MAKING DIAGNOSTIC SLIDES


In the studies of arbuscular fungi conducted by the author of the website, intact and crushed spores mounted in polyvinyl alcohol-lactic acid-glycerol (PVLG) and a mixture of PVLG and Melzer’s reagent (1:1 v/v) were used to make their permanent vouchers. Most spores of the genera Glomus and Gigaspora were crushed by applying light to moderate pressure on the cover slip with the end of a needle. Spores of the genera Acaulospora, Archaeospora, Entrophospora, Pacispora, and Scutellospora were frequently also first placed in a drop of lactic acid and then slightly crushed with the end of a needle. Such crushed spores were stored for 12-24 h and then used to prepare permanent slides, as described below. The earlier crushing loosens adherent layers, especially those of germination walls and, thereby, makes them to be better visible.

To make permanent slides, the following procedure is used:

1. Two small drops of PVLG and PVLG+Melzer’s reagent are placed on the surface of a clean microscope slide, allowing space on one end for a label.

2. About 10-20 spores are added to each drop, depending on the spore size. The mountant is allowed to set for 3-5 minutes to become more viscous before adding a cover slip.

3. Clean cover slips are placed on drops of PVLG and PVLG+Melzer’s reagent at a ca. 45o angle.

4. Spores are crushed by applying light to moderate pressure on the cover slip with the end of a needle. This is done under either a dissecting microscope or a light microscope. Crushing of spores under a light microscope enables to precisely exhibit all significant diagnostic structures and place them in a position needed to clearly show a given structure in a picture.

5. The slides are labeled.

6. Slides are placed in an incubator at 65oC for 12-24 h to clean the spores and the mounting medium from oil droplets and air bubbles. Additionally, the heating decreases the space between the lower surface of the cover slip and the upper surface of the microscope slide and, thereby, makes easier to take pictures of the subcellular structures of the spores mounted.

REFERENCES

Koske R. E., Tessier B., 1983. A convenient, permanent slide mounting medium. Mycol. Soc. Am. Newsl. 34, 59.