147.5—The microagglutination test for pullorum-typhoid.
Routinely, the microagglutination test is applied as a single-dilution test and only a single 18-24 hour reading is made.
(a)
The procedure for the collection and delivery of blood samples in the microagglutination test is the same as that described in § 147.1(a). A method that has proven advantageous is to transfer the serum samples from the blood clot to a microplate as described in “Applied Microbiology,” volume 24, No. 4, October 1972, pages 671-672. The dilutions are then performed according to paragraphs (d) or (e) of this section.
(b)
Stained microtest antigen for pullorum-typhoid is supplied as concentrated stock suspension and must be approved by the Department. 4 Directions for diluting will be provided with the antigen. The stock as well as the diluted antigen prepared each day should be kept sealed in the dark at 5 ° to 10 °C. when not in use.
Code of Federal Regulations
Footnote(s): 4 Information as to criteria and procedures for approval of concentrated stock suspension of stained microtest antigens may be obtained from the National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 200, Conyers, GA 30094.
(c)
Available data indicate that a 1:40 dilution for the microagglutination test is most efficient for the detection of pullorum-typhoid agglutinins in both chickens and turkeys. In all official reports on the blood test, the serum dilutions shall be indicated.
(1)
Add 100 microliters (0.10 cc.) of 0.85 percent physiological saline to each well of the microplate.
(2)
Using a microdiluter or a multimicrodiluter handle fitted with twelve 10 microliter microdiluters, transfer 5 microliters (0.005 cc.) of the serum sample from the collected specimen to the corresponding well of the microplate. This is accomplished by touching the surface of the serum sample with the microdiluter and then transferring and mixing with the diluent in the microplate well. The microdiluter is removed, blotted, touched to the surface of the distilled water wash, and again blotted. Other acceptable methods of serum delivery are described in “Applied Microbiology,” volume 21, No. 3, March 1971, pages 394-399.
(3)
Dilute the microtest antigens with 0.50 percent phenolized saline and add 100 microliters (0.1 cc.) to each microplate well.
(4)
Seal each plate with a plastic sealer or place unsealed in a tight incubation box as described in “Applied Microbiology,” volume 23, No. 5, May 1972, pages 931-937. Incubate at 37°C. for 18-24 hours.
(1)
Add 50 microliters (0.05cc.) of 0.85 percent physiological saline to each well of the microplate.
(2)
To the wells representative of the lowest dilution in the titration, add an additional 50 microliters (0.05 cc.) of 0.85 percent physiological saline making a total of 100 microliters in these wells.
(3)
Transfer each serum sample as described in § 147.5(d)(2) of this section to the first well containing 100 microliters (0.10cc.) in the titration, which represents the lowest dilution.
(4)
Make twofold serial dilutions of each serum by transferring 50 microliters (0.05cc.) of diluted serum from one well to the next using twelve 50 microliter microdiluters fitted in a multimicrodiluter handle. When transfers have been made to all of the wells of the desired series, the 50 microliters remaining in the microdiluters are removed by blotting, touching the microdiluters to the surface of the distilled water wash, and blotting again.
(5)
Dilute the desired microtest antigen with 0.50 percent phenolized saline and add 50 microliters (0.05 cc.) to each microplate well.
(6)
Seal each plate with a plastic sealer or place the unsealed microplates in a tight incubation box and incubate at 37 °C. for 18-24 hours.
(3)
S, or ? (suspicious) when the microplate well has a small button. Suspicious reactions may tend to be more positive than negative [±] or vice versa [∓] and can be so noted if desired.
(Approved by the Office of Management and Budget under control number 0579-0007)