Types of Tissue and Preparation of Samples for Isozyme Analysis of Fungi

Fortunately, virtually any type of fungal tissue can be used for isozyme analysis. This could include sections of sporocarps collected in the field or produced in culture, mycelia from plates or liquid cultures, or even conidia washed from lesions or cultures. The main requirement is that the tissue must be living so the enzymes are functional. Old cultures that are dead or dying will not give satisfactory results. This precludes dried herbarium material as the activity of most enzymes will be destroyed during the drying process. However, living material that has been lyophilized and stored at — 80°C will maintain enzyme activity for many months and possibly for years. Lyophilization also may be a good option to save material until a sufficient quantity is available for analysis if only limited amounts of material can be collected at a time.

Regardless of source or amount, the fungal material must be macerated to release the enzymes. Lyophilized material can be frozen quickly with a small quantity of liquid nitrogen and ground in a mortar after the nitrogen has evaporated. Small amounts of tissue can be ground in microcentrifuge tubes using a glass rod as a pestle. Disposable plastic pestles designed specifically for use in microcentrifuge tubes also can be used. Fresh tissue can be crushed with a disposable plastic pestle attached to a variable-speed electric drill. Adhering liquid and tissue should be wiped from the pestle between samples, but there is no need to clean the pestle completely as

Figure 1 Isozymes of glucose-6-phosphate isomerase in 12 isolates of the barley scald pathogen, Rhynchosporium secalis. This haploid fungus yields a single band in each lane corresponding to one of the three putative alleles 87, 100, or 114.

a small amount of cross contamination will not affect the results due to the relatively low sensitivity of the technique. To maintain enzyme activity, all samples should be kept on ice whenever possible. Tissue extracts also can be frozen at — 80°C for several weeks if necessary with little or no enzyme degradation. However, this should be tested for each system specifically before used on a large number of samples.

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