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SAM 307.07

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 307

Supplemental Assay Method for the Titration of Feline Rhinotracheitis Virus 

in Cell Culture

Date: October 30, 2014

Number: SAM 307.07

Supersedes: SAM 307.06, May 20, 2014

Standard Requirement: 9 CFR 113.315

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 06Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 09Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09Jan15

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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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 Test Vaccine

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 the Titration of Feline Rhinotracheitis Virus in Cell Culture

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro titration method for assaying 

modified-live feline rhinotracheitis virus (FRV) vaccines for viral content. The method uses 

plaque-forming units (PFU) in a cell culture system for titration of FRV.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Syringe, self-refilling, repetitive, 2-mL

2.1.2 Micropipettor, 200-μL, and tips (Rainin Pipetman®) or Handistep

2.1.3 Blender

2.1.4 Media bottle, borosilicate glass with screw-top lid, 1000-mL

2.1.5 Incubator, 36°± 2°C, high-humidity, 5% ± 1% CO2 (Model 3158, Forma 

Scientific Inc.)

2.1.6 Water bath

2.1.7 Inverted light microscope (Model CK, Olympus America Inc.)

2.1.8 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.9 Pipette-aid

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 FRV Positive control, C-27 strain

2.2.2 Crandall feline kidney (CRFK) cell culture, free of extraneous agents as 

tested by title 9, Code of Federal Regulations (9 CFR), by section 113.52
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2.2.3 Minimum essential medium (MEM) (National Centers for Animal Health 

[NCAH] Media #20030

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)

3. Dissolve with 900 mL deionized water (DI). 

4. Add 5.0 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Add to Step 3 with constant mixing.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add 50 μg/mL gentamicin sulfate.

8. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add:

a. 100 mL gamma-irradiated fetal bovine serum (FBS)

b. 10 mL L-glutamine (200 mM)

3. Store at 2°- 7°C.

2.2.5 2X Medium

1. 19.22 g MEM with Earles salts without bicarbonate

2. 2.2 g NaHCO3

3. Dissolve with 900 mL DI.

4. Add 5.0 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Add to Step 3 with constant mixing. 
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5. Q.S. to 1000 mL with DI, and adjust pH to 6.8-6.9 with 2N HCl.

6. Sterilize through a 0.22-μm filter.

7. Store at 2°- 7°C.

8. Prior to preparing the Overlay Medium (Section 3.3.3), aseptically add

100 μg/mL gentamicin sulfate.

2.2.6 2% Tragacanth Gum (Trag)

1. 20 g Trag

2. 1000 mL DI

3. Add small amounts of Trag at a time and vigorously mix with a 

blender set on high.

4. Pour 500 mL into individual glass 1000-mL media bottles.

5. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

6. Store at 2°- 7°C.

2.2.7 7.5% Sodium Bicarbonate (NCAH Media #41009)

1. 7.5 g NaHCO3

2. Q.S. to 100 mL with DI

3. Sterilize by autoclaving at 15 psi, 121°± 2°C for 30 ± 10 minutes.

4. Store at 2°- 7°C.

2.2.8 70% Ethyl Alcohol (NCAH Media #30184)

1. 73 mL ethyl alcohol (95% (195 proof))

2. 27 mL DI

3. Store at room temperature.
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2.2.9 Crystal Violet Stain (NCAH Media #30012)

1. 7.5 g crystal violet

2. 50 mL 70% Ethyl Alcohol

3. Dissolve crystal violet in alcohol; add remaining ingredients.

4. 250 mL formaldehyde

Note: According to 29 CFR 1910.1048, precautions for the use of 

formaldehyde are as follows: Toxic by inhalation and if swallowed. 

Irritating to the eyes, respiratory system, and the skin. May cause 

sensitization by inhalation or by skin contact. Risk of serious damage 

to eyes. Potential cancer hazard; repeated or prolonged exposure 

increases the risk.

5. Q.S. to 1000 mL with DI.

6. Filter through a Whatman®#1 filter.

7. Store at room temperature.

2.2.10 Tissue culture plates, 4-well

2.2.11 Polystyrene tubes, 12 x 75-mm

2.2.12 Pipettes, 25-mL

2.2.13 Needles, 18-gauge x 1 1/2-inch

2.2.14 Syringes, (tuberculin) 1-mL

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience in the preparation and maintenance of cell culture as 

well as in the propagation and maintenance of animal viruses and the quantification of 

virus infectivity by plaque formation.
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3.2 Preparation of equipment/instrumentation

On the day of test initiation, set a water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 Preparation of CRFK tissue culture plates (Test Plates)

Cells are prepared from healthy, confluent CRFK cell cultures that are maintained 

by passing every 3 to 4 days. Two days prior to test initiation, add 8.0 ml/well of 

approximately 105.0 to 105.3 cells/mL suspended in Growth Medium into all wells 

of the 4-well tissue culture plate. Prepare one Test Plate for the FRV Positive

Control and one for each Test Vaccine. Incubate at 36°± 2°C in a CO2 incubator. 

3.3.2 Preparation of FRV Positive Control

1. On the day of test initiation, rapidly thaw a vial of the current FRV 

Positive Control virus in the water bath.

2. Dispense 1.8 mL of MEM into sufficient 12 x 75-mm polystyrene 

tubes to bracket the expected endpoint according to the CVB Reagent 

Data Sheet; appropriately label (e.g., 8 tubes, labeled 10-1through 10-8, 

respectively). 

3. Transfer 200 μL of the FRV Positive Control virus to the tube labeled 

10-1; mix by vortexing.

4. Using a new pipette tip, transfer 200 μL from the 10-1-labeled tube to 

the 10-2 tube; mix by vortexing.

5. Repeat Step 4 for each of the subsequent dilutions, transferring 200 

μL of the previous dilution to the next dilution tube until the tenfold 

dilution series is completed.

3.3.3 Overlay Medium Preparation

On the day of test initiation, prepare approximately 35 mL per plate of Overlay 

Medium. Volumes listed are for 1 liter of Overlay Medium.

1. Aseptically add to 500 mL of 2X Medium:

a. 10% gamma-irradiated FBS

b. 10 mL of 7.5% Sodium Bicarbonate
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c. 50 μg/mL gentamicin sulfate

d. 50 mL of 2% Trag

2. Mix and warm the Overlay Medium in a 36°± 2°C water bath for 

60 ± 10 minutes prior to performing the procedure in Section 4.5.

3.4 Preparation of the Test Vaccine

3.4.1 The initial test will be with a single vial (a single sample from 1 vial). On 

the day of test initiation, using a 1.0-mL syringe and an 18-gauge x 1 1/2-inch

needle, rehydrate a vial of the Test Vaccine with the provided diluent by 

transferring 1.0 mL for a 1-mL-dose vaccine, 0.5 mL for a 1/2-mL-dose vaccine, 

etc., into the vial containing the lyophilized Test Vaccine; mix by vortexing. 

Incubate for 15 ± 5 minutes at room temperature.

3.4.2 Dispense 1.8 mL MEM into each of 6, 12 x 75-mm polystyrene tubes 

labeled 10-1through 10-6respectively, using a 2-mL self-refilling, repetitive 

syringe.

3.4.3 Transfer 200 μL of the Test Vaccine to the tube labeled 10-1; mix by 

vortexing.

3.4.4 Using a new pipette tip, transfer 200 μL from the tube labeled 10-1to the 

10-2tube; mix by vortexing.

3.4.5 Repeat Section 3.4.4 for each of the subsequent dilutions, transferring 

200 μL of the previous dilution to the next dilution tube until the tenfold dilution 

series is completed.

4. Performance of the Test

4.1 On the day of test initiation, label the Test Plates and remove the Growth 

Medium. 

