Dehydration means the removal of water.  The process is used in Histotechnology during both processing and staining techniques.  During processing procedures, dehydration is used to remove the free water molecules and, if performed correctly, leave the molecularly bound water.  Dehydration is normally accomplished using alcohol solutions; most commonly ethyl, denatured or isopropyl; occasionally methyl; or butyl for plant and animal tissue.  Other solutions such as acetone and various universal solvents can also be used.

If specimens are improperly dehydrated and water is left in the specimen, the clearant and infiltration medium will not penetrate the tissue and it will be soft and mushy.  Excessive dehydration will remove the bound water, causing shrunken, hard, brittle specimens that require excessive soaking before sectioning.

NOTE:  When an alcoholic solution follows a solution of 10% neutral buffered formalin it should be 70% or less to prevent precipitation of the buffer salts.

Dehydrating Agents, Alcohol

Ethyl alcohol is a clear, colorless, flammable liquid.  Since it is consumable, it is strictly controlled by the federal government and extensive record keeping is required.  Ethanol is rapid and efficient.  Exposure should be minimal to avoid progressive removal of bound water from carbohydrates and proteins, causing hard and brittle specimens.  It is normally a poor lipid solvent except under microwave processing conditions.

Reagent Alcohol (CAT# 6900) is composed of 95% specially denatured ethanol and 5% isopropanol.  It is the most popular substitute for pure ethanol and is ideal for all processing and staining procedures.  It is made using ultra high filtration and quality control techniques, ensuring consistent high quality.  100% and 95% solutions are available in 1 gallon bottles, 4/case.

Promet Alcohol™ (CAT# 69100) is a patented blend of A.C.S. grade isopropyl and methyl alcohol that is an economic alternative to pure ethanol.  Utilized in processing procedures it produces specimens that section with ease.  In histology and cytology staining procedures, it produces specimens that exhibit brilliant staining characteristics.  100% and 95% solutions are available in 1 gallon bottles, 4/case.

Isopropyl alcohol, 99%, (CAT# 36420) is an ASC grade alcohol that produces outstanding results when used in routine and microwave processing procedures.  It causes less shrinkage and hardening effects than other alcohols because it has less affect on bound water molecules.  Specimens can remain in the solvent for extended periods without harm.  It is completely miscible with water, most organic solvents and melted paraffin wax, and is readily expelled with heat.  It is a solvent for some lipid-soluble dyes, but rarely used for staining because many other dyes are insoluble in this solvent.  It is available in 1 gallon bottles, 4/case.

Methanol, 99% (CAT# 40380) is a good ethanol substitute that is readily miscible with water, ethanol and most organic solvents.  It is not a particularly good lipid solvent.  It is widely used as a cytology fixative.  It is available in 1 gallon bottles, 4/case.

Clearing

“Clearing”, which is used in both processing and staining, originally received its name because many of the reagents used for this purpose have a high refractive index and will render the exposed specimen transparent.  Although useful in determining an end point, this characteristic is not necessarily a requirement for a clearing agent used in processing.

Clearing agents used for processing must be miscible with both the dehydrating agent and the infiltration medium.  They are also referred to as “dealcoholization” agents since their primary purpose is to remove the alcohol used for dehydration and prepare the specimen for the infiltration medium.  Inadequate clearing, which can be caused by water remaining in the specimen or inadequate exposure times, will result in soft, mushy specimens due to poor paraffin infiltration.  Excessive exposure will produce hard, brittle specimens caused by the denaturation of the tissue proteins that is very similar to the effect of excessive dehydration.

Xylene

Xylene has been the most widely used clearant for many years.  It is an aromatic hydrocarbon that rapidly replaces alcohol and has a refractive index capable of rendering the tissue transparent.  Exposure times should be limited and closely monitored to prevent excessive brittleness and over-hardening of tissues, especially fibrous, muscular, central nervous system, or cartilaginous tissues.  It is a flammable reagent that should only be used with adequate ventilation, and skin contact should be avoided.  It turns cloudy in the presence of water.

Xylene, Laboratory Grade, (CAT# 8400) is quality controlled to ensure consistent high quality and filtered to remove the impurities found in industrial grade xylenes.  Used during processing, its high solvency factor allows maximum displacement of alcohol and enhances paraffin infiltration.  In staining procedures, its excellent dewaxing and clearing capabilities contribute to brilliantly stained slides.  It is available in 1 gallon, easy to pour bottles, 4/cs.

Xylene Substitutes

Xylene Substitutes, either limonene reagents or short chain aliphatic hydrocarbons (alkanes), are rapidly gaining popularity.  When substituting these solutions for xylene during processing it is necessary to evaluate and possibly adjust the exposure times, number of stations and replacement schedules.  The alcohol immediately preceding a xylene substitute must be anhydrous.  Compatibility with various mounting media should be determined when used for clearing in staining procedures.

