Scale preparation

With belly-harriers the species classification is traditionally done by the shape of the scales. Therefore, species identification is often only possible if one can accurately assess the scale shape of the animals. Unfortunately, the scales of gastrotrichs are often difficult to see. Even with DIC - although here the weak birefringence of the scales further enhances the contrast - live observation is not sufficient for species diagnosis in many species. Therefore I would like to present here a possibility for the preparation of the scales and thus for the (mostly) safe species diagnosis.
As an example I have chosen Lepidochaetus zelinkai . Due to the distinctive appearance of this gastrotrich, a scale analysis for species identification is actually not necessary, but some scale details are controversially discussed in the literature, so I wanted to make up my own mind.

L. zelinkai is a medium sized gastrotrich with a distinctive spiny coat:

L. zelinkai : Overview

Even with higher magnification not all details of the scaling become clear:

L. zelinkai : Scales of the ventral interfield

To prepare the scales free, the animal must be macerated. To do this, fix the animal - preferably with a simple Vaseline copressorium - with the coverslip and draw a drop of glacial acetic acid under the coverslip. I stained the glacial acetic acid in advance with very little eosin - this stains the scales at the same time as maceration. The glacial acetic acid dissolves all soft parts of the animal within a few minutes, while the scales of the gastrotrichs are acid-resistant and therefore remain intact. Depending on the scale structure, the scales remain as a cohesive shell or separate from each other. In L. zelinkai , the scales are apparently not interlocked, so the scales disperse:

This type of preparation is quite simple and allows the scale shapes to be accurately assessed. For example, in the above images, the ridges in the scale surface that extend parallel to the leading edge are noticeable. In the literature, these “double edges” of the scales are usually considered to be an optical artifact produced by the double intersection of the focal plane through the curved scales. The above scale analysis clearly proves that these conspicuous ridges really exist and represent a thickening of the scales.

Microaquarium

By microaquaria I mean slide-sized preparations that can be observed directly with a microscope. The optical quality of the microaquaria (especially the thickness of the slides) should be good enough to use any possible magnification. In addition, the life span of the organisms inside should be at least a few weeks to be able to study the behavior or development of the objects. Last but not least, the method must be easy and fast to use - a cumbersome method that requires a lot of equipment is not practical in my eyes in the amateur field.
A few years ago I came across a preparation method on the Internet, which I was able to develop into a long-term microaquarium. Such a microaquarium is constructed as follows:

Construction of a microaquarium

The life of a “normal” temporary wet preparation can be increased by effectively preventing the water from evaporating. This can be accomplished by sealing the cover glass with a Vaseline rim to make it watertight. Vaseline has a number of ideal properties for this purpose:

• it is absolutely waterproof
• Vaseline is permeable to air (known from Vaseline-based moisturizers)
• Vaseline is easy to apply due to its consistency
• it can be found inexpensively in every drugstore

To apply a Vaseline edge to the coverslip, apply a thin (!) film of Vaseline to the heel of your hand and wipe a thin ridge off the skin with each coverslip edge. The coverslip can then be placed directly on the slide (with specimen) with one edge. From this edge, lower the coverslip and slowly press the coverslip to the desired thickness using a wooden stick. Excess water is thus pressed out from under the coverslip and can then be easily removed.

To create a long-lasting microaquarium, it is necessary to include a relatively large amount of water in the preparation. If this water buffer is too small, the organisms would die after a few days. Therefore, a microscope slide with a hollow section is used for a microaquarium. To increase the area that can be used for observation, I use a coverslip that is as large as possible (e.g. 24mm x 32 mm or 24mm x 60mm). This maximizes the area outside the hollow grounded section - the area with the optimal slice thickness.

A microaquarium created this way has many advantages:

• Lifespan of organisms up to 6 weeks
• great mechanical stability:
  • cleaning is easy due to the fixed cover glass
  • vertical storage in normal preparation boxes (transparent cover!)
  • absolutely stable conditions also for long-term video recordings
  • Use of immersion oil is unproblematic: cover does not slip, oil does not penetrate under the cover glass
• additional effort for preparation only a few hand movements

But the main advantage in my eyes is that the organisms behave “naturally” after an acclimatization period of about one day in the micro aquaria. The typical great restlessness known from the “normal” temporary wet preparations has subsided and the enclosed animals go about their “daily business”. Most ciliates calmly graze their food or even stand in the same spot for hours, swirling their food in a relaxed manner. Gastrotrichs calm down and emerge from their hiding places after some time and graze - optimally observable - the underside of the cover glass.
To demonstrate these relaxed conditions in a microaquarium, I would like to show the following - almost unedited film - taken in several microaquariums a few days after their creation:

Please click on the picture to start the movie!