Note: Presence of confluent cells is necessary in order to distinguish plaques due to 

infection by FRV. If cells are not confluent at two days incubation, start of the test 

may be delayed an additional day in order to obtain confluency.
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4.2 Inoculate 1 well/dilution with 200 μL from dilutions 10-6through 10-3of the Test 

Vaccine. Inoculate 1 well/dilution from dilutions 10-8through 10-6of the FRV Positive

Control. Change tips between each unique sample (i.e., each Test Vaccine), but tip 

changes are not necessary between each dilution in a series when pipetting from the most 

dilute to the most concentrated (e.g., 10-8through 10-6). Gently rotate the plates to evenly 

disperse the inoculum.

4.3 One uninoculated well on a Test Plate serves as a negative cell control.

4.4 Incubate the inoculated Test Plates at 36°± 2°C for 75 ± 15 minutes in a CO2

incubator. To evenly disperse the inoculum, gently rotate the plates at intervals of 

25 ± 5 minutes.

4.5 Add 8 mL/well of the Overlay Medium to the plates with a 25-mL pipette. 

Discard any unused, warmed Overlay Medium.

4.6 Incubate the Test Plates undisturbed at 36°± 2°C in a CO2 incubator for 

4 days ± 1 day.

4.7 After incubation, without removing the Overlay Medium, pipette 5 mL of the 

Crystal Violet Stain into each well of the Test Plates with a 25-mL pipette; mix by gentle 

rotation. Note the precaution statement in Section 2.2.9(4).

4.8 Allow Test Plates to incubate at room temperature for 25 ± 5 minutes.

4.9 Wash the Overlay Medium and the Crystal Violet Stain from the cell monolayers 

by dipping each plate several times in a container overflowing with running water from 

the cold water tap until the water is clear. Allow to air dry.

4.10 Count the number of FRV plaques for each well and record observations. If FRV 

and feline calicivirus (FCV) plaques are counted together in a combination vaccine, the 

FRV plaques will contrast markedly from FCV. The FCV plaques are large, clear circular 

areas (averaging 3 to 4 mm in diameter) with fuzzy edges. The FRV plaques are visible 

as small, clear plaques approximately 1 mm diameter with distinct edges. If the plaques 

are not distinguishable by size, use an inverted light microscope with 100X magnification 

to differentiate the plaque edges.

4.11 Record results as the number of FRV plaques for each dilution of a Test Vaccine

and the FRV Positive Control. 

4.12 Calculate the FRV titers of the Test Vaccine and the FRV Positive Control from 

the dilutions with plaque counts containing 10-100 plaques. The titer may be expressed as 

PFU per dose of vaccine.
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Example:

During the titration of a Test FRV vaccine, the plaque count obtained at dilution 10-3 was 

65. Calculation of the titer of this vaccine is done according to the following formula:

Test Vaccine Titer = X + p, where

X = Log10 of plaque count

p = Log10 of dilution counted 

In the above example: 

Log10 of plaque count (65) = 1.8

Log10 of dilution counted (10

-3

) = 3.0

FRV Test Vaccine Titer = 1.8 + 3.0 = 4.8

Adjust the titer to the recommended Test Vaccine dose as follows:

A. Divide the Test Vaccine Dose by the Inoculation Dose

Test Vaccine Dose = manufacturer’s recommended vaccination dose (for this test 

FRV vaccine is 1 mL)

Inoculation Dose = amount of diluted Test Vaccine added to each well of the 

Test Plate (for this test FRV vaccine, the inoculation dose is 0.2 mL)

 1 mL dose = 5 

0.2 mL inoculum

B. Calculate log10 of value in A and add it to the Test Vaccine Titer as 

illustrated below:

Log of 5 = 0.7

FRV Test Vaccine Titer = 4.8 + 0.7 = 5.5

Therefore, the titer of the FRV Test Vaccine is 105.5 PFU50/mL.

