Sample Sorting and Identification

A good dissecting microscope with magnification in the range of 10-40x is essential. A preliminary sorting will separate collembola, mites, and other microarthropods. The latter category includes the few tiny spiders, small beetles, and other insects (adults and larvae) that can usually be identified with the dissecting scope. Some may require slide mounts (see later discussion). Collembolan and mite specimens can be transferred to sorting dishes with a fine-tipped pipette (such as a Pasteur pipette), a camel's hair brush trimmed to 3-4 lashes, or a flattened, curved dissecting needle.

Collembolans: Identification of springtails almost always requires high magnification (400x or greater) of cleared specimens, using a good phase contrast microscope. Christiansen (1990) recommends clearing specimens and observing them in temporary mounts; heavily pig-mented forms may require more clearing than the mounting medium provides. He suggests the following reagents:

TABLE 9.1. Procedure for Flotation Extraction of Microarthropods from Soil Samples, Using Saturated Sugar Solutions

Step one Place the soil core in a plastic bag; crumble it gently.

Step two Transfer the crumbled core into a 1-liter wide-mouth jar. Wet it with distilled water.

Step three Add saturated sugar solution, leaving approximately 3 cm of headspace.

Step four Cover the jar with a lid, shake it gently, and let it stand for 2 hours to allow organic matter to float to the surface.

Step five Decant the solution through a number 200-mesh sleeve into a large bowl to trap the organic matter (do not include silt from the bottom of the jar).

Step six Rinse the organic matter with distilled water, then wash it from the sieve into a sample jar containing 95% ethyl alcohol.

Step seven Return the sugar solution to the bowl; return to step four.

Step eight Repeat steps four to seven for three iterations, then combine all organic matter into one jar.

After Snider and Snider, 1997.

1. Potassium hydroxide, a 5% solution, for brief periods only.

2. Lactic acid, an excellent clearing agent; collembolans tolerate long exposure to it. May be mixed with an equal portion of glycerine.

3. Andre's fluid (40 cc chloral hydrate, 30 cc glacial acetic acid, 30 cc distilled water). Clears rapidly but may cause damage to specimens.

4. Bleach. A5.35% solution of sodium hypochlorite will clear heavily pigmented specimens, but is destructive to cuticles.

Christiansen (1990) further recommends the use of depression slides for study of cleared specimens, because weight of the cover glass may crush the arthropod. Slide-mounted specimens are convenient for reexamination and for reference specimens (see later discussion about preparation of temporary and permanent mounts of microarthropods).

Mites: Preliminary sorting of mites into subgroups (Prostigmata, Mesostigmata, adult Oribatei, immature Oribatei, and Acaridida) can be accomplished successfully with experience. Even so, slide mounts will be necessary for confirmation of these identifications. In the sorting dish, the mites are separated into morphospecies and representatives are mounted on microscope slides, depending upon the group. Mesostig-mata, Acaridida, and Prostigmata are usually mounted in Hoyer's medium (see description below), with preliminary clearing for large or heavily pigmented specimens. Oribatids require special consideration because their heavily pigmented and brittle exoskeleton is easily crushed by a coverslip.

Clearing agents for mites are similar to those used for collembolans. Apopular one is lactophenol (Krantz, 1978):

Lactic acid 50 parts Phenol crystals 25 parts Distilled water 25 parts

Specimens may be left in lactophenol at room temperature for several days, or heated for more rapid action. Larger specimens may need to be punctured. After soaking, large mites such as trombidiids may be pressed with a flattened dissecting needle before mounting them. Andre's fluid (described previously) is also recommended for mites as well as collembolans. Nesbitt's fluid (40 g chloral hydrate, 25mL distilled water, and 2.5 mL concentrated acetic acid) is useful for specimens that do not respond to milder clearing agents. Oribatids stored in lactic acid for a few weeks are usually satisfactorily cleared for study.

Most permanent or semipermanent mounting media used for mites (and collembolans as well) are aqueous, in that they contain, or are soluble in, water (Krantz, 1978). Gum arabic and chloral hydrate are the principal ingredients. Hoyer's medium is one of the most popular:

Distilled water 50 ml

Gum Arabic 30 g

Chloral hydrate 200g

Glycerine 20ml

Clear crystals of gum arabic are preferred. Powdered gum arabic is difficult to wet but may be dissolved in alcohol, which is then allowed to evaporate (R. A. Norton, personal communication). If slide mounted specimens are given gentle heat (40°C) for a few days, considerable clearing of the specimen will take place. Slide mounts using Hoyer's medium, if ringed, will last for some years but eventually deteriorate. Canada Balsam is not satisfactory for mites or collembolans because the refractive index of the medium is so similar to that of the cuticle (Christiansen, 1990). Permount is suitable but requires that specimens be dehydrated and mounted from xylene (Adl, 2003). Generally speaking, mite and collembolan specimens should be archived in 70% alcohol, although "constant vigilance" is necessary to guard against evaporation of the preservative (Christiansen, 1990).

As noted above, many specimens of oribatid mites cannot be mounted on slides in the usual manner without crushing them and thus obscuring their features. Following clearing, specimens may be examined in depression slides, partially covered with a cover slip, in lactic acid or glycerin and manipulated with a fine needle. R. A. Norton (personal communication) recommends slide mounts using a procedure with a small cavity drilled into a microscope slide. A small drop of fluid is placed next to the tiny hole and allowed to flow into it. The mite is positioned and allowed to partially dry; then mounting medium and coverslip may be added. Norton further recommends a 50-50 mixture of Hoyer's medium and Nesbitt's fluid for preliminary clearing of oribatids.

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