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Revival - do it yourself experiment (Part 1)

For all newcomers we present an  Echiniscus  tardigrade from the concrete sidewalls along the Isar river, city of Munich, Germany. At this location lives a complex waterbear population composed of a rich variety of different ethnic waterbear groups: coming from the Bavarian mountains they have been floating as passengers on any kind of wooden material and finally ended up as Munich city residents.

[ tardigrade from the city of Munich, Germany ]

Echiniscus tardigrade, resident of the multi-cultural Munich city waterbear community. Please note the red eyes and the green moss juice within the stomach-intestine area.
Length ~ 0.25 mm.

Herewith we follow numerous requests by teachers to offer once more, in detail, an experimental procedure that can be used also in classroom in order to demonstrate waterbear revival after desiccation.

Though some of you might think that we obiously are overdoing and behaving by far too sentimental in a totally barbarian world: please behave generously and put the tardigrades back on a wet moss cushion after your experiment - no need to discuss here whether your motive should be fair play, religious feeling, general respect against nature or simply the pleasure of irrational, totally non-economic compassion: just do it!

The procedure is as follows: a small moss cushion, preferrably from an old stone wall or pavement grooves, is put upside down into a petri-dish (*) with either good quality tap water or pond-water. Demineralized water is less ideal but will do as well as the moss sample contains the lacking minerals and in the end everything will end up again in mineral harmony ...
In case of doubt you might as well use still mineral water, one with relatively low mineral content - just compare the chemical analyses on the labels.
The setup is shown   here  . Take care to avoid high soil contents in the moss. The petri dish with the moss should stand still (cool environment, no sunlight) for a few hours or over night. Afterwards you should remove the moss cushion. Most of the tardigrades from the moss will have been fallen to the bottom of the petri dish by now. You can examine the residue in a good dissecting microscope. An optimum dissecting microscope has good quality optics with a magnification of 20x to 30x and wide field eyepieces. Once you have found the tardigrades you can pick up one or two of them up by means of a standard pipette (*) and transfer them to our recommended  micro aquarium .
The ideal surrounding water quantity should allow the water to evaporate slowly, with the micro aquarium almost totally closed, so that the residual water will vanish during a long period, e.g. 6 to 7 hours. Quicker evaporation will kill the tardigrades as the time will not be sufficient for the tardigrades to prepare their special desiccation chemistry (synthesizing the trehalose sugar and positioning it carefully around the delicate protein molecules).
Alternatively you might as well offer an extremely tiny moss leaflet as a substrate. When the water recesses the tardigrades will look out for appropriate shelter and clinge to the tiny moss leaf in order to have optimum survival conditions. An optimum tardigrade dry 'tun' looks roundish, symmetrically wrinkled:

[ Anhydrobiosis of tardigrades (1) ]

Various tardigrade species, dry state, clinging to a moss leaf. In the very center a crystal clear Eutardigrade tun, surrounded by coloured  Echiniscus  tuns.

The tuns are very small and it is not easy to see them after desiccation with the bare eye.

[ Anhydrobiosis of tardigrades (2)  ]

The small water droplet has evaporated completely and, apart from our water bear there is only some salt residue from the water spread out over the diameter of the water droplet.

Loupe magnification (10x) is sufficient in order to find the tuns on the ground of the micro aquarium, a dissection microscope will even serve better.

[ Anhydrobiosis of tardigrades (3)  ]

Salt residue from the water with tardigrade tun, at loup magnification.
The tun shown here is a tun of the tardigrade Milnesium tardigradum , an older individual with conspicuos reddish colour.
Beware of dust, pullover fibres etc.

When looking at the tuns under a normal microscope, at medium magnification (100x) we will notice the typical horizontal striae and sometimes a protruding claw, filament or even the buccal tube in the core of the tardigrade tun.

[ Anhydrobiose der Bärtierchen (5) ]

Tun of Milnesium tardigradum as seen in the light microscopes (incident light).
Length 0.15 mm.

The other half of the experiment - the revival - has to take into account some requirements as well. It is understandable that the revival process consumes lots of energy and oxygen. In particular we will have to avoid elevated temperatures and will have to provide enough volume for the tardigrade to stretch and to start movements. This will be explained in more detail in our next issue.

(*) Equipment:
petri dishes and pipettes can be bought via Ebay or - sometimes - at your local pharmacy. Optimum pipettes are made of glass and end with a long thin tip.

© 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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