4.13 Conversion from PFU50/mL to TCID50/mL.

One plaque represents a single infective unit (IU), whereas one, 50% tissue culture 

infective dose (TCID50) is statistically equivalent to a theoretical 0.69 IU. A TCID50

endpoint per dose will be 1.44 times those expressed as PFU per dose. Therefore, to 

express a PFU titer as a TCID50 titer, multiply the plaque count by 1.44. In the example 
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above, multiply 65 times 1.44 resulting in 93.6. Convert 93.6 to log10 resulting in 1.97. 

Add the log for the dilution counted (3) and the conversion factor for dose (0.7) which 

results in a log10 TCID50/mL of 5.67. In practical use, 0.16 (the log of 1.44) may be added 

to the log10 value of the PFU titer per mL to convert to TCID50/mL. In the above 

example, the titer of the Test Vaccine is 105.66 TCID50/mL.

5. Interpretation of the Test Results

5.1 Validity requirements

5.1.1 The calculated titer of the FRV Positive Control must fall within plus or 

minus 2 standard deviations (± 2 SD) of its mean titer, as established from a 

minimum of 10 previously determined titers.

5.1.2 The uninoculated cell control cannot exhibit any plaques, cytopathic effect 

(CPE), or cloudy media that would indicate any contamination.

5.1.3 For endpoint determination, only those tenfold dilutions of the Test 

Vaccine and the FRV Positive Control inducing 10 to 100 plaques are included in 

the plaque counts.

5.2 If the validity requirements are not met, then the assay is considered a NO TEST

and may be retested without prejudice.

5.3 If the validity requirements are met and the titer of the Test Vaccine is greater 

than or equal to the titer contained in the Animal and Plant Health Inspection Service 

(APHIS) filed Outline of Production for the product under test, the Test Vaccine is 

considered SATISFACTORY.

5.4 In a valid test, if the titer of the Test Vaccine is less than the titer contained in the 

APHIS filed Outline of Production for the product under test, the Test Vaccine may be 

retested in accordance with title 9, Code of Federal Regulations, section 113.8(b).

6. Report of Test Results

Results are reported as PFU50 or TCID50 per dose of Test Vaccine.
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7. References

7.1 Title 9, Code of Federal Regulations, parts 113.8(b) and 113.315, U.S. 

Government Printing Office, Washington, DC.

7.2 Title 29, Code of Federal Regulations, part 1910.1048, U.S. Government Printing 

Office, Washington, DC.

7.3 Cottral, GE, Manual of standardized methods for veterinary microbiology. 

Comstock Publishing Associates. Ithaca, New York, 1978, pg. 731.

7.4 Davis, Dulbecco, Eisen, and Ginsberg, ed., Microbiology including Immunology 

and Molecular Genetics, 3

rd ed. Harper and Row, Hagertown, Maryland, 1980, pg. 880.

8. Summary of Revisions

Version .07

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

to keep the same next review date for the document.

Version .06

 The contact information has been updated.

Version .05

 The Contact information has been updated.

 FRV Positive control, C-27 strain is no longer available from the CVB and all 

references to it have been removed from the document.

 References to NVSL have been changed to NCAH throughout the document.

Version .04

 The Contact has been changed from Victor Becerra to Alethea Fry.

 2.2.9: A precautionary statement for use of formaldehyde has been added.

 4.13: Conversion for PFU50/mL to TCID50/mL has been clarified.

 Use of reference control terminology has been changed to positive control throughout 

the document.
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Version .03

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:

 1.2 “Key Words” has been deleted.

 2.2.3.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.

 2.2.5.8 50 μg/ml gentamicin sulfate has been changed to 100 μg and usage of 

penicillin and streptomycin has been deleted.

 4.6 Ninety-six hours has been changed to 4 days ± 1 day.

 4.12 The determination of virus titer for each Test Vaccine has been rewritten to 

correct errors in calculations using the example given.

 5.1.3 The endpoint determination of plaques has been clarified.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.

 “Test Serial” has been changed to “Test Vaccine” throughout the document.

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.

 The footnotes have been deleted with any pertinent references now noted next to the 

individual items.