In this issue of the Water Bear web base we will describe the favourite homes of terrestric water bears, how to get hold of the animals within and how to study them under a microscope in the soft way i.e. without doing harm to them.

What kind of equipment do we need?

Well, first of all some glassware: we should have a petri dish, a glass pipette, slides and cover glasses. Furthermore - and what seems most important to me - a good dissecting microscope with about 25 x magnification (fig. 1) is needed to screen the petri dish, in particular when there are only a few tardigrades within the sample. Otherwise you might seek without end. When you will have found a water bear and will have transferred it onto the slide by means of the pipette, a rather primitive microscope, even the toy microscope in fig. 2 will give you a first visual impression. Just keep in mind that the typical water bear body length is about 0.3 - 0.5 mm, so you need above all a    low    magnification (e.g. 40 - 50 x) to start with.

Fig. 1: Dissection microscope - it will be much easier with this low magnification (20 x ... 30 x) microscope to find the water bears within your sample under incident light (light coming from above). The field of view should be as wide as possible, do not waste your precious leisure time with those "light at the end of the tunnel" devices.

Fig. 2: Once you have found the water bears, get hold of them one by one by means of a fine glass pipette (a clean eye-dropper tube might do as well), transfer them to a slide and then watch them in a regular microscope. Even the toy depicted above will give you a first visual impression.

The best localities to start with when searching tardigrades

Collect sun-dried moss cushions from white rocks, from natural stone walls and from terracotta roof tiles. Many tardigrades prefer calcitic stones as they need some calcite to build up their stiletto teeth. Mosses from forests are less appropriate - many tardigrades prefer mosses which become completely dry every few days and this is not the case with forest mosses. You will come across tardigrades from time to time also when screening freshwater samples from ponds. But as a rule the population density there will be much lower than in mosses. Avoid smelly and permanently damp mosses. Water bears like mosses which are free of bacteria and funghi. As a consequence it is advisable to store the collected mosses in a way that they can dry completely, e.g. you might expose your finds some time to direct sunlight or keep them in paper bags at a dry place.

How to find the water bears in the samples

The following procedure is recommended by most authors:

Choose a moss cushion which just fits into the petri dish you have. Remove most of the loose earth particles if possible and don't panic in case some of the less microscopic moss inhabitants should flee and crawl across your kitchen table!

Place the moss cushion upside down into the petri dish and fill with tap water, rain water or de-ionized water. After some time the moss will be soaked and you will have to add some more water. When the moss will be fully soaked after a few minutes the water level in the dish should still be a few millimeters. Refill some water if necessary. Leave the dish with the moss like it is for at least some hours or overnight.

Take the wet moss cushion out of the dish und look out for the tardigrades by means of a dissecting microscope at a magnification of at least 20 x.
In case you should have no dissecting microscope you might try a good mineralogist's loupe (10 x). A black underground gives the best contrast and the light, e.g. from a torch, should come from the side. Look out for little animals which seem to move like puppies. Some of the water bears are red like bricks, most of the bigger ones whitish or transparent.

To find something unknown for the first time in life is always a problem. So it is helpful to have an idea how the tardigrades look like at low magnification. Once you will have found one of them it will be easier for you to recognize the rest. Beginners will need some patience and possibly will find no tardigrades at all for some time. It is difficult to decide for them whether they might have just overlooked the tardigrades or whether the sample simply contains no tardigrades at all. As a guideline for this phase of learning I have placed an image below which illustrates the impression which you might have when looking at a petri dish on black ground with the light from a torch coming from the side and with no more technical equipment than a 10 x loupe. I have chosen a cheap loupe with some edge distortion to be as realistic as possible. The circle on the left side indicates the presence of two big water bears (Macrobiotus sp., ca. 0.5 mm overall body length). In the center of the circle on the right side is a much smaller red   Echiniscus  tardigrade similar to those shown on the above logo of the   Water Bear web base . Note that the short legs are barely visible at this magnification. Sometimes the tardigrades clinge to minor debris or earth particles and therefore might be even more difficult to detect.

View through a 10 x loupe on a petri dish with tardigrades in water.

How to study the water bears in detail

By means of a pipette it is possible to extract a detected tardigrade out of the petri dish and to transfer it on a slide. You might check the drop on the slide afterwards to see whether the transport procedure was ok. For low magnifications (until to the 10x microscope objective) it is not absolutely necessary to use a cover glass whereas for higher magnifications (20 x objective and higher) you can't work without a cover glass. Furthermore in my opinion it is absolutely necessary to have one cover glass on the right and left side of your droplet both of which will function as spaceholders and guarantee a minimum height of 0.16 mm which is necessary to keep the tardigrades alive.
Perhaps you are lucky and stumble across one of those marvellous   Echiniscus  water bears which really deserve the term "water bear":

Portrait of an  Echiniscus  tardigrade. Image width about 100 µm

As we are highly motivated amateurs and real enthusiasts it goes without saying that we should place the tardigrade back onto its moss cushion after the investigation. It is also worth while for ourselves, as we can have the moss cushion redried and the tardigrade revived later once again. So after some time you will get a collection of various mosses inhabited by various tardigrades.

Good bye formaldehyde and see you, primitive insect needle ...

Those readers who consider the localities described in this issue as too primitive and boring might look forward to the more exotic localities which will be subject of the next  Water Bear web base  issue. We will look over the shoulder of the renowned tardigrade scientist Eveline du Bois-Reymond Marcus and discuss her finds from Lat. 43° 4´ N, Long. 31° W. Do you already have an idea where this might be?

© Text, images, and video clips by  Martin Mach  (webmaster@baertierchen.de).
Water Bear web base is a licensed and revised version of the German language monthly magazine  Bärtierchen-Journal . Style and grammar amendments by native speakers are warmly welcomed.

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