Cutting sections and mounting - The world under the microscope

A microtome is a tool specially designed to cut very thin slices of tissue ( coupes). A histological coupe has a thickness of 1μm to about 7μm (1μm = 1/1000mm). There are various types of microtomes. On the page 'microtomes' different types of microtomes are described in detail. On the 'download'-page a manual of the A&O 820 and LKB 2218 Historange can be downloaded in .pdf format.

After the coupes have been cut on a microtome, they must be mounted on an absolutely clean object glass. The slightest contamination is followed by partial or even total loosening of the cuttings during further dehydration or staining steps. If pre-cleaned slides are purchased, they too will not be sufficiently clean. The slides can be cleaned in a mixture of ether alcohol or benzol-alcohol (mixture ratio 1:1) [1]. A cheap alternative is acetone alcohol (mixing ratio 1:1). A good method of cleaning is to place a stock of glasses in a staining tray that may remain there for a few hours in the acetone alcohol (weeks of impurities). Immediately before the glass is actually used, take it out of the liquid and wipe well with e.g. lint-free tissues.

Allow the clean slides to dry on a heat plate to allow the last film of acetone to evaporate. Only now the slides are clean enough.

Various methods have been developed for attaching paraffin coupes to slides. A good method is to use a hot water stretching bath. Ordinary tap water is not recommended because of the many impurities that are carried between the coupe and the glass. Demineralised or distilled water is better. The water should remain at a constant temperature between 35-40°C. If the water is too cold, a large coupe will not stretch properly. Water that is too hot will expel the cut (the paraffin becomes too soft and the tissue begins to deform). Tissue fixated with picric acid (Bouin) or trichloracid must not swim too long in the warm water, otherwise the connective tissue in the coupe will begin to swell[1].

The coupes can be thrown on the warm water with, for example, tweezers. It is very important that the shiny side of the coupe (this is the side facing the knife) lies on the water[1]. This requires some skills. The trick is to allow the coupe to stretch without disturbing folds. After a few seconds, the coupe has stretched and can be taken up on a clean slide. This technique has to be practiced a lot for a good result. Click on the image to load a video showing the pickup of the coupes. After pickup, the slides should dry thoroughly. This can be done on a heat plate or drying cabinet at 42-45°C. Drying time is a few hours, but may be longer and is not harmful[1]. An alternative is drying in a warm place, e.g. in a petri dish or sample folder. After drying, the coupe must be matt on the glass side. Glossy parts indicate air between the paraffin and glass. There is a good chance that during deparaffining or moisturizing, the paraffin will partially detach or even completely swallow off the glass. For the vast majority of cuts, this method of adhesion bonding is sufficient for a good result, however, there are tissues that have a rapid tendency to detach, or tissues that are fixed with solutions containing e.g. potassium dichromate can also detach easily[1]. A good solution for this is to use protein glycerin. In various literature it can be read that the protein has to be applied to the disposable glass. However, the author's experiences are not so good because too much is applied quickly, which is detrimental when staining later on. For example, the dye eosine quickly tends to attach itself to the protein. A good tried and tested method is to mix a few drops of protein glycerin through the warm stretch water. In this way, a small amount of protein is absorbed which is just enough to allow the coupe to adhere well to the glass.

Protein glycerine can be bought ready-to-use but is also easy to make yourself. Required: An egg, glycerine and thyme (preservative). The glycerine (a few ml at most) is heated in a test tube or possibly large spoon with some thyme (one or two knife points). Stop as soon as the glycerine starts boiling. Filter and collect the clear yellowish or brownish liquid. Break an egg and let some of the very liquid egg white run into a cup or dish. Absolutely no splash of yolk and preferably not the tougher egg white part. This is very easy by putting the egg in a tea strainer. The very liquid egg white then drips through. Now mix one part of cooled glycerine with three parts of protein and mix well (e.g. 6 ml protein with 2 ml glycerine). Foam will appear. Filter the mixture again. The part that runs through it quickly is suitable. This protein glycerine, can be kept in the refrigerator for a few weeks. If turbidity occurs (shake for a moment) it should be replaced by fresh ones. The smell will quickly become unpleasant (wet dog smell), but that is not a problem.

References:

[1] Prof. Dr. Peter Böck (1989, 17., neubearbeitete auflage), Romeis Mikroskopische Technik, München. Verleger Urban & Schwarzenberg. Chapter 7, 'Aufziehen der Schnitte und Vorbereitung zur Färbung' blz 168, 169 en 170.