1. Download Canon EOS Utility to your computer: Download link
   (Listed as “EOS Digital Solution Disk Software”)
2. Ensure photo setup is as pictured (in the flow hood in Adam’s office on the 2nd floor):
   
3. Hook your computer up to the camera to enable live shooting and direct photo downloads.
4. Turn on the hood blower and wipe down all surfaces with 70% ethanol.
5. Set the camera dial to M (Manual) and adjust to the following settings:
   Manual focus
   Large resolution (use Raw for poster photos)
   Shutter 1/100
   F9.0
   ISO 400
   Spot metering
   Flash white balance setting
   WB Shift: B5 G2
   As it appears on the EOS Utility:
   
6. Set a plate open-side-up underneath the camera and manually focus the camera to the top of the agar.
7. Add a piece of paper displaying the underlying plate number in the photo.
8. Proceed taking photographs of the top and bottom of each plate. Settings do not need to be altered between plates/photographs.
9. Save photographs to our lab drive (Forest Entomology:\Plate photos) and name them according to plate number (eg: “plate number XXXX.jpg”)

10. Keep the plate at 4-10° C until ready for discard. Plates should be discarded into an autoclave bag.

    **Note on batteries for flash: Only use Panasonic eneloop batteries. All batteries should be equally charged. Disconnect batteries after use. Battery charger is in camera drawer in Hulcr lab.

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:

Small culture slants have the advantage of easy storage, integration with our database system, and safe shipping to collaborators.

1. Sterilize 2mL cryotubes by autoclaving or prolonged exposure to UV light.
2. Under a biosafety hood, arrange the open tubes in a rack so that the open end of the tubes face almost horizontally (about 15° ).
3. Expose the tubes to UV light again if necessary to ensure they are not contaminated.
4. Add about 1mL of autoclaved media (usually PDA) to the bottom of the tube.
5. Let the media harden and cool UNDER AN ANGLE in the hood before adding the tube caps.
6. Store in the fridge.

Following this, you may culture the fungus on the slant media, label it with a scolytos vial number, and place it in the incubator, slightly open with parafilm, to grow. After the media has a culture, you may add 10-20% glycerol to fill the vial, close it completely and use the “Mr. Freeze” jar to slowly bring it down to freezing in the -20C. It can then be moved to the -80C for long term storage.

For sending the tubes to collaborators:

1. Keep the culture for a couple days before shipping to make sure the fungus is growing and isn’t contaminated.
2. Wrap the lid-tube joint with parafilm, so that the lid doesn’t get loose.
3. Tape the tube to a piece of paper so that it doesn’t roll around in the envelope during shipping.
4. Make sure it is sufficiently padded and that the tube and envelope are labeled.
5. Ship IMMEDIATELY: there is very little oxygen for the fungus to breathe.