2.3.1. Sample preparation
Morphological analyses are best carried out on mite specimens that have been mounted on glass microscope slides. For this, a specimen must first be cleared of its soft tissues before being mounted on a slide. This is achieved as follows.
1. Remove specimen from preserving medium.
2. Immerse specimen in Nesbitt’s Solution (see recipe below) in the depression of a concave slide.
3. Wait until the specimen becomes saturated with Nesbitt’s Solution, and then push it under the surface of the solution to make it sink to the bottom, using a fine needle.
4. Place a cover slip over the depression of the slide.
5. Warm the slide in an oven for 1 hour at 45 °C.
The specimen should become free of body tissue and appear transparent, but older specimens may require further clearing in the oven for several hours or overnight.
This procedure can be speeded-up by warming the slide over a flame or hotplate for a few seconds, instead of placing it in an oven. However, extreme care should be taken to avoid boiling the Nesbitt’s Solution, which will destroy the specimen. Laboratory gloves and coat should be worn when clearing specimens.
The cleared specimen is then mounted as follows:
6. Remove specimen from the Nesbitt’s Solution and transfer it to a drop of Hoyer’s Mounting Medium (see recipe below) on a glass microscope slide.
Note: the drop should be just large enough to form a thin layer when a cover slip is placed on top, without overflowing around the edges of the cover slip.
7. Push the specimen down through the Hoyer’s so that it rests on the slide, using a fine needle.
8. Gently lower a cover slip (thickness No. 1, diameter 16 mm) over the drop of Hoyer’s, starting from the edge of the drop and letting it slowly settle over the drop under its own weight, spreading the Hoyer’s as it goes.
9. Place the slide horizontally to cure in an oven at 45 oC for at least 2 weeks.
10. Label and store slide.
Hoyer’s medium does not completely harden and remains water-soluble, so that the slide can be reheated and specimen floated off the slide for dissection or re-mounting. For long-term storage or for transporting, the edges of the cover slip should be sealed with some water-resistant material, such as clear fingernail varnish. Laboratory gloves and coat should be worn when mounting specimens.