The small size of mites and larval ticks means that their identifying structures need to be viewed at high magnification (up to about 1000x) under a compound microscope. Phase¬ contrast and differential interference contrast (DIC) (= Nomarski interference contrast, NIC) are illumination systems on compound microscopes that are very useful for studying Acari. They influence the paths of light waves passing through an unstained specimen and the material surrounding it. As a result, structures can be seen in greater relief than with ordinary compound microscopy.
Specimens are mounted on microscope slides for examination. Killing mites in alcohol sometimes causes the chelicerae to withdraw into the body and the legs to curl up. Dropping live specimens into boiling water has the opposite effect. This makes it much easier to mount specimens so that structures important for identification are clearly displayed. Be aware that the mounting process causes flattening of specimens. Sometimes the dorsal and ventral body surfaces come into such close contact that they appear superimposed upon one another (e.g., Figure). When searching for diagnostic characters, it is therefore important to keep checking which surface is in focus.
Because compound microscopy relies on light being transmitted through an object, specimens need to be cleared, i.e., the tanning of the exoskeleton reduced (= maceration) and the internal tissue removed. Effective clearing agents are lactic acid (50-60 per cent by volume, diluted with distilled water) and lactophenol (lactic acid 50 parts, phenol crystals 25 parts, distilled water 25 parts). Both agents double as temporary slide mountants and their use is described below.
These have the advantage of allowing specimens to be orientated during examination. Also, mounted material can be returned directly to alcohol if a permanent preparation is not needed.
1) Put a drop of 50-60 per cent lactic acid or lactophenol in the centre of a flat glass microscope slide (a thickness of 0.8/1.0 mm is recommended). If greater scope for re-orientation is wanted, use a cavity slide.
2) Position the specimen/s in the middle of the drop using a fine paintbrush or needle and push it/them down to the surface of the slide.
3) Prop a coverslip (ca. 19 mm diameter, No. 0 thickness) at the edge of the mountant and carefully lower it.
4) Warm the slide on a hot-plate or photographic dish-warmer at 60-70°C (about hand hot) until the body contents have dissolved. This takes about 20 minutes for the most weakly sclerotized mites (usually white or pale yellow). These forms will also often clear if left overnight at room temperature. Heavily sclerotized specimens (mid- to dark brown) need 60 or more minutes of heating.
5) Position the specimens as required by gently pushing the edge of the coverslip with, for example, a mounted needle.
The gut contents of blood-engorged specimens often obscure morphological features. Piercing the body will aid clearing, and at least some of the blood can be squeezed out by gently pressing the coverslip. Such specimens may need to be treated in a stronger clearing agent than lactic acid. A 10 per cent aqueous solution of sodium hydroxide or potassium hydroxide is usually more effective.
Structures can be highlighted by adding a drop of selective chitin stain such as lignin pink (Evans & Browning 1955) or Chlorazol Black (Coineau 1974) to the mountant.
Although temporary mounts are intended for short-term use, it is possible to keep them for many months. Remove excess mountant from around the coverslip and seal the edge with, for example, EuparalTM, GlyceelTM (= ZutTM) or nail varnish.
These keep specimens in good condition over many years and so are recommended for building up reference collections.
Mountants widely used for mites and larval ticks are based on
Berlese fluid is a commercially available gum chloral mountant.
Hoyer's medium
Is a frequently used variant, cannot be bought ready-made.
Prepare it by mixing the following at room temperature until all solids have disappeared:
distilled water 50 parts
crystalline gum Arabic/acacia 30 parts
chloral hydrate 200 parts
glycerol 20 parts
Pass the solution through bolting silk or filter paper to remove impurities. Then store it in an airtight container.
The amount of chloral hydrate in both Berlese fluid and Hoyer's medium is sufficient to clear very weakly sclerotized mites. Larval ticks and other mites need to be cleared before mounting, as described in Temporary slide mounts. Rinse specimens transferred from lactic acid or lactophenol several times in about 70 per cent alcohol. This prevents subsequent crystallization of the permanent mountant.
Ideally, mount specimens with their body parallel to the short axis of the slide. It is easier to interpret morphology and follow identification keys when specimens are perpendicular to the observer. If a sample contains several examples of the same species, mount some ventral side and some dorsal side uppermost so that there are unimpeded views of both surfaces. Specimens can be dissected in the mountant by teasing them apart with a pair of mounted needles.
The mounting procedure is the same as for temporary preparations except that smaller (ca. 13 mm) diameter coverslips are normally used. Make final adjustments to the orientation of the specimen by gently moving the coverslip. Then heat the slide for 10-15 minutes on a hot-plate at about 45°C. During this time, check that the specimen has stayed in the right position. If necessary, re-orientate. This initial heating causes the mountant to expand, which in turn tends to straighten out the appendages. Drying is completed in an oven at 40°C for about 10-14 days.
Because chloral hydrate-based mountants are hygroscopic, they absorb moisture from the air unless sealed after drying. GlyptalTM insulating varnish and GlyceelTM are effective sealants (Travis 1968, Brown 1997). With a fine paintbrush, apply three coats so that they cover the edge of the coverslip. Allow each coat to dry before adding the next. Nail varnish is also sometimes used but is prone to chipping. A ringing turntable (available commercially) enables the rings to be applied evenly and quickly, and is particularly useful when large numbers of slides need to be sealed.
If necessary, specimens can be rescued for remounting by soaking off the coverslip in warm water (gum chloral mountants) or glycerol (PVA mountants). Scratching the sealant rings with a needle or scalpel will speed up the process. Use only the minimum depth of liquid necessary to cover the slide and check progress regularly in case the specimen floats away unnoticed.
Like alcohol-stored samples, slide preparations must bear full specimen details. A label fixed at one end of the slide gives the identity, sex or life stage of the specimen/s, the name of the identifier, the date of identification and the type of mountant. The latter is included so that the correct method is used if the specimen needs to be remounted. The label at the other end gives the collection data, including place of origin, host/habitat, collector and date of collection. Labels are normally positioned so that the writing/printing is parallel to the slide’s long axis and their top is near to the same edge as the posterior end of the specimen, or majority of specimens. Because the image is inverted during compound microscopy, this arrangement means that specimens can be viewed in the best position for character interpretation (i.e., anterior end away from the observer) and the data still easily read.