Glycerol gelatine
Glycerol gelatine ("GG") melts at about 60 oC. Since multiple melting cycles decreas the quality melt GG only once with hot water and then distribute the GG in small jars, which must be closed carefully. That way you can use small quantities each time. To avoid air bubbles during preparation remove the molten GG with a glass rod and not with a dropper pipette. The frequently recommended method to melt small blocks of GG directly on the object slide produces numerous air bubbles. Never heat GG with a flame or allow it to boil!
Adequate objects: Desmids, filamentous algae, moss, pollen, spores, protonemata, reproductive organs of moss, small crustaceans, rotifers with stable cuticle, small insects, small spiders, mites, ticks.
Preprocessing: The objects are covered with a solution of 4% formalin (10 ml of water plus 1 ml of formalin conc.) for one day. After having removed the chemical fixative the objects are transferred into a flat dish and are covered with at least 30 ml of a solution of 5 grams of glycerol in 100 ml water and then they are put in a dust-free place. After about one week most of the water will have evaporated. Occasionally the objects must be moved into the centre of the dish to avoid desiccation! This preprocessing is necessary to avoid shrinkage. Finally pure glycerol (80% for medical use) is added. In pure glycerol (anhydrous glycerol is unnecessarily expensive and less useful as well) the objects are stable and can be stored.
Sometimes it is preferable to transfer the objects after they have been washed directly from the fixative to an objectslide and to cover them with glycerol-water. When the water has evaporated they must be covered with molten GG; that way the GG will not be diluted by glycerol.
Staining: Only alizarinviridin and nuclear-fast-red when applied together with special mordants render stable stainings: In case of alizarinviridin chromium aluminium sulfate must be used, in case of nuclear-fast-red aluminium sulfate. Such solutions are for sale. The objects are covered with the diluted solution (about 1:10) for one day. Afterwards they are washed several times and then covered with glycerol-water. The objects will never be over-stained as these dyes mainly stain the surface and are not concentrated in the tissue.
Mounting: You need small coverslips (12 mm) and large coverslips (20 mm). Round coverslips lead to more beautiful prepared slides but are much more expensive. In addition a mounting medium solved in toluene or xylene is needed. Very sharp "watchmaker´s tweezers" are very helpful.
The objects are positioned on the small coverslide and then covered with molten GG. If the GG becomes solid, heat it very carefully with a small spirit lamp. Now the coverslip is turned upside down and placed on the larger coverslip to form a sandwich-structure. If GG oozes out this is no problem. Let the coverslips dry for some weeks and check them every few days. In case of shrinkage add some more GG.
After drying remove all GG from the rim of the larger coverslip with a razor blade and cleanse the surface with some alcohol. After that, put a big drop of mounting medium, solved in toluene or xylene, on an object slide and place the sandwich - the smaller coverslip facing downwards - on this drop. Store the prepared slide horizontally and allow the mounting medium to dry.
Notes: If the GG still should contain too much water, it will evaporate after some time, for no sealing in this world is totally impermeable! This drying process will inevitably spoil the prepared slide by sucking mounting medium into the GG. So allowe the GG to dry completely! The mounting medium cannot seal the inner part of the slide hermetically, it is there to prevent the intrusion of bacteria and fungi.
Mounting with glycerol: Professionals often use glycerol instead when applying the "sandwich-method" explained here, because then the time-consuming drying process can be omitted and the prepared slides can be finished immediately. But if glycerol is used the glycerol layer between the coverslides must be very thin, otherwise drops of the mounting medium will penetrate. In addition, the mounting medium must show a sufficient viscosity and the solvent must evaporate quickly in order to prevent the penetration of the resin into the glycerol. So resins solved in toluene are preferable. Such slides must be kept strictly horizontal, of course.