturers’ instructions, and 

monitor in compliance with current corresponding standard operating procedures or 

equivalent.

3.3 Preparation of reagents/control procedures

Prepare reference viruses in the same manner as sample preparation.

3.4 Preparation of the sample

3.4.1 AE

Rehydrate 500 doses of vaccine with 10.0 mL sterile purified water. Mix 

thoroughly. Transfer 1.0 mL of this vaccine to a 50-mL sterile glass centrifuge 

tube containing 9.0 mL sterile purified water. Mix thoroughly. Add 10.0 mL

Freon. Mix on a Vortex mixer for 3 separate 30-second intervals. Centrifuge at 

600 X g for 10 minutes. The aqueous phase is considered the 100concentration of 

virus and contains 1 dose in 0.2 mL. Transfer 0.5 mL of the aqueous phase (upper 

layer/supernate) to a test tube containing 4.5 mL sterile purified water. Make 

further tenfold dilutions through 10-5using sterile purified water.
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3.4.2 Pox

Rehydrate 500 doses of vaccine with 10.0 mL sterile purified water. Mix 

thoroughly. Transfer 0.5 mL of this vaccine to a sterile test tube containing 

4.5 mL of TPB. This is considered the 100concentration and contains 1 dose in 

0.2 mL. Make further tenfold dilutions, transferring 0.5 mL vaccine to 4.5 mL

TPB, up through 10-6.

4. Performance of the Test 

4.1 AE

Inoculate dilutions 10-1through 10-5into the yolk sac using 10 embryos per dilution. 

Inoculate 0.2 mL per embryo. Also have 20 uninoculated negative controls. Incubate the 

embryos and calculate the titer according to the criteria specified in title 9, Code of

Federal Regulations (9 CFR), section 113.325(c)(2)(i).

4.2 Pox

Inoculate dilutions 10-2through 10-6onto the dropped chorioallantoic membrane (CAM) 

using at least 6 embryos per dilution. Inoculate 0.2 mL per embryo. Incubate the embryos 

and calculate the titer according to the criteria specified in 9 CFR 113.326.

5. Interpretation of the Test Results

5.1 Controls

Titrate a known positive reference virus with each group of titrations. The titer of the 

positive reference virus must be within the established range for the test results to be 

valid. 

5.2 Calculating the titer

Determine the log 10 EID50 titer using the method of Reed and Muench. This dilution and 

inoculation procedure allows for a direct readout on a per-dose basis. Round to 1 decimal.

5.3 Retests

Conduct retests as required by 9 CFR 113.8(b) and requirements of minimum release in 

the firm’s current Outline of Production, Part V.
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5.4 Evaluation of test results

5.4.1 The 9 CFR 113.8(b) defines the criteria for a satisfactory/unsatisfactory 

serial.

5.4.2 The firm’s requirements of minimum release/stability titers for each 

vaccine are listed in the current Outline of Production, Part V, for the specific 

product code.

6. Report of Test Results

Titers are reported out as EID50 per bird dose.

7. References

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

Washington, DC.

7.2 Reed, L.J., and H. Muench. 1938. A simple method of estimating 50% endpoints. 

Am. J. Hyg. 27:493-497.

8. Summary of Revisions

Version .05

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

to keep the same next review date for the document.

Version .04

 The contact information has been updated.

Version .03

 The document number has been changed from VIRSAM0409 to SAM 409.

 The Contact information has been updated.
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Version .02

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

Biologics and to provide additional detail. While no significant changes were made that impact

the outcome of the test, the following changes were made to the document:

 2.2.9/3.4.1 Chloroform has been changed to 1,1,2-Trichloro 1,2,2-Trifluoroethane 

(Freon)

 2.2.11(2) 1.775 g has been changed to 500 units/ml of penicillin and 100 g has been 

changed to 2 mg/ml of streptomycin

 2.2.11 The “Normal Saline” formula has been deleted.