For me this kind of preparation is meanwhile the most satisfying way to observe the wonder world in the pond. Of course it is not possible to show the last anatomical details of the organisms. But you get the possibility to observe the behavior or the development of the organisms over a long period of time and without stress.

Coverslip Compressorum

To observe living gastrotrichs in detail microscopically, it is necessary,

• to minimize the slice thickness, as this is the only way to ensure optimal optical conditions for objectives with high magnifications
• to keep the animals immobile
• to minimize the drying of the specimens to the extent that a comfortable observation time can be achieved
• prevent the penetration of immersion oil into the specimen

For this purpose I have developed the coverslip compressorum, which allows to achieve these conditions very easily and quickly.

First, as always, it is necessary to isolate the observation object with a thin pipette under the stereo microscope and apply it to a cleaned slide with as small a drop of water as possible. Care should be taken to ensure that the water droplet is small enough so that the layer of water under the coverslip to be applied does not reach the edges of the coverslip at the desired thickness. Some experience is required for this…

Coverglass compressorium: vaseline bars in yellow.

Some vaseline is rubbed on the heel of a hand. With the upper and lower edge of a rectangular cover slip (I usually use the size 18x18mm) one strips a thin bar of vaseline from the ball of the hand. The vaseline serves as a spacer around the edge of the cover glass. The coverslip is placed on the slide with one edge at an angle - the Vaseline holds the coverslip in this position. Using a small wooden stick, the protruding edge of the coverslip is carefully pressed towards the slide until it reaches the water drop and also rests with the upper side on the slide. The water drop now fills a small section of the coverslip with a relatively large layer thickness.

In this state, the gastrotrich trapped in the water droplet is still free to move and can be conveniently observed with weaker objectives. The small area of the water drop makes it easy to find the object.

Under microscope control, the layer thickness of the water can now be carefully reduced by pressing the coverslip edges just enough so that the gastrotrich is no longer movable and is gently pressed through the coverslip onto the slide. The two Vaseline edges provide sufficient resistance to manual pressure to reduce the coating thickness in a very controlled manner. The two opposing Vaseline edges ensure that

• drying of the preparation is greatly delayed, since evaporation of the water only occurs on the exposed coverslip edges without Vaseline and is therefore reduced.
• the adjusted layer thickness remains almost unchanged when the preparation dries out, since the water drop evaporates only at the edges and only its diameter changes.
• penetration of immersion oil is impossible at the sealed coverglass edges, and there is usually no contact with the sample water at the exposed ones.
• a “pull-through” of chemicals for dyeing etc. is possible without problems because of the open cover glass edges on the opposite side.
• a later manual reduction of the layer thickness until the object is flattened (e.g. for scale analysis is possible at any time in a controlled manner.

With a little experience, the creation of a cover glass compressorum is easy and possible within a few seconds.

I have been using this preparation method for high-resolution observations for years and would not want to do without it!

Double Coverslip

Gastrotrichs are animals in which the upper and lower sides differ greatly. It is often necessary to observe both the ventral and dorsal sides closely. When observing an animal with a coverglass compressorium, usually only one side can be observed accurately. The other side is only accessible when focusing through the animal. However, this results in great optical quality losses - especially with high-resolution lenses. One can then only try to rotate the animal by moving the cover glass. Unfortunately, this method only has a chance of success with round objects and usually ends lethally with gastrotrichs… :cry:

diagonal double cover glass vaseline bridge in yellow

To allow bilateral observation, one can enclose the object between two coverslips and turn the entire coverslip stack on the slide if needed. To do this, use two different sized coverslips. The larger coverslip (e.g., 20x20 mm) is pinned to a slide with a small drop of distilled water. The coverslip should be oriented diagonally so that it can be easily grasped at the opposite corners if necessary. A second, smaller coverslip (18x18 mm) is fitted with a vaseline ridge on two edges (as described in the article coverslip compressor and pressed onto the lower coverslip with the sample in a controlled manner, thus holding the object between the two coverslips.

The object can now be observed with the usual optical quality on the top side. Once observation from this side is complete, a drop of immersion oil is placed on the coverslip (if not already done) and the coverslip stack is detached from the slide at the protruding corners. The stack is rotated and placed with the oiled side on the slide. Now the observation can be made from the now upper, former bottom side. The use of different coverslip sizes ensures that only one coverslip is touched when the coverslip stack is rotated and that the coverslips do not shift against each other.

Equipment

• Flash Light
• Lateral stereo microscope illumination
• Rotatable object guide