Processing in histopathology

Processing

Histology technique necessitates that thin sections of the fixed tissue be cut. In order that these thin sections may be cut, the fixed tissues must be of suitable hardness and consistency when presented to the knife edge. The desired hardness and consistency can be imparted by infiltrating and surrounding (embedding) the tissue with paraffin wax, celloidin or low viscosity nitrocellulose (LVN); various types of resins; or by freezing. Each of these methods has its own special use. Celloidin or LVN is mainly used when dealing with tissues of the central nervous system and organs with greatly differing textures such as eyes, embryos and sometimes bone. Plastic embedding media such as acrylic, polyester and epoxy resins are used for sectioning of undecalcified bone; preparation of ultra thin sections (50–80 nm) for electron microscopy. They are increasingly being used for semi-thin sections (1-2 um) for light microscopy, especially of lymph nodes, renal biopsies and bone marrow biopsies. Frozen sections are prepared for urgent diagnostic work, the demonstration of fats and certain enzymes and for some staining techniques in which free floating sections are required. Paraffin wax embedded material is used for practically all routine diagnostic work. This infiltration is possible only after the fixed tissue has been dehydrated and cleared. Processing is the term used to describe the various stages between fixation and cutting of sections.

The stages are:

1. Dehydration 

2. Clearing

3. Infiltration and embedding 

The procedures in these stages do not apply to frozen sections. Preparation of frozen sections is described separately. 

Selection of Tissue Blocks for Sectioning

Following fixation, the selection of tissue blocks for sectioning is done by the pathologist who briefly describes the gross specimen. The description is recorded by, usually, a histotechnologist who also numbers the gross specimen and prepares a tag (small piece of quality paper e.g. Whatman filter paper) on which the number is written in pencil (ball point pen ink is soluble in alcohol). The tag is placed in a perforated cassette with a snap cover along with the tissue block. The block taken can be up to 3 x 2.5 cm in area and about 4 mm thick. The histotechnologist notes the number of blocks taken and whether or not the whole gross specimen (e.g. curretings) has been taken. The rest of the gross specimen is placed back in the container of fixative and stored as long as is necessary.

Some tissues may require special attention, and for easy identification of such tissues, the tag or identification label should be marked with asterisk, and the special information noted on the back of the working card. If it is necessary to cut a block from a particular surface, then the surface is easily identified by passing a thread through one corner of the opposite surface of the tissue to that which is to be cut. The whole procedure of processing can be carried out either manually or by automated machines.

DEHYDRATION

Dehydration of tissue occurs when the water constituent of the tissue is removed. Dehydration is important because most embedding media are not miscible with water, and the removal of water facilitates the subsequent impregnation with the embedding media. Naturally, dehydration is carried out by using the same reagent that mixes with both water and the ante-media. 

Alcohol The best agent for dehydration is ethyl alcohol which has the advantage of not being poi sonous. The dehydration process is carried out by the use of series of increasing strengths of alcohol. Routinely, 70% is dilute enough for the first bath. Transfer of tissue directly from formalin to higher grade of alcohol could lead to distortion. For delicate tissues like brain and embryos, dehydration should be more gradual, beginning with 50% alcohol, with smaller intervals between the different grades of alcohol.

To check for the presence of water in the final bath, add a small amount of dried white copper sulphate. If water is present, the copper salt will show atinge of blue. Alternatively, a layer of anhydrous copper sulphate covered with filter paper is kept at the bottom of the jar used. The salt acts as an indicator, turning blue when water is present. When this happens, the alcohol should be replaced.

It is essential to use high quality top grade alcohol. The alcohols which are in general use in histopathology are ethanol (ethyl alcohol), industrial alcohol (methylated spirit), isopropanol, amylalcohol, rebutanol, tertiary butanol and polyvinyl alcohol. Ethanol and methylated spirit are the most popular. In the absence of a high grade alcohol, isopropanol is the next choice of alcohol for dehydration

It has been claimed by some workers that the time necessary for dehydration may be shortened when processing is carried out at 37°C instead of at room temperature. This makes the method ideal for urgent work.

