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Serology
This test makes use of the antigen-antibody reaction. An antigen is any substance which induces the production of antibodies upon introduction into the blood stream of an animal. The antigenic substances are proteins or polysaccharides and bacteria can also serve as an antigenic substance. Antibodies are proteins produced by plasma cells in response to antigens with the unique capability of binding specifically to the antigen which induced their formation. The bacterial cell may contain many antigenic components.
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Those antigens which are present on the surface or in the cell are known as somatic or 'O' antigens while those present in the flagella are known as flagellar or 'H' antigens. Somatic antigens may be either heat-labile or heat-stable, the latter mostly found on the surface of cell and are considered to be strain specific. When identical antigens are present in bacterial strains, they are known as serologically identical bacteria.
If bacterial strains contain some antigens common among them, they are designated as serologically related. On the other hand, serologically unrelated bacterial strains possess unidentical antigens. A series of laboratory tests are available to distinguish bacterial strains for the above-mentioned relationships and they come under the broad category of agglutination, precipitation and complement fixation.
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Among these tests, the precipitation or precipitin reaction is commonly used to distinguish between strains of rhizobia. In this test, a reaction takes place between a soluble antigen and a solution of its homologous antibody which is manifested by the formation of a visible precipitate at the interface of the reactants. Greater accuracy and efficient separation of components in mixtures of antigen and antibodies can be obtained by allowing the reactants to diffuse together on an agar gel.
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For this purpose, a double-diffusion method in two dimensions devised by Ouchterlony has been commonly used. In this method, wells are cut on agar in Petri dishes and the reactants diffuse from wells. When the reactants meet midway, they react and form precipitin bands. Based on the characteristics of bands, the homologous or heterologous nature of the bacterial strains could be determined. The outline of the procedure, as followed in the Division of Microbiology, Indian Agricultural Research Institute, New Delhi, for distinguishing rhizobial strains is detailed below:
1. The mother culture is grown in yeast extract mannitol agar or liquid medium at 28°C ± 1°C for 5 to 8 days, depending on whether the bacterium is slow or fast growing.
2. The bacterial cells are harvested in sterile physiological saline (0.85% NaCl) and the cell suspension centrifuged at 15,000 r.p.m. to obtain a pellet.
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3. The pellet is transferred to a screw capped tube in minimum amount of physiological saline and stored below 0.C. This will serve as the antigen.
4. The antigen is diluted so as to carry 10-15 µg protein/ml or 106
cells/ml and one ml mixed thoroughly in 1/2 ml of Difco Baco adjuvant or sterilized paraffin oil.
5. The antigen is injected intramuscularly into a healthy rabbit (minimum weight 1.5 kg), at the upper part of the hind leg. Similar injections are repeated thrice at weekly intervals.
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6. Four weeks after the first injection, 1 ml of the antigen (106 cell/ml) is again injected intravenously without the adjuvant, through the marginal vein of the ear to boost the titre value of the antibody.
7. After 1 week, the rabbit is made to bleed through the marginal vein of the other ear and 5 ml of blood collected in a small beaker. The blood is incubated at 35°C fur an hour to coagulate and stored in a refrigerator above 4°C overnight when the serum stands as a supernatant.
8. The supernatant (straw coloured) is separated and centrifuged at 3000 r.p.m. for 5 minutes. This is the antiserum which should be tested initially against the reference antigens (that is, the antigen described in columns 1 to 3 above). If bands due to precipitin reaction are formed (see procedures in column 10-14), the rabbit may be immediately bled and sizeable amounts of blood collected, coagulated, antiserum separated and stored in vials (0.2 to 1 ml aliquots) in a deep freezer, until further use.
9. At this stage, agar plates are prepared for immunodiffusion tests, as follows: One hundred ml of agar suspension(1.5%) is prepared in physiological saline by steaming in an autoclave and an equal volume of 0.05% sodium azide solution is also prepared in physiological saline without heating. Both the solutions are mixed gently (to avoid air bubbles) in a measuring cylinder and the volume made up to 200 ml with distilled water. This will mean that the mixture will contain: agar: sodium azide: sodium chloride in the ratio of 0.75%: 0.025%: 0.85%. The mixture is poured in Petriplates and allowed to solidify on a uniformly levelled table so as to obtain a gel of 4 mm thickness.
10. Equidistant holes are punched in a hexagonal array on agar in Petriplates with the help of a cork borer of 4 mm. diameter and the agar plugs removed under mild suction by a Pasteur pipette using a water pump. The bottoms of the holes are sealed by pouring droplets of molten agar through a Pasteur pipette.
11. The centre well of the Petri plate is filled with the reference antiserum and the reference antigen filled into two wells across the centre well. In each of the remaining wells, antigens of other isolates whose serological relationships have to be determined are filled. To differentiate thermostable surface antigens from thermolabile flagellar or intracellular antigens, the cell suspension is steamed for 30 minutes in an autoclave and used.
12. The Petri plates are incubated in a moist air-tight plastic box at constant temperature (below 25°C) for 4 to 6 days. The formation of precipitin bands around the centre well is observed. If the bands formed due to the reference strain and a strain under test are identical in nature, position, and happen to coalesce without the formation of spur, it may be concluded that the strain under test is serologically identical with the reference strain, failing which the strain, under test may be either partially related or totally unrelated to the reference strain.
Immunodiffusion reaction in gel showing precipitation bands. Antiserum appears in centre well and antigens in surrounding wells. From Somasegaran and Hoben, 1985 of NifTAL, USA
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