IN THIS LESSON WE WILL DISCUSS:

1) MICROSCOPY TECHNIQUES
2) BEST PRACTICES

Step 4: Squash mounts

*note that you’ll need a micrometer + ImageJ for fungal measurements

*Always create sub packets with fungal fruitbodies to place inside your larger specimen packet (e.g., there are #8 fruit bodies on this Polytrichum commune stem)

  • First isolate a fruit body and mount it on a glass slide. Then carefully place a cover slip

  • Note that pursuing these interactions can often feel like scanning a sea of green for geologic inference

  • Gently press down the cover slip with your forceps. If this doesn’t work, try tapping with your forceps

  • In order to get thin squash mounts, it’s recommended to cut a well developed fruit-body out of the infected leaf and remove adjacent host tissue before squashing

  • This process of squashing allows the fungal fruit body to rupture and release critical morphological characters like asci, ascospores, and interascal filaments that are required for discerning taxonomic and physiological information

  • These characters can then be measured using the oil immersion lens at 100x magnification on a compound microscope

  • It is important to note that fruit bodies are frequently immature or only presenting their anamorphic state

    *In this case, the morphological characters required to ID the fungus are not present

    *If possible, use phase contrast for greater resolution

Step 3: Microsite identification

  • Newly acquired microsite knowledge can now be implemented for locating known bryofungi microniches on the host bryophyte

  • In Polytrichalean mosses, it is essential to screen lamellae as the microniches present there represent the greatest diversity of ascomycete diversity of all known microniches across bryophyte hosts

  • Next, remove fruit bodies from infected tissue using dissecting forceps and prepare for squash mounts

Step 2: Detritus removal

  • Remove excess water droplets with a towel and then place specimens under a stereomicroscope for preliminary analysis and preparation. Each shoot should be screened for the presence of bryophilous fungi and species-specific microniches

  • Bryophyte hosts previously known to contain greater fungal counts can be more carefully analyzed for fungal fruit body detection

Step 1: Preliminary screen

  • First take a random sampling of moss stems and individual leaves for the initial bryophilous screen. Next, rinse the specimen in gently flowing tap water to loosen debris such as pollen grains, algae, invertebrate eggs, and sand particles (Döbbeler, 1978)

Step 5: Staining

  • The stain lactophenol cotton blue (LCB) is typically used by mycologists to improve the visibility of certain fungal structures *see ink-vinegar staining method for alternative staining options here

  • Iodine reactions should be proven with Lugol’s solution (IKI) (Baral, 1987)

  • Older specimens are sometimes treated with 5% potassium hydroxide (KOH)

  • Measurements should always be first taken in tap water

    *Permanent slides can be made with: Lactophenol cotton blue, glycerol, commercially available mounts, and clear nail varnish around slide