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The methods for making specimens can differ slightly depending on the producer, but the same principles, such as that detailed structures should not be hidden by glue or other body parts, applies to all specimens.
Specimens should be easy to use and have a low risk of damage. It is better if the specimen has aesthetic value.
Most insects are stored as dry specimens, but if the body is too soft or dry and might be damaged, an immersion specimen can be used. If it is required to store it permanently or observe it in detail with an optical microscope, make it into a slide specimen.
Although the most convenient method to make and preserve specimens is by immersion, it is very inconvenient to observe them. So it is preferable to make dry specimens for observation, if possible. However, if the body is too soft to dry, or detailed observation with a microscope is required, dry specimens are inappropriate. In this case, a slide specimen is produced. A slide specimen can be divided into temporary and permanent ones depending on the duration and whether the specimen could go back to an immersion state. The process of making slide specimens can be very different depending on the mounting medium used.
Storage bottles should be chosen depending on the specimen size. For example, they should not be too large so that small specimens such as mites can be found easily.
When preserving specimens in a state of immersion, a good method is to fill the bottle with liquid and remove gas completely after putting paper in the bottle so the specimen does not move easily, which prevents damage of the specimen. In some cases, cotton is placed on the bottom of the container or the bottle can be stored upside down to prevent the evaporation of alcohol. But if social insets are made into immersion specimens, individuals from several classes are stored in one bottle for future convenience. In many cases, there may be a need to turn immersion specimens into dry and slide specimens. So always keep this in mind.
Some insects with a fragile body only need to be put on a mounting medium, but when the insect is dark colored, non-fragile or its reproductive organs need to be observed, the specimen must go through a clearing process before putting it on the mounting medium. KOH is used most often for clearing. Nesbitt’s solution is also used for lice, collembola and mites. On the other hand, KOH can be used widely for almost all arthropods. Therefore, we plan to deal only with KOH in this section.
Fixation
When making a slide specimen of an insect immediately after it dies or while it is alive, put the collected insect into 90-96% ethanol and boil it in a double boiler to fix it securely to prevent the soft body from being damaged during the preparation process. If the insect has been stored in 70% alcohol for a long time, this process can be omitted.
Removing coloration and softening
To make a slide specimen, it is essential to soften the solidified insect and remove coloration appropriately for convenient observation of the characteristics to be used for classification and identification. These occur simultaneously with the KOH treatment.
Color removal and softening is done until the collected insect becomes partially transparent, so the time required differs depending on the insect. In addition, the time required for color removal and softening may also differ according to temperature of the KOH. In a boiling state, the rate is very fast and it takes mere minutes, but in cold temperatures it may take from several hours to several days. But there is also a risk of damaging the specimen by boiling it, and it is not always preferred.
When a hole to remove contents in the insect is made using an ophthalmic scissors or a sharp insect needle before treating it with KOH, the risk that inner substances will leak when mounting it on the cover glass can be reduced. Take care to prevent the major characteristics used for classification and identification from being damaged. KOH must be removed after completing color removal and softening by immersing the specimen in distilled water for a long time.
When there is a risk that the specimen could be damaged during KOH washing, sink it by adding ethanol to KOH and wash it with ethanol again.
Purification
Then, after leaving it in 50% lactic acid for at least one day or boiling it in lactic acid in a double boiler at 85-90 ℃ for several minutes, the specimen is cleaned and stored in 50% lactic acid prior to mounting.
If you have a large insect collection to be made into specimens, it is convenient to use the following instruments. Prepare a glass tube that is 100 mm in length and about 10 mm in inner diameter and block one end with cotton gauze. Put a paper containing information about the collection on top of the tube. Place the tube upright to prevent the paper from getting wet. In order to protect the specimen, the gauze should not be unwound and not touch the ground. For that, it is necessary to install a special device with a legged-ring shape.
Although there are many substances that can be used for mounting, none of these is perfect; use an appropriate substance according to the type and purpose of the specimen.
Water+glycerin+lactic acid
This mounting medium is usually used to make temporary slide specimens. This medium not only has the advantage of being easy to process, but also has a low refractory angle to allow detailed observation of smaller insects. For example, it is possible to even observe the characteristics of the first instar of a smaller insect.
Alternatively, it has the disadvantage of evaporating fast, and concave slides are needed to compensate for this. Fix two 20x20 mm pieces of cover glass on each side of the cavity and leave about 2 mm of space between them (Figure 40). Supply distilled water to the space and refill it when the specimen gets dry. If it is necessary to temporarily discontinue observation, store it in a very humid place.
Most water-soluble mounting media have the merit of having a low refractory rate (below 1.4) from the characteristic of being a water-soluble substance. Several types are available, but PVA is most preferred. It must be noted that PVA decreases in volume by 50%.
Canada balsam
Canada balsam has been used as a mounting medium for more than 100 years. It is safe and causes a reduction in volume of only about 10% when dehydrated, but has the fatal demerit that the refractive index is 1.54-1.55. Therefore, it is advisable to use PVA on a normal specimen and use Canada balsam for important specimens, such as type specimens.
To mount a specimen on Canada balsam, organize the collection in 50% alcohol and put it into clover oil, passing through 96% alcohol and 100% alcohol. When the body is expanded to a certain degree, apply one drop of Canada balsam on the slide glass and close with a cover glass.
How to perform mounting with PVA
If the slide or cover glasses are not clean, wipe them with alcohol and dry them with a soft paper prior to mounting. Osmosis may damage the chitin by using PVA, and small holes should be made in an appropriate location on the abdomen. If the abdomen is full of fat-soluble substances that cannot be dissolved in PVA, press the abdomen to remove them completely prior to mounting. Then, put the insect on the PVA. If individuals of the same species in different growth stages are placed together on the same slide, the taxonomic value is increased. Pop air bubbles with a needle. Lay a glass over it slowly by completely attaching one side on the slide glass using a clock repair tweezers. After drying it for two days at 55-60℃, it is possible to use it for microscope observation and drying for about a week is sufficient.
If individuals of the same species in different growth stages are placed together on the same slide, the taxonomic value is increased. Pop air bubbles with a needle. Lay a glass over it slowly by completely attaching one side on the slide glass using a clock repair tweezers. After drying it for two days at 55-60℃, it is possible to use it for microscope observation, and drying for about a week is sufficient.
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