Many natural oils and resins belong to a group of chemicals called terpenes which are produced by numerous plants and some animals. They are used for many purposes, mounting media (Canada balsam), perfumes, aromatherapy, flavourings and so on. The name is derived from “turpentine” which was one of the first to become available.
The basic building block of these oils is isoprene, shown to the left. The isoprene units are combined in various ways to produce the individual compounds making up the oils. Some of these natural oils have been used as clearing agents. As a group they tend to be gentler in action, but they are not as freely miscible with dehydrants as some other fluids. This means they may be slower in action. This does depend on the particular fluid, of course, and some are more rapid as clearants than others.
Cedarwood oil
Perhaps the most well known natural wood oil is cedarwood oil. In the two diagrams to the left, the one on the left is of cedrol, that on the right, cedrene. These two compounds are the major constituents of cedarwood oil. They are obtained from juniper and cypress species, both of which are within the general descriptions of being “cedar” trees. Their major use is as perfume ingredients for earthy type soaps and perfumes. Comparatively little is used histologically.
As a natural product obtained from several sources it does differ in characteristics and quality. For histological processing a minimally viscous variety is required. The clearing time will vary, with more viscous oils taking longer than less viscous oils. The major advantage to this oil is that it causes almost no damage to the tissue. However it does take significantly longer, and is significantly more expensive than the usually used alternatives. Some technologists wash tissues quickly in xylene when they use it, as, although it is miscible with paraffin wax, any excess oil on the tissue will inhibit its access. If a xylene wash is given it should not be prolonged, but used only long enough to remove excess oil. The actual time will depend on the viscosity of the oil, the temperature, and so on.
Some of the other natural oils, such as clove oil, have also been used histologically but they are usually even more expensive than cedarwood oil and, since there is little advantage to using them in preference to cedarwood oil, they have never been popular.
Limonene
Another terpene clearing agent is limonene. This is a natural oil found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as lemon or orange zest. The name is derived from Citrus limonum, the lemon tree. Limonene is obtained industrially by the steam distillation of orange peel which is a byproduct of the orange juice industry. Since xylene has been seen as an undesirable and possibly toxic chemical, one of the substitutes recommended is limonene, which goes by several trade names. It is a clear, colourless fluid with a distinctly citrus aroma, not unpleasant to most people, although some do not like it.
Although effective for clearing, albeit a little more slowly than xylene, it does have one serious disadvantage. Some people are allergic to it and develop skin rashes when they come in contact with it. This issue is not easily resolvable and the choice is either to stop using it or to assign the employee to another area where they do not come in contact with it.
Limonene is often sold as a xylene replacement and some technologists substitute it for xylene in other uses, but this is not universally successful. When used as the clearant immediately prior to coverslipping there are some reports that the mounting medium, usually dissolved in either toluene or xylene, does not mix well with the limonene and flow under the coverslip is affected. In such cases, replacing the limonene with xylene or toluene, or quickly dipping the section in either one just prior to coverslipping should be effective. This does, of course, defeat the purpose of the replacement to a degree.
