Comparison of cleaning methods for delicate insect specimens for scanning electron microscopy

Abstract

The objective of the present study was to compare cleaning methods for delicate insect specimens for investigations with scanning electron microscopy (SEM). As typical specimens we used aquatic larvae of mosquitoes, springtails, larvae of mayflies and caterpillars because they are very fragile and large parts of their body consist of soft tissue. Additionally their cuticle is very often covered with dirt, soil particles or other materials. Cleaning with ultrasonic sound, as the most common cleaning method used for SEM, will destroy fragile insects. Therefore we tested different procedures to remove the dirt particles. In a first approach we compared cleaning with Potassium hydroxide (KOH), Proteinase K, and Triton X in aquatic larvae of flies, which were available in numbers and kept under the same conditions. As our results showed that the treatment with KOH gives the best results we treated in a second approach springtails, larvae of mayflies and caterpillars only with KOH. The springtails and caterpillars were largely free of particles after treatment with KOH; however, the larvae of mayflies were still covered with remnants of diatoms and precipitates of calcium carbonate of the algae. KOH dissolves organic impurities, on the other hand silicon dioxide and lime crusts are not solved. With this limitation, treatment with KOH is a simple technique for routine use as cleaning method for fragile insect specimens for SEM.

The specialized thoracic skeletomuscular system of the myrmecophile Claviger testaceus (Pselaphinae, Staphylinidae, Coleoptera)

“…The protocol recommended by Schneeberg et al (2017) was modified to clean the beetles: the specimens were transferred from FAE into 70% ethanol, followed by 0.5% Triton X100 (14 h), 5% KOH (14 h), glacial acetic acid (3 × 15 min), distilled water (multiple times until the specimens appeared clean), and finally 70% ethanol. Subsequently, they were dehydrated and dried in an Emitech K850 at the critical point.…”

Morphological study of the labial sensilla in Nabidae (Hemiptera: Heteroptera: Cimicomorpha)

“…All of the Nabinae material (heads of insects) were dissected and cleaned in detergent using an ultrasonic cleaner. Due to the large amount of dirt, the Prostemma guttula specimen was cleaned according to the protocol for fixation and was cleaned with KOH (Schneeberg et al 2017). Then, the method described by Kanturski et al (2015Kanturski et al ( , 2017 was followed: dehydration using serial baths of 80%, 90%, and 96% ethanol for 20 min each and two baths of absolute ethanol for 30 min each.…”

Walaphyllium subgen. nov., the dancing leaf insects from Australia and Papua New Guinea with description of a new species (Phasmatodea, Phylliidae)

“…All specimens were sputter coated with silver (SPI Module Sputter Coater with Carbon Module, Structure Probe, Inc., West Chester, PA) and mounted on a circular specimen-stage. The specimen in Figure 10B was cleaned using a potassium hydroxide protocol developed by Schneeberg et al (2017), and allowed to air-dry for 24hours before sputter coating and imaging. This method unfortunately made the antennae brittle and warped after cleaning.…”