Protocol

Materials

• 1000ul and 100ul pipettes
• pipettes tips
• two pairs of fine hard forceps
• petri dishes for beetle dissection
• microscope in hood
• vials
• vial rack
• sterile 1X PBS
• alcohol-resistant marker

General notes

1. Wash the beetle with a surfactant and/or saline, to get most of the dirt off. You don’t need to get every last fungal spore off the surface (we will dilute those away). You also don’t need to use any toxic solutions, often used in older works. Some people use ethanol for surface sterilization; it probably helps in removing some contaminants, but there is also a high percentage of the mycangial symbiots that die. We have tested it…
2. Don’t grind up the whole beetle – focus on the right body part. If the mycangium is in the head, use the head (most Xyleborini). If it’s in the prosternum (for example, Xyloterini, Corthylini) or pronotum (Platypodinae), use the prothorax. If it’s in the mesonotum (the Xylosandrus–Anisandrus clade of Xyleborini) it gets a bit trickier, but with a little practice you will learn how to excise that general part of the body out as well. The main point is to avoid most of the surface, the alimentary canal (particularly the hind gut which is full of yeasts) and the space under elytra, which also hosts many unwanted associates (including nematodes). Yes – the space under the elytra is dirty. When you want to study the microbial associates of people’s mouth, you also don’t grind up the whole person.
3. Dilution plating! This is the ESSENTIAL part of the process. Plate several dilutions of your inoculum, and use the lowest one where some Colony Forming Units end up growing in the end. The goal here is to dilute away all the non-specific associates which are likely present in lower abundances, and only capture the abundant symbionts. Those are typically present in thousands of cells, so you have a good chance of getting mostly those in the lowest dilution.

Dilution plating

1. Prepare two tubes per each sample, label them “0.1” and “0.01”, fill each with 500 ul of water or PBS.
2. Suspend mycangium in the tube “0.1” and vortex.
3. Plate 50 ul of the suspension on media. Record the plate as “0.1 dilution” in [PLATES].[note] in the Isolations database.
4. Transfer 50 ul of the initial suspension to the second tube (“0.01”) and vortex.
5. Plate 50 ul of the second suspension on second media plate, and record that plate as “0.01 dilution”.
6. Plate 5 ul of the second suspension on a third plate, and record it as “0.001 dilution”.

Quantititive extraction for fungus community characterization:
See Calculating colony-forming units