Fluorescent Antibody

Rhizosphere and the Phyllosphere
Rhizosphere Introduction
Rhizospher Effect
Nitrogen Fixation in the Rhizosphere
Alteration of Rhizosphere Microflora
Associative and Antagonistic Activities in the Rhizosphere
Root Exudates
Organic Compounds Detected in Plant Root Exudates
Fungistasis
Techniques of Rhizosphere
The Soil Dilution and Plate Count Method
The Soil Plate
The Soil Box
The Contact Slide Technique
Artificial Rhizosphere
Model System

Fluorescent Antibody

	Fluorescent Antibody The technique is based on the specificity of antigen-antibody reaction which is widely used -in medical microbiology and pathology. Small amounts of flurorescence can be detected in bacteria if a fluorochrome dye is attached to them. This is accomplished by coupling an antibody (Ab) to a fluorescent dye and allowing the complex to react with its antigen (Ag). The microscopic preparations are then examined under a fluorescence microscope. Commonly known as FA technique, this precise technique has paved the way for an elegant method of observing or detecting a microorganism in its natural environment.
Special skill and equipments are needed for its successful operation which can 'be briefly outlined as follows: The antigen (Ag) providing organism is injected into rabbits and serum collected by standard procedures. The serum is fractionated to give gamma globulin which is dialysed and diluted to 1% protein level. A fluorochrome dye, fluorescein isothiocyanate (FITC) is conjugated to immune globulin under carefully controlled pH and buffer conditions. The conjugated complex is separated from the dye on Sephadex column and dilutions prepared until the desired dilution is obtained which will act as the correct indicator of good antibody activity avoiding non-specific background staining. The FA thus prepared can be preserved for many years at 20°C.
The soil sample or the sample of any ecological microhabitat to be studied (natural material) is placed on a microscopic slide. The latter may as well as allowed to be in contact with the material for long periods to obtain a 'contact slide'. The slide is heated and a gelatin-rhodamine isothiocynnate (RhITC) conjugate is applied to the slides with the object of not only suppressing non-specific antibody absorption but also to serve as a counterstain. FA is finally applied to the dried gel-RhITC layer and incubated, for reaction for one hour. The preparation is washed in buffer, air dried and examined under fluorescence microscope.
One proven example of the application of FA technique, was the detection of an efficient strain of Rhizobium japonicum as brightly stained rhizobia-like rods in field soils containing myriads of R. japonicum strains. The survival and competition of rhizobial strains in soils have also been studied by the use of this technique. Despite many inherent limitations, the technique has many potentialities in studies on soil ecologyin laboratories where the required facilities for this type of work are available. Specific detection of Rhizobium japonicum strain 61A72 by immunofluorescence preparation made from slides that had been in contact with field soil inoculated with R. Japoniccum 61A72. In Some areas cells are seen in association with soil particles

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