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SAM 602.02

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 602

Supplemental Assay Method for the Evaluation of 

Salmonella pullorum Antigens

Date: January 28, 2010

Number: SAM 602.02

Supersedes: SAM 602.01, April 11, 1986

Standard Requirement: 9 CFR Part 113.407

Contact: Anna C. Dash, (515) 337-6838

Approvals:

/s/Michel Y. Carr Date:

Michel Y. Carr, Acting Section Leader

03Feb10 

Biotechnology, Immunology, and Diagnostics

/s/Byron E. Rippke Date:

Byron E. Rippke, Director

08Feb10 

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date:

Rebecca L.W. Hyde, Section Leader

09Feb10 

Center for Veterinary Biologics

Quality Management

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 the Evaluation of Salmonella pullorum Antigens

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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 samples

4. Performance of the Test

4.1 Formalin content

4.2 Hydrogen ion concentration

4.3 Density

4.4 Sensitivity

4.5 Homogeneity

5. Interpretation of the Test Results

5.1 Formalin content calculation

5.2 Hydrogen ion concentration determination

5.3 Density calculation

5.4 Sensitivity determination

5.5 Homogeneity determination

6. Report of Test Results

6.1 Formalin content

6.2 Hydrogen ion concentration

6.3 Density

6.4 Sensitivity

6.5 Homogeneity

7. References 

8. Summary of Revisions
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Supplemental Assay Method for the Evaluation of Salmonella pullorum Antigens

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1. Introduction

This Supplemental Assay Method (SAM) describes how Salmonella pullorum antigens are 

analyzed by using the following procedures: a percent concentration of formalin by 

spectrophotometric determination; hydrogen ion concentration by a pH meter; bacterial density by 

spectrophotometric determination; sensitivity by a rapid agglutination test; and homogeneity by 

microscopic examination. These procedures are applicable to S. pullorum stained K polyvalent 

antigens, as required in the Code of Federal Regulations, Title 9 (9 CFR), Part 113.407.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Spectrophotometer or colorimeter

2.1.2 Micropipettors

2.1.3 Pipette tips 

2.1.4 Sterile serological pipettes

2.1.5 pH meter

2.1.6 Graduated cylinder, 100-mL

2.1.7 Timers or stop clocks with 1 second subdivisions

2.1.8 Minnesota testing box containing a glass plate with perpendicular etched 

lines forming 3-cm x 3-cm squares

2.1.9 Aluminum mixing device designed for use with the slide agglutination test

2.1.10 Glass slides and cover slips

2.1.11 Light microscope

2.1.12 Vortex mixer

2.1.13 Cuvettes
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2.1.14 Centrifuge tubes, 15-mL and 50-mL

2.1.15 Volumetric flasks, 10-mL and 100-mL

2.1.16 Centrifuge

2.2 Reagents/supplies

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

2.2.1 10% Formalin solution (Fisher SSF98-4) - Must be non-neutral buffered

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.

2.2.2 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) 

(Sigma #129739)

2.2.3 Ferric chloride/sulphamic acid solution

10 g Ferric chloride

16 g Sulphamic acid

Q.S. to 1000 mL with distilled water

Store at 2o- 8oC.

2.2.4 pH buffer solutions

2.2.5 1N Hydrochloric acid 

82.8 mL Hydrochloric acid

917.2 mL distilled water

Store at 20o- 25oC.

2.2.6 Distilled water (H2O)

2.2.7 S. pullorum serums--A total of at least 12 positive serums shall be used. 

This shall include 3 definitely positive (high titer) serums--regular (R), intermediate 

(I), and variant (V), 3 weakly positive (low titer) serums--R, I, and V, and 3
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negative serums A panel of 12 positive and negative serums are produced at the 

Center for Veterinary Biologics (CVB), and can be requested for this assay.

2.2.8 S. pullorum stained K antigen reference to compare product on test to a 

known reference antigen. Available by request from the CVB.

2.2.9 McFarland standard No. 1 (Scientific Device Laboratory #2350)

2.2.10 Immersion oil

3. Preparation for the Test

3.1 Personnel qualifications/training

Laboratory personnel must be trained in the operation of instruments and equipment used

prior to performing this test. Personnel must be familiar with proper use of test reagents

and biological materials, and have knowledge of safe operating procedures and polices.

3.2 Preparation of equipment/instrumentation

Turn on the spectrophotometer to 'warm up' for at least 10 minutes.

3.3 Preparation of reagents/control procedures

Allow samples, serums, and reagents to warm to room temperature before conducting the 

tests.

3.4 Preparation of the samples

Samples are S. pullorum stained K polyvalent antigens.

4. Performance of the Test

4.1 Formalin content

4.1.1 Prepare a 0.5N HCL solution from 1N HCL solution and distilled water. 

4.1.2 Prepare a 0.5g/L solution of MBTH

4.1.3 Centrifuge 5 mL of reagent (sample) at 3000 rpm for 30 minutes in a 

15-mL centrifuge tube.
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4.1.4 Remove supernatant and place in a new 15-mL centrifuge tube.

