Fungal endophyte test.

CFR · 2024-01-01 · US · Title 7

A fungal endophyte test may be used to determine the amount of fungal endophyte ( (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1 percent aniline blue solution with 2 parts of 85 percent lactic acid (C (3) Use stain as-is or dilute with water if staining is too dark. (b) Procedure for determining levels of fungal endophyte in grass seed: (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind under consideration. (2) Digest seed at room temperature for 12-16 hours in a 5 percent sodium hydroxide (NaOH) solution or other temperature/time combination resulting in adequate seed softening. (3) Rinse thoroughly in running tap water. (4) De-glume seeds and place on a microscope slide in a drop of endophyte staining solution. Slightly crush the seeds. Use caution to prevent carryover hyphae of fungal endophyte from one seed to another. (5) Place coverglass on seed and apply gentle pressure. (6) Examine with compound microscope at 100-400x magnification, scoring a seed as positive if any identifiable hyphae are present. (7) Various sample sizes may be used for this test. Precision changes with sample size; therefore, the test results must include the sample size tested. (c) Procedure for de

official document · source cfr-2024 · id 7:§ 201.58d