Limonene reagents, which are derived from citrus fruit, do not tend to harden the tissue as much as xylene but they contaminate the paraffin faster, necessitating more frequent changes.  They are considered biodegradable but, because they are not water soluble, they cannot be disposed of down the drain.

Citra-Clear™ (CAT# 6400) is a high purity, completely organic compound.  It is redistilled and extensively filtered to minimize any citrus odor.  This formulation exhibits low toxicity and is not considered a sensitizer.  Exposure to air should be minimized to prevent degradation impurities.  It is available in 1 gallon bottles, 4/cs.

Aliphatic hydrocarbons penetrate specimens faster and remove fat more effectively than xylene.  They are very gentle and do not cause hardening of the tissue. They are low in toxicity and reactivity, and considered nonirritating and nonsensitizing.  They are, however, completely intolerant of water and require careful handling in high humidity areas.  They can be used for processing and staining techniques.  They are not always compatible with all mounting media; therefore, use on automated coverslippers should follow manufacturer’s recommendations.

XS-3™ (CAT# 7400) is a synthetic aliphatic solvent formulated to provide an alternative to xylene that offers increased safety for the user and does not over-harden the tissue.  It is odorless, non-greasy and non-sensitizing.  It is classified by DOT and OSHA as a combustible liquid instead of flammable, which can significantly reduce freight costs.  It is available in 1 gallon bottles, 4/cs.

Other Clearants

Benzene, toluene, chloroform and the essential oils can also be used but they are not as common.

Infiltration and Embedding Medium

Paraffin is a relatively inert mixture of hydrocarbons produced by the cracking of petroleum.  Tissue should remain in paraffin for the shortest time necessary for good infiltration because long exposures cause shrinkage and hardening of the specimen.  Infiltration temperatures on a routine automatic processor should remain between 2° to 4° above the melting point.  Infiltration is enhanced by vacuum, but caution should be exercised when using vacuum and heat on fragile biopsy specimens.  The final infiltration paraffin must be free of contamination by the clearant.  If microwave processing techniques are used, the high temperatures necessary to evaporate the alcohol will not have a detrimental effect on either the paraffin or the specimen.

Commercial paraffin contains additives to reduce the crystal size, enhance penetration, improve ribboning characteristics or alter the hardness and/or melting point.  Generally, the hardness of the solidified paraffin will vary with the melting point.  A high melting point usually indicates harder paraffin that provides additional support for hard tissue, makes thin sectioning easier and ribboning slightly more difficult.  Conversely, a low melting point usually indicates softer paraffin that allows better ribboning but makes obtaining thinner sections slightly more difficult.

Although quite common, it is not necessary to use the same paraffin formulation for both infiltration and embedding.  The paraffin used for infiltration can be selected for its ability to facilitate penetration while the paraffin used for embedding can be chosen because it more closely matches the hardness of the tissue.  The temperature at which the paraffin will be sectioned should also be considered.

Paraplast® Tissue Embedding Medium (CAT# 1006) is a refined mixture of highly purified paraffin that can be used for infiltration and embedding.  It contains plastic polymers to increase its hardness, making it an ideal support medium for hard specimens. Thin sections are easily obtainable, compression is minimal and its elasticity assists in producing wrinkle-free sections.  The melting point is 56° to 57°C.  It is available in 1kg bags, 8 bags/case.

Paraplast® Plus Tissue Embedding Medium (CAT# 2004) is the same as Paraplast (listed above) with the addition of dimethyl sulphoxide (DMSO) to enhance specimen penetration.  The sectioning qualities and melting point are unaffected by the DMSO.  It is available in 1kg bags, 8 bags/case.

Paraplast® X-tra Tissue Embedding Medium (CAT# 3002) can also be used for infiltration and embedding.  The melting point is 50° to 54°C but it retains the hardness necessary to support hard tissue and cut x-tra thin sections.  It has a low viscosity, permitting rapid infiltration of all specimen densities.  It is available in 1kg bags, 8 bags/case.

Polyfin™ (CAT# H-PF) is a mixture of highly refined paraffin waxes and co-polymer alloys.  This unique non-sticky formulation provides exceptional support for soft and hard specimens and allows thin, compression free sections.  Its viscosity allows rapid penetration.  The melting point is 55°C.  It is available in 1kg bags, 8 bags/case.

Tips for Proper Paraffin Use

·       Melt and store paraffin at least 2° to 4° above its melting point to assure that the additives go into solution.  Heavier additives, such as polymers, could settle to the bottom if the temperature is too low.

·       Molten paraffin should be stirred before it is dispensed.

·       Set the embedding center controls so that the paraffin dispenser remains on overnight.  Empty and clean it often.

·       Always be certain that the paraffin is filtered when leaving the dispenser.

·       Most paraffin contains stabilizers but it can still degrade due to oxidation.  Degradation is easily detectable by color, exhibiting a straw to yellow discoloration.  Further deterioration will create a foul odor.

·       For stability during microtomy, add enough paraffin to the mold to fill the cassette at least half way after the block solidifies.