4.1.5 Dilute supernatant of sample 1:4 with 0.5N HCL and allow to decolorize.

4.1.6 Prepare standards (std) in 10-mL volumetric flasks. Add the following 

amounts of 10% formalin to appropriately labeled flasks. Q.S. to volume with 

0.5N HCL.

std 1120 μL

std 2180 μL

std 3240 μL

std 4300 μL

std 5360 μL

4.1.7 Label 15-mL centrifuge tubes for standards, blank and samples.

4.1.8 Add 5.0 mL 0.5g/L MBTH solution to each tube.

4.1.9 Add 500 μL of standards and samples to appropriately labeled tubes. Add 

500 μL of 0.5N HCL to the blank tube.

4.1.10 Allow tubes to stand for 60 minutes.

4.1.11 Add 1.0 mL of ferric chloride/sulphamic acid solution to each tube.

4.1.12 Allow tubes to stand for 15 minutes.

4.1.13 With a spectrophotometer measure the absorbance of all tubes at 628 nm. 

Subtract the blank from each of the standard and sample readings.

4.1.14 Record results.

4.2 Hydrogen ion concentration

4.2.1 Standardize the pH meter with standard buffer solution pH 4.0 and pH 7.0 

according to the equipment manual.

4.2.2 Dispense 5 mL of product into a 50-mL screw-cap centrifuge tube.

4.2.3 Read product pH by inserting electrode into the tube containing the 

product.

4.2.4 Record results.
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4.3 Density

4.3.1 Allow all reagents to warm to room temperature. Prepare a 0.5N HCL 

solution.

4.3.2 Vortex sample for 10 seconds.

4.3.3 Dilute sample 1:50 with 0.5N HCL (1 mL sample + 49 mL HCL) in the 

100-mL volumetric flask. Mix well. Allow to decolorize for 2 hours.

4.3.4 Add 10 mL of distilled water to the sample for a 1:60 dilution. Mix well.

4.3.5 Zero the spectrophotometer using a wavelength of 420 nm and adjust the 

transmittance to 100% with distilled water.

4.3.6 Record transmittance of McFarland Standard No. 1.

4.3.7 Read and record transmittance of 1:60 diluted sample.

4.3.8 Add 1 mL of distilled water to the volumetric flask, mix well, and read and 

record transmittance.

4.3.9 Continue to add distilled water 1-5 mL at a time to the volumetric flask 

until the transmittance of the diluted sample is equivalent to the McFarland 

Standard No. 1 ± 3%.

4.3.10 Record results.

4.4 Sensitivity

4.4.1 Allow all reagents to warm to room temperature.

4.4.2 Vortex each S. pullorum serum and the S. pullorum antigen sample for 10 

seconds.

4.4.3 Set the electronic timer for 2 minutes.

4.4.4 Using a micropipette, place 20 μL of each S. pullorum serum within 2 

separate squares on the Minnesota box.

4.4.5 Add 1 drop of the sample in each square next to the serum.

4.4.6 Quickly mix the serum and sample with the aluminum mixing device using 

a circular motion, wiping the mixing device between each set of samples.
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4.4.7 Start the timer.

4.4.8 At the end of the incubation, gently swirl the liquid in a circular motion to 

read and record the result.

4.5 Homogeneity

4.5.1 Prepare a wet mount slide.

4.5.2 Examine the slide under the 100x objective.

4.5.3 Record all observations.

5. Interpretation of the Test Results

5.1 Formalin content calculation

A formalin content of 1.0 ± 0.2% is a satisfactory result. The absorbance reading shall fall 

between the readings of std 2 and std 4.

5.2 Hydrogen ion concentration determination

Stained K antigens must have a pH of 4.6 ± 0.4. 

5.3 Density calculation

5.3.1 The density is defined as the reciprocal of the amount of distilled water 

required to dilute the product to the equivalent of McFarland Standard No. 1.

5.3.2 The density must be 80 ± 15 times McFarland Standard No. 1.

5.4 Sensitivity determination

5.4.1 Agglutination (definite clumping with clearing of fluid) shall occur in at 

least 5 of the 6 positive serums.

5.4.2 No agglutination shall be observed in any of the 6 negative serums.

5.5 Homogeneity determination

5.5.1 There shall be no autoagglutination.
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5.5.2 There shall be no unusual appearance such as the presence of flakes, specks 

or a preponderance of filament forms.

6. Report of Test Results

6.1 Formalin content

Record the spectrophotometic reading of the sample and the readings of the two standards 

the sample reading fall inbetween, and final test conclusion. 

6.2 Hydrogen ion concentration

Record the pH of the product tested, and the final test conclusion.

6.3 Density

Record the density results, in terms of number of times the McFarland Std 1, and the final 

test conclusion.

6.4 Sensitivity

Record the number of negative serums that were negative and the number of positive 

serums that were positive, and the final test conclusion.

6.5 Homogeneity

Record the result as homogeneous or heterogeneous, and the status of autoagglutination, 

and the final test conclusion. 

7. References

Code of Federal Regulations, Title 9, Part 113.407, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

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

Biologics and to provide additional detail. While content changed significantly, no significant 

changes were made that impact the outcome of the test.