Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Paraffin wax: crystals, additives and cutting
What are the best polymers or other additives for reducing crystal size and improving the cutting propereties of paraffin wax?
Paraffin wax is a mixture of (virtually) straight chain hydrocarbons. Note the word "mixture". Unless you go to enormous lengths (of purifying or searching for a fine chemical supplier), you will ALWAYS have a mixture. There is a relationship between hydrocarbon chain length and melting point, but as the waxes are always mixtures, melting points are never exact, either in the compounding or the measuring, but that is another story!
Perhaps more important than the melting point is the "plastic point," but that is virtually ignored by our suppliers. The plastic point occurs about 10 C below the melting point and its meaning should be fairly obvious - try softening a piece of physiotherapy wax in your hands and that should explain all you need to know. The reason the plastic point is important is related to the sectioning properties of the wax, but we will come to that later! Crystal size is important in the wax surounding the tissue and in the tissue spaces, but not in the tissue per se. Molten wax infiltrates the specimen; the size and shape of crystals will be influenced by the tissues as the molten wax solidifies - i.e. crystalises. So we cannot have "small crystals" infiltrating although smaller crystals will result from solification in denser tissues.
Some of the theory behind this suggests that wax crystalises first as flat "plates," the higher melting point hydrocarbons crystalising first. As successively lower melting points deposit further plate crystals, they pile up upon one another. Distortion due to these dynamic events forces the edges or corners of the most well developed plates to curl and roll. Eventually, that gives rise to needle shaped crystals, which some "experts" consider most ideal for microtomy. All this will be contingent upon the boundaries imposed upon the process by cell and tissue structures. During microtomy, essentially two types of forces are exerted in the cutting process. Flow shearing and point-to-point shearing. Flow shearing is, as
you might expect, the smoother and prcedes ahead of the edge of the blade. Point to point shearing has forces seeking the line of least resistence ahead of the blade and these result in a section of uneven thickness - not that you would notice this microscopically.
Imagine the difference between cutting through a jelly and cutting through a beefburger. Now you can imagine where the importance of the plastic point (as opposed to the melting point) comes in. Additives to paraffin waxes are intended to minimise the point-to-point shearing and improve the plastic flow. The association between the words "plastic" and "polymers" should now be awakening. Additives to paraffin wax are usually polymers (of know chain length, for they are synthesised exactly), with a major role in "harmonizing the consistency," in part at least by filling in beteen the wax crystals.
I use pure paraffin wax with no additives, in the belief that proper processing and a SHARP blade are the central features of good microtomy. (I just wish I could practise as well as I
can preach!) I have only ever come accross one wax with crystalline structure significantly different from others, and that is Ralwax, which can be helpful when cutting decalcified specimens, etc.