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Identification of fungi by the fluorescent antibody technique Johnson, Gary Clifford

Abstract

The fluorescent antibody technique was investigated as a means of facilitating the recognition and identification of the fungal components of a western red cedar (Thuja plicata Donn) heartwood flora in situ. Fungi isolated from the heartwood were grown in bulk and prepared for two different injection trials. In one trial the antigen was the particulate matter of the cell that could be centrifuged into a pellet after the hyphae were destroyed by a tissue grinder. In the second trial the hyphae were ground up and ultrasonically disintegrated. Only the cytoplasm and small wall fragments were retained for injection. After antisera collection the indirect staining method was employed. Unlabeled specific antiserum was layered over the antigen, allowed to incubate and washed off before fluorescent sheep anti-rabbit globulin was applied to form the final layer. All attempts to detect specific antibodies to the fungal antigens failed. This was probably due to not using antigens rich enough in protein. Successful production of precipitating antibodies to fungal antigens has been shown by other workers to be more likely when the antigen contains greater than 10 milligrams of protein per milliliter of antigen solution. It has also been found that in some cases fresh antigen must be prepared for each diffusion test and injection as it can't be preserved even at -20°C. It is hoped that if fresh, high protein antigen were to be used this study could be successfully completed.

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