147.1—The standard tube agglutination test. 1
Code of Federal Regulations
Footnote(s): 1 The procedure described is a modification of the method reported in the Proceedings of the U.S. Live Stock Sanitary Association, November 30 to December 2, 1932, pp. 487 to 491.
(1)
The blood samples should be taken by properly qualified and authorized persons only, and in containers provided by the laboratory. The containers should be stout-walled test tubes, preferably 3/8 by 3 inches, without lip, or small well-selected medicine vials, which have been thoroughly cleaned and dried in a hot-air drying oven. If stoppers are used, they should be thoroughly cleaned and dried.
(2)
Sufficient blood should be procured by making a small incision in the large median wing vein with a small sharp lancet and allowing the blood to run into the tube, or by the use of a small syringe (with 20 or 21 gage needle) which is properly cleansed between bleedings with physiological saline solution. To facilitate the separation of the serum, the tubes should be placed in a slanted position until the blood has solidified. After the blood has completely clotted, they should be packed and shipped by mail (special delivery), rapid express, or by messenger, to the laboratory. All labeling must be clear and permanent, and may be done with a suitable pencil on etched portions of the tube, or by means of fast-gum labels.
(3)
The blood samples must reach the laboratory in a fresh and unhemolyzed condition. Hemolyzed samples should be rejected. It is imperative, therefore, to cool the tubes immediately after slanting and clotting, and unless they reach the laboratory within a few hours, to pack them with ice in special containers, or use some other cooling system which will insure their preservation during transportation. In severe cold seasons, extreme precautions must be exercised to prevent freezing and consequent laking. The samples must be placed in cold (5 °to 10 °C.) storage, immediately upon arrival at the laboratory.
(b)
The antigen shall consist of representative strains of S. pullorum which are of known antigenic composition, high agglutinability, but are not sensitive to negative and nonspecific sera. The stock cultures may be maintained satisfactorily by transferring to new sloped agar at least once a month and keeping at 18 °to 25 °C. (average room temperature) in a dark closet or chest, following incubation for from 24 to 36 hours at 37 °C. The antigenic composition and purity of the stock cultures should be checked consistently.
| Water | 1,000 cc. |
| Difco beef extract | 4 gm. (0.4 percent). |
| Difco Bacto-peptone | 10 gm. (1.0 percent). |
| Difco dry-granular agar. | 20 gm. (2.0 percent). |
| Reaction—pH 6.8 to 7.2. |
(d)
Large 1-inch test tubes, Kolle flasks, or Blake bottles should be streaked liberally over the entire agar surface with inoculum from 48-hour slant agar cultures prepared from the stock cultures of the selected strains. The antigen-growing tubes or bottles should be incubated 48 hours at 37 °C., and the surface growth washed off with sufficient phenolized (0.5 percent) saline (0.85 percent) solution to make a heavy suspension. The suspension should be filtered free of clumps through a thin layer of absorbent cotton in a Buchner funnel with the aid of suction. The antigens of the separate strains should be combined in equal volume-density and stored in the refrigerator (5 °to 10 °C.) in tightly stoppered bottles.
(e)
Thiosulfate-Glycerin (TG) medium may be used as an alternate medium for the preparation of tube agglutination antigen. The TG medium, formerly used for the preparation of stained, whole-blood antigen, is described in more detail in the article by A. D. MacDonald, Recent Developments in Pullorum Antigen for the Rapid, Whole-Blood Test, Report of the Conference of the National Poultry Improvement Plan, pages 122-127, 1941. This medium provides a tube antigen of excellent specificity and greatly increases the yield of antigen from a given amount of medium. The TG medium has the following composition:
| Beef infusion | 1,000 cc. |
| Difco Bacto-peptone | 20 gm. (2.0 percent). |
| Sodium thiosulfate | 5 gm. (0.5 percent). |
| Ammonium chloride | 5 gm. (0.5 percent). |
| Glycerin, U.S.P. (95 percent) | 20 cc. (2.0 percent). |
| Difco dry-granular agar. | 30 gm. (3.0 percent). |
| Reaction—pH 6.8 to 7.2. |
Large 1-inch test tubes, Kolle flasks, Blake bottles, or Erlenmeyer flasks should be seeded over the entire agar surface with inoculum from 24-hour beef infusion broth cultures prepared from the stock cultures of the selected strains. The antigen-growing tubes or bottles should be incubated 96 hours at 37 °C., and the surface growth washed off with sufficient phenolized (0.5 percent) saline (0.85 percent) solution to make a heavy suspension. The suspension should be filtered free of clumps through a thin layer of absorbent cotton in a Buchner funnel with the aid of suction. The antigen then should be centrifuged. The mass of bacteria should be removed from the centrifuge tubes or bowl and resuspended in saline (0.85 percent) solution containing 0.5 percent phenol. After the bacterial mass has been uniformly suspended in the diluent, it should be again passed through a cotton pad in a Buchner funnel without the aid of suction. The antigens of the separate strains should be combined in equal volume-density and stored in the refrigerator (5 °to 10 °C.) in tightly stoppered bottles.
(f)
The diluted antigen to be used in the routine testing should be prepared from the stock antigen by dilution of the latter with physiological (0.85 percent) saline solution containing 0.25 percent of phenol to a turbidity corresponding to 0.75-1.00 on the McFarland nephelometer scale. The hydrogen-ion concentration of the diluted antigen should be corrected to pH 8.2 to 8.5 by the addition of dilute sodium hydroxide. New diluted antigen should be prepared each day and kept cold. The diluted antigen may be employed in 2 cc. quantities in 4 by 1/2 -inch test tubes, or 1 cc. quantities in smaller tubes, in which the final serum-antigen mixtures are made and incubated. The distribution of the antigen in the tubes may be accomplished by the use of long burettes, or special filling devices made for the purpose.
(g)
The maximum serum dilution employed must not exceed 1:50 for chickens, nor 1:25 for turkeys. The available data indicate that 1:25 dilution is the most efficient. In all official reports on the blood test, the serum dilutions shall be indicated. The sera should be introduced into the agglutination tubes in the desired amounts with well-cleaned serological pipettes or special serum-delivery devices which do not permit the mixing of different sera. The antigen and serum should be well mixed before incubation. The serum and antigen mixture must be incubated for at least 20 hours at 37 °C.
N, or − (negative) when the serum-antigen mixture remains uniformly turbid.
P, or (positive) when there is a distinct clumping of the antigen, and the liquid between the agglutinated particles is clear.
S, or ? (suspicious) when the agglutination is only partial or incomplete.
M, or missing, when samples listed on the original record sheet are missing.
H, or hemolyzed, when blood samples are hemolyzed and cannot be tested.
B, or broken, when sample tubes are broken and no serum can be obtained.
(Some allowance must always be made for the difference in sensitiveness of different antigens and different set-ups, and therefore, a certain amount of independent, intelligent judgment must be exercised at all times. Also, the histories of the flocks require consideration. In flocks where individuals show a suspicious agglutination, it is desirable to examine representative birds bacteriologically to determine the presence or absence of S. pullorum.)
(Approved by the Office of Management and Budget under control number 0579-0007)