Acetone Acetone may be used and has been found to be a good dehydrant, cheap but very volatile. It 

hardens tissue more than alcohol and much greater volume of it is required than ethanol. Dehydration time is 1/2 hour to 2 hours with resultant shrinkage, and for this reason it is not quite suitable for routine work. Furthermore, only small pieces of tissues should be treated in acetone. 

Dioxane (diethylene dioxide) Dioxane is miscible with water, alcohol, xylene, balsam and paraffin wax. This makes it a unique dehydrating agent. There is very little shrinkage to the tissue. But it is not recommended for routine use because it is a dangerous substance with little warning odor and a cumulative toxic action.

• CLEARING

This stage of tissue processing can conveniently be referred to as de-alcoholisation--the removal of alcohol from blocks or sections of tissue by immersing them in a fluid that will dissolve the wax with which the tissue must be impregnated. This fluid is the ante-medium. In addition to removing the alcohol from the tissues, most of the well known ante-media have the property of making the tissues transparent. 

Therefore this stage of tissue processing is referred to as clearing stage and the anti-media as clearing agents. It should be pointed that some ante-media (e.g. chloroform) have no clearing property. A good clearing agent must be miscible with alcohol and wax, should remove alcohol quickly and clear rapidly without over hardening of the tissue. It should not evaporate too quickly in the wax bath or dissolve aniline dye while staining. Clearing fluids should be used in amounts not less than 10 times the volume of the tissue. The most popular clearing agents are:

Xylene (Xylol) This is a very good and true clearing agent. It tends to make tissues such as brain and blood containing tissues excessively hard and brittle. Tissues should never be left in xylene for more than 2 hours. Biopsies and tissue blocks not more than 3 mm thick are cleared in 2030 minutes. When dehydration is not complete, xylene becomes milky when the sections or tissues are immersed into it. Xylene is cheap and is usable with celloidin sections. It is highly inflammable. 

Toluene (Toluol) As an ante-medium for routine work, toluene is slower than xylene in action. Otherwise in general properties, it is similar to xylene. It has practically superseded benezene because its fumes are less toxic. 

Benzene It has properties very similar to xylene. It is not recommended for usage as it is known to be carcinogenic.

Chloroform It is the most ideal clearing agent for the central nervous system tissues, lymph nodes and embryos because it causes little shrinkage and does not harden tissues excessively. It is good for large blocks. It is not inflammable. It is not a classical clearing agent as it does not render tissues transparent, and as a result its end-point is difficult to assess. De-alcoholization time for tissues 3-5 mm thick is 5-20 hours. Precautionary measures are necessary when handling chloroform as its vapour is toxic and anaesthetic. It must be used in a well ventilated room and in a tightly closed container. It is relatively expensive. 

Cedar wood oil Due to the high cost and very slow action of cedar wood oil, it is rarely used for routine clearing purposes. It is very gentle and causes little or no damage to the most delicate tissues. It is ideal for research work especially in the study of embryos. This reagent imparts proper consistency to dense fibrous tissues and skin tissues which allows easy cutting of the sections. It also imparts transparency to the tissues. One major disadvantage is the difficulty in eliminating the oil from the wax oven. It must be noted that cedar wood oil clearing agent is different from the more viscous type employed as immersion oil in microscopy 

Other clearing agents Other agents that can be used but are not of great importance are carbon tetrachloride, carbon disulphide, paraffin oil, petrol, clove oil, and methyl benzoate (used in conjunction with celloidin for double impregnation of tough or fragile tissues). A number of proprietary reagents for clearing are commercially available. Most of them are cheap, non-toxic derivatives of good grade materials. One such reagent is Histoclear. These reagents have shown good promises and may become very popular in future.

IMPREGNATION AND EMBEDDING

These two stages in the processing schedule involve the impregnation of the tissues with a medium that will fill all natural cavities, spaces and interstices of the tissues and then solidify to form a sufficiently firm consistency that can give considerable support during the cutting of sections without distortion to or alteration of the tissue and cellular constituents. The specimen is surrounded with a mass or block of embedding medium thereby allowing the specimen to be handled and attached to the microtome.

The paraffin wax, celloidin and LVN (low viscosity nitrocellulose) methods of embedding are the most popular. Other embedding media include a host of other waxes such as esterwax, carbowax, polyester wax and microcrystaline wax: resins such as acrylic, epoxy resin and urea formaldehyde; gelatine; and agar.

Paraffin wax infiltration Paraffin wax is obtained by distillation of shale which gives liquid oil that is separated by refrigeration and then purified to give the final product of paraffin wax. When clearing is completed, the tissues are transferred to molten paraffin wax in the embedding oven. The molten wax diffuses into the tissues to replace the clearing agent which diffuses into the surrounding medium. The penetration of the tissues by paraffin wax occurs more readily if the cell proteins have been coagulated. This is why protein coagulating fixatives are preferred for paraffin wax embedding. 

Impregnation occurs by immersing the tissues in a succession of molten wax baths accompanied by agitation on an automatic tissue processor. The number of changes of wax and the time required for the tissues in each bath depend on the nature of the clearing agent, the size and the density of the block of tissues. But generally, blood and grossly congested tissues, muscles and fibrous connective tissues tend to become excessively hard if left for more than 3 hours in the wax. Brain, skin, dense bone tissues and materials from female breast hav. ing a large amount of retained secretions in dilated ducts, require longer time in wax. Such tissues are best processed using blocks of tissues not more than 3 mm thick.

The melting point (MP) of paraffin wax ranges between 54°C and 60°C. It is kept molten at a tem perature 2-5°C higher than the melting point. Some workers claim that mixtures of waxes of different MPs give better result than a single wax. A good and popular such mixture is the paraplast which is a mixture of highly purified paraffin wax and several synthetic plastic polymers. Its MP is 56-57°C. It gives a more uniform block than any other medium. With paraplast, there is no need for rapid cooling or embedding. Ribbon sectioning is made easy; it is more resilient than paraffin wax and allows large blocks and dense bone blocks to be cut fairly easily. Embeddol is mainly a synthetic wax substituent that is similar to and as good as paraplast. Its MP is 56-58°C.

EMBEDDING

Embedding is the process of casting the tissues in paraffin wax and allowing them to set to facilitate cutting of sections.

Embedding or Blocking out Moulds

The moulds come in various shapes, sizes and makes.

Leukhart's embedding moulds These are more or less the traditional embedding moulds. They consist of two pieces of L-shaped heavy metal such as brass and a base made of copper or brass or a plate glass. The L-shaped pieces are usually purchased in pairs of various sizes. They are laid on the base plate and arranged to form a mould of desired size. The mould is filled with molten wax. When the wax has completely solidified, the moulds and the encased blocks are removed from the base plate. The two L pieces come away easily from the wax block and are ready to be re-used. The Leukhart's moulds are simple to use. They give even blocks with parallel sides. However, they are cumbersome when processing a large number of tissues.

Plastic trays These are convenient for a busy laboratory. The mould consists of disposable plastic tray divided into separate compartments. Various sizes are available. Each compartment is filled with molten wax. A good substitute is the ordinary ice tray that comes with domestic refrigerators.

Waxed paper cups these are also suitable. They are made of thin or stout paper.

Embedding Procedure

It is essential to use clean high grade paraffin wax. The wax is kept molten in a wax oven at 2°C higher than the melting point of the wax. It is possible to take down the final beaker of wax on the automatic processing machine to the work bench and plug to an electrical outlet to keep the wax molten. The small perforated metal