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SAM 408.06 

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United States Department of Agriculture

Center for Veterinary Biologics 

Testing Protocol 

SAM 408

Supplemental Assay Method for Titrating Tissue Culture Adapted Vaccine 

Strains of Infectious Bursal Disease Virus 

Date: November 28, 2014

Number: SAM 408.06

Supersedes: SAM 408.05, May 20, 2014 

Standard Requirement: 9 CFR 113.331 

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

Approvals: /s/Geetha B. Srinivas Date: 22Jan15 

Geetha B. Srinivas, Section Leader 

Virology 

/s/Byron E. Rippke Date: 25Jan15 

Byron E. Rippke, Director 

Policy, Evaluation, and Licensing 

Center for Veterinary Biologics 

/s/Rebecca L.W. Hyde Date: 26Jan15 

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 Tissue Culture Adapted Vaccine Strains of 

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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

5.1 Controls

5.2 Calculating the titer

5.3 Retests

5.4 Evaluation of test results

6. Report of Test Results

7. Summary of Revisions
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Supplemental Assay Method for Titrating Tissue Culture Adapted Vaccine Strains of 

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1. Introduction

This Supplemental Assay Method (SAM) describes the titration of vaccine strains of tissue 

culture adapted infectious bursal disease virus (IBDV) in primary chick embryo fibroblast cell 

cultures (1°CEF).

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Water-jacketed incubator with a humidified 5 ± 1% CO2 atmosphere and 

temperature set at 37o± 2°C (Forma Scientific, Model No. 3158)

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

2.1.3 Microliter pipette (Rainin Pipetman, P1000)

2.1.4 Laminar Flow Biological Safety Cabinet (NuAire Inc., Labgard)

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 24-well tissue culture treated plates, planted with 1 mL of 1°CEFs 

suspension per well at a concentration of approximately 400,000 cells/mL. The 

growth medium is M-199/F10 with 2-3% FBS, 1% L-glutamine, and 2 μg/mL 

Fungizone. 1°CEFs are from susceptible specific pathogen free (SPF) 

embryonating chicken eggs. 

2.2.2 M-199/F-10 media (National Centers for Animal Health (NCAH) Media 

#20012):

M199/F10 Formula

Medium 199 47.74 g

1x F-10 Nutrient Mixture 5000 mL

Bacto Tryptose Phosphate Broth 14.8 g

Penicillin G 0.613 g

Streptomycin 1.0 g
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Sodium Bicarbonate 22 g

HEPES 59.85 g

Sterile, distilled, or deionized water 10.5 L

All ingredients are mixed, pH is adjusted to 7.2, and the solution is filter 

sterilized. The pH is checked again after sterilization. The solution is then

dispensed into appropriate containers.

2.2.3 Sterile distilled or deionized water, 100 mL aliquots in 100-mL serum 

vials with rubber stoppers and aluminum caps

2.2.4 Needle, 1 1/2-inch x 18-gauge 

2.2.5 LuerLok disposable syringe, 5- or 10-cc 

2.2.6 Sterile pipette tips, Rainin P100-P1000

2.2.7 Sterile glass test tubes, 16 x 125-mm

2.2.8 Fetal Bovine Serum (FBS)

2.2.9 L-Glutamine (200 mM)

2.2.10 Pipettes, various volumes

2.2.11 Fungizone

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 policies/procedures 

(SOPs) or equivalent.
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3.3 Preparation of reagents/control procedures

Prepare reference viruses in the same manner as sample preparation (see Section 3.4).

3.4 Preparation of the sample

Preparation of vaccine for titration: rehydrate or dilute vaccine in 100 mL of sterile 

deionized water. Mix thoroughly. If necessary, further dilute the vaccine so that 1 dose is 

contained in a volume of 0.1 mL.

4. Performance of the Test

To prepare the dilution and inoculate the plates, use maintenance media consisting of M199/F10 

media with 1% FBS, 1% L-Glutamine, and 2 μg/mL of Fungizone to make dilutions for both the 

vaccine and positive control virus. Make tenfold serial dilutions of the vaccine and the positive 

control virus encompassing the range of the expected titer. Use at least 4 dilutions for a titration. 

Inoculate 5 wells with 0.1 mL per well for each dilution. The sixth well of each row remains 

uninoculated and serves as a cell control. Incubate in a humidified atmosphere of approximately 

5 ± 1% CO2 at 37o± 2°C.

5. Interpretation of the Test Results

5.1 Controls

The titer of the positive control must be within the established range for the test results to 

be valid. Uninoculated negative control cells are maintained to monitor the integrity of 

the cell culture system.

5.2 Calculating the titer 

Microscopically examine the plates daily and track the development of typical IBDV 

cytopathology including refractile cells. The negative control wells must remain normal 

throughout the test. On day 7 postinoculation, calculate the 50% endpoint of infectivity 

using the Reed-Muench method. This value will represent the titer per dose.

5.3 Retests

Conduct retests as required by title 9, Code of Federal Regulations (9 CFR), section 

113.8(b), and also by the requirements 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 IBD

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 Tissue Culture Infective Dose 50% Endpoint (TCID50) per bird dose. 

Report the results as satisfactory or unsatisfactory.

7. Summary of Revisions 

Version .06

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

to keep the same next review date for the document. 

Version .05

 The Contact information has been updated.

Version .04

 The Contact information has been updated.

Version .03

 The Contact has been changed from Scott Taylor and Karen Wineland to Sheridan 

Booher and Danielle Koski.

 2.2.1: Information has been included that pertains to the concentration of FBS, 

L-Glutamine, and Fungizone that should be added to the growth media.

 2.2.2: The M199/F10 formula has been added.

 2.2.8: This section has been removed because it was a duplicate listing of equipment

in Section 2.2.1.
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 3.3: The phrase “see Section 3.4” has been added.

 3.4: “Or diluted” has been added to encompass those vaccines not lyophilized.

 4.1: Clarification has been added on what the maintenance media consists of.

 5.3: The phrase “also by” has been added and the phrase “of minimum release” has

been removed.

Version .02

This document was rewritten to clarify 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:

 The introduction has been revised for clarity.

 2.1 Many items listed were unnecessary to list and have been removed from the 

document for clarity.

 2.2 Changes in the reagents/supplies used have been made to the items previously 

listed for clarity.

 2.2 The Dulbecco’s PBS and trypsin solution (0.25%) have been removed. The 

growth medium has been removed and replaced with a maintenance medium.

 2.2.16 The section on cell cultures has been deleted from the document.

 4.1 The use of the maintenance medium for the vaccine and positive reference virus 

has been added for clarity.

 5.2 This section has been revised for clarity.

 6. This section has been revised to provide clarification of reporting test results.