Paraffin wax is the most commonly used wax for infiltration during tissue preparation. This is composed of straight chain or n-alkanes with a carbon chain length of between 20 and 40. It is solid at room temperature but melts at temperatures up to about 65°C or 70°C. It is invariably a mixture, so the melting point is not sharp. It is inflammable, but not dangerously explosive, and is the wax used to make common candles. Normal precautions make it reasonably safe to use.
Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56°C–58°C, although a somewhat higher temperature may be used in tropical countries, and a slightly lower for very delicate tissues. At its melting point it tends to be slightly viscous, but this decreases as the temperature is increased. The traditional advice is that it should be used at about 2°C above its melting point, but technologists often increase the temperature to above 60°C or 65°C in practice to decrease viscosity. This does cause some extra damage to the tissue, but facilitates more thorough infiltration.
It should be melted as required rather than kept in the molten state for weeks. Hot molten wax slowly deteriorates and eventually develops a greasy feel and may be discolored. It is then unsuitable for infiltration. In normal use this is not a serious concern, but in the past, when manual processing was the norm, it was common practice to keep a container of molten wax in an oven for months at a time and this would eventually deteriorate, particularly if stored at elevated temperatures.
Plain paraffin wax can be used as an infiltration medium, but the resulting blocks have poor sectioning qualities. Ordinary paraffin wax is not very “sticky”, and the block will often crack around the tissue-wax interface. When sections are floated out the outside wax may expand away from the tissue and leave it free floating. The edge of the tissue may be damaged in the process. In addition, the crystal size of plain wax does not allow very thin sections to be cut, the thinnest being at about 5 microns, with skill.
These two defects were corrected by adding other materials to the paraffin wax. In the past there were several innovative approaches, such as heating the wax with raw natural rubber or asphalt to increase “stickiness”. There were several such formulas and they were reasonably successful.
The crystal size of the wax was modified by the addition of other waxes having much smaller crystal sizes, such as microcrystalline wax. Microcrystalline waxes are solid alkanes with branching chains. They are harder, have smaller crystal sizes and higher melting points than a corresponding unbranched alkane. They can be blended with the plain paraffin wax to both increase stickiness and decrease overall crystal size. The usual explanation was that the smaller crystals could enter the spaces between the larger crystals and attach to the tissue. In addition, bayberry wax (derived from berries of bayberry plants), spermaceti wax (from the sperm whale) and ceresin (a naturally occurring microcrystalline wax) have been used.
Modern paraffin waxes are generally propriety formulations, and the specifics of their manufacture are not freely available. They are plastic copolymer waxes, that is, they are paraffin wax with plastic copolymer additives to improve their characteristics. Likely, they also still contain microcrystalline waxes. The plastic copolymers used are not given but are probably ethylene-vinyl acetate, although the proportions of the additives are not published either. Many technologists find the different formulations to be quite similar in their qualities, but it must be acknowledged that they are an improvement on what used to be available. Using a good quality modern copolymer wax a skilled technologist can cut a true 3 micron section, compared to the 5 micron section with plain paraffin wax.
How Infiltration Works
As with dehydration and clearing, infiltration depends on simple fluid replacement, this time of the clearant by molten wax. In addition, the heat may cause some of the clearant to evaporate, depending on whether it vaporizes at the temperature being used. Carbon disulfide, for instance, can be seen as bubbles streaming from the tissue. With others it may not be so noticeable, but it may be a factor in the infiltration. As the clearant evaporates, wax may be drawn in to fill the space. The wax is very tolerant of clearing agents, and often is miscible with them in all proportions. It is completely intolerant of water and tolerates dehydrating agents poorly, with some exceptions. In practice, a properly dehydrated and cleared tissue sample of reasonable size will infiltrate fairly rapidly at the usually recommended temperatures of a few degrees above the melting point, more rapidly at a little higher temperatures.
Number of Changes
A minimum of two changes is required, usually at least an hour each. This can leave the blocks with a somewhat greasy feel, so a third change will be found very advantageous.
Agitation
Due to the viscosity of molten paraffin wax some form of gentle agitation is highly desirable. It serves to remove the clearant from around the tissue and permit the access of uncontaminated wax. Once again it has to be emphasized that this should be gentle agitation, as if it is too vigorous particles could detach from one tissue and attach to another, resulting in a misdiagnosis.
Vacuum
Vacuum infiltration is quite common, although not essential. Infiltration will be successful without it but using it increases the completeness and can help reduce the time required. It is accomplished by reducing the air pressure within the vessel holding the molten wax and tissues. Reducing the air pressure lowers the boiling point of fluids, so any clearant will evaporate more readily and will be more easily replaced by wax. In addition, any small air bubbles trapped within the tissue are more likely to be released as the pressure declines.
When the air pressure is reduced it is done gradually by using an air pump. When releasing the reduced pressure, it must also be gradual so as to avoid collapse and distortion of fine membranes. It should take a few minutes for the air pressure to return to ambient, rather than be instantaneous. Pressure should be slowly reduced from ambient air pressure by between 400 and 500 mm mercury (about 15-20 inches). Since standard air pressure is 760 mm mercury (about 30 inches), this means the pressure within the retort should be between 260-360 mm mercury (about 10-15 inches).
In modern vacuum infiltration systems an electrical air pump is used in conjunction with an oven or retort designed specifically for it, so it is very convenient. Vacuum infiltration may be a standard feature on modern automatic tissue processors, in which case it is advised to ensure it is used.
Shrinkage
Hot paraffin wax is the major cause of shrinkage during paraffin processing. The majority of it occurs during the first 15 minutes or so of infiltration in the first wax. Generally, the hotter the wax the more shrinkage, and increasing the temperature of infiltration too much can have a noticeable effect on the tissues. The temperature of the molten wax should be kept to the minimum possible consistent with complete infiltration during the time available. The length of time the tissue stays in the wax has little effect on the degree of shrinkage and tissues can safely be left for several hours, even overnight. Excessive time in hot wax may make the tissue more brittle.
Disposal
Paraffin wax is not a hazardous material and can usually be disposed of in municipal waste, although local regulations should be checked. It takes so long to deteriorate that it is non-degradable to all intents and purposes. A useful encapsulation medium to assist in the disposal of more hazardous material has been found. Since paraffin wax is used for making candles it may be tempting to filter the used wax and reuse it to make them. This is not advisable. The wax used for candles contains no additives and burns without sputtering. Used paraffin wax not only has additives which may affect how it burns, but also contains traces of clearing agent, which may increase black smoke. Used wax may also contain traces of human or animal fats from incomplete defatting, a rather unaesthetic factor.
