17.2.3. Microscope slide mounting
The features that need to be seen for the identification of many of the smaller insects (and their immature stages) often can be viewed satisfactorily only under the higher magnification of a compound microscope. Specimens must therefore be mounted either whole on glass microscope slides or dissected before mounting. Furthermore, the discrimination of minute structures may require the staining of the cuticle to differentiate the various parts or the use of special microscope optics such as phase- or interference-contrast microscopy. There is a wide choice of stains and mounting media, and the preparation methods largely depend on which type of mountant is employed. Mountants are either aqueous gum-chloral-based (e.g. Hoyer’s mountant, Berlese fluid) or resin-based (e.g. Canada balsam, Euparal). The former are more convenient for preparing temporary mounts for some identification purposes but deteriorate (often irretrievably) over time, whereas the latter are more time-consuming to prepare but are permanent and thus are recommended for taxonomic specimens intended for long-term storage.
Prior to slide mounting, the specimens generally are “cleared” by soaking in either alkaline solutions (e.g. 10% potassium hydroxide (KOH) or 10% sodium hydroxide (NaOH)) or acidic solutions (e.g. lactic acid or lactophenol) to macerate and remove the body con- tents. Hydroxide solutions are used where complete maceration of soft tissues is required and are most appropriate for specimens that are to be mounted in resin-based mountants. In contrast, most gum-chloral mountants continue to clear specimens after mounting and thus gentler macerating agents can be used or, in some cases, very small insects can be mounted directly into the mountant without any prior clearing. After hydroxide treatment, specimens must be washed in a weak acidic solution to halt the maceration. Cleared specimens are mounted directly into gum-chloral mountants, but must be stained (if required) and dehydrated thoroughly prior to placing in resin-based mountants. Dehydration involves successive washes in a graded alcohol (usually ethanol) series with several changes in absolute alcohol. A final wash in propan-2-ol (isopropyl alcohol) is recommended because this alcohol is hydrophilic and will remove all trace of water from the specimen. If a specimen is to be stained (e.g. in acid fuchsin or chlorazol black E), then it is placed, prior to dehydration, in a small dish of stain for the length of time required to produce the desired depth of color.
The last stage of mounting is to put a drop of the mountant centrally on a glass slide, place the specimen in the liquid, and carefully lower a cover slip onto the preparation. A small amount of mountant on the underside of the cover slip will help to reduce the likelihood of bubbles in the preparation. The slides should be maintained in the flat (horizontal) position during drying, which can be hastened in an oven at 40—45°C; slides prepared using aqueous mountants should be oven dried for only a few days but resin-based mountants may be left for several weeks (Canada balsam mounts may take many months to harden unless oven dried). If longer-term storage of gum-chloral slides is required, then they must be “ringed” with an insulating varnish to give an airtight seal around the edge of the cover slip. Finally, it is essential to label each dried slide mount with the collection data and, if available, the identification (section 17.2.5). For more detailed explanations of slide-mounting methods, refer to Upton (1991, 1993) or Brown (1997) under Further reading.