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After our recent discussion of  senile  and  young  tardigrades we will come to a more technical topic: how to produce slides with permanent preparations.

As you will have noticed the  Water Bear web base  has a primary focus on living tardigrades. Due to the nature and special abilities of the tardigrades we can keep them for years in dry form on the shelf, re-animate them with a droplet of water any time and re-dry them again and again.
For this reason we normally do not need any chemical fixation of our findings. Nevertheless we might come across a rare tardigrade egg or an exotically formed cuticula which can be transformed easily into a permanent slide.

The standard system for permanent slides of our objects is called Hoyer's medium, sometimes  Berlese mixture. Though the recipees seem to be a little bit different in the various literature sources the media are basically of the same type. The system is straightforward to use, also for beginners. The specimens can be placed directly into the medium without further preparative steps. Moreover the refractive index of 1.48 provides clear images of the cuticular structures of the tardigrades.

[ Berlese mixture in glass vial ]

We will need some chemicals as specified below:

--  Gum arabic (a water-based natural glue, a solid substance):  1.3 g
--  De-ionized water:  2.2 ml
--  Glycerol (i.e. 1,2,3 propane-triol, a colourless liquid):  0.7 ml
--  Chloral hydrate (i.e. 2,2,2-trichlor-1,1-ethandiol, a poisonous, wax-like material) :  8.8 g

The preparation of the Berlese mixture

Put the gum arabic in a small glass vial and add the water. It will take some time until the gum will fully dissolve. But even bigger gum particles will dissolve with time. When you are in a hurry (everbody is) you might grind the gum to small pieces before dissolving it and furthermore you might stir with a glass rod - but take care to avoid air bubbles and do not heat in order to accelerate the procedure.
Add the glycerol and stir a little bit. Finally the chloral hydrate is added. It should dissolve completely after some time. As a result you should receive a clear, slightly yellow liquid, similar in consistency to honey. It must be stored in a hermetically sealed vial for chemicals (see below, in the red box) and should be kept in a dark place.

How to use the Berlese mixture

The object, e.g. a tardigrade egg, is placed on the center of a slide together with a really minute amount of water, just enough that it will not dry out immediately. Let a small drop of the Berlese mixture fall on the object and put a small, round cover glass on it (you might check out the appropriate amount to fill out the space under the cover glass before, without any object, on a second slide). Keep the slide in horizontal orientation and let it dry in a clean place for one or two days. After this period the gum should be strong enough to keep the cover glass in place. If you want to be a little bit more perfect you might preserve the edges of your specimen with nail varnish (nail polish) at this moment. But also without nail varnish the specimen will last for at least some months, possibly years.

Important hints

a) Chloral hydrate is a poisonous narcotic. Please be careful when handling this substance and above all, keep it away from children. Store it in an appropriate glass vial for chemicals, with a clear inscription and a poison symbol on it.
b) In the literature you will find complaints that the gum doesn't dissolve properly, that the solution must be filtered afterwards, that the preparation will take at least one week of your precious time, that you will have to wait until the 'sediment' has settled etc.
All this might happen in case you have bought poor quality gum arabic. Even in pharmacies you might get terrible quality gum with lots of dirt in it. Be clever and buy only gum arabic in form of transparent pieces, the bigger the better and avoid fine gum arabic powder.
c) Do not heat the gum arabic in order to accelerate the dissolving process. It will finally dissolve in the specified amount of (cold) water.
d) Though the quantity of 8.8 g chloral hydrate might look like a typewriting error at first glance: this quantity will sink in and finally dissolve in the small volume of the gum arabic solution - a real miracle.

[ tardigrade cuticula ]

Preparation in Berlese mixture:
Empty tardigrade skin left back by a tardigrade after moulting.
Image width ca. 200 µm.

Natural tardigrade colour - like the vivid red of some echiniscus tardigrades - tends to bleech markedly within a few days and will finally completely vanish in Berlese mixture. Furthermore the Berlese mixture is an aqueous, semi-stable system which might become too dry and might crystallize some day. In order to get more permanent preparations you will have to apply some lacquer seal around the edges (e.g. nail varnish or black acrylic lacquer).

But, as you are a microscope amateur, try not to stick too much to permanent preparations. Just keep in mind that tardigrades are  living marvels:

[ tardigrades; tardigrade echiniscus ]

Young echiniscus tardigrade - alive.
Please note that the youngsters have only two claws on each leg.
Length ca. 150 µm.

And, above all, do not miss the next issues of the Water Bear web base:

June (#24): what kind of microscope can be used for tardigrade watching? - with 3 real images of the same object taken through 3 different types of microscopes.
July (#25): issue 25 - a small jubilee. We will look at the habits of our audience, where they come from, how long they use to stay on the website etc. - full privacy is guaranteed, of course.
August (#26): a really strange tardigrade - Cornechiniscus.


Udo Sellenschlo: Die Berlese-Mischung - ein fast vergessenes Einschlußmittel.
Mikrokosmos 70 (1981) 239-240.

Otto Kraus: Hoyers Gemisch statt Polyvinyl-Lactophenol.
Mikrokosmos 73 (1984) 54-55.

© 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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