The process of designating morphotypes is probably the most important step in distinguishing which fungi are associated with beetles. For this reason, careful assignments should be made from observations collected at different times and DNA data collected from multiple isolates per morphotype and per beetle.
After isolation of fungi from beetles, fungi need to grow to sufficient size to allow for elucidation of macroscopic characteristics (ie color, size comparison/growth rate, texture, etc). Additional methods for differentiation morphotypes may be deployed at this step, including culturing duplicate dilutions in darkness vs. fluorescent (+blue-black-blue) light.
Subculture: 5-10 days post isolation
Before subculturing, create preliminary morphotypes by recording the original isolation plate # and the phenotype on the back of a subculture plate. Indicate which CFU(s) will be sampled on the isolation plate, and photograph them (front & back) without opening. For each morphotype, subculture two different CFUs per plate
DNA extraction and RAPD: 7-14 days post subculture
Once subcultures have grown sufficiently, single colony subcultures should be photographed prior to DNA extraction. In pure culture/at a larger size, subcultured fungi may display slightly different phenotypes than those observed on isolation plates, and may allow for re-assessment of preliminary morphotypes (or grouping order in RAPD gel).
After DNA extraction, perform a RAPD PCR reaction (m13 primer & cycling) by grouping similar morphotypes next to each other on the gel. If the results are unexpected, a single-direction PCR of a more variable locus (or loci) can be used to test the accuracy of the RAPD result.
Database entry and CFU quantification
New or existing morphotypes can now be entered into the database. This will also create a new “final” plate number for subcultures, which needs to be recorded on the plate (see photo). CFU counts can be calculated using isolation plate photos as a reference. Relevant isolates should be subcultured to cryo-tube slants and preserved.
Final subcultures should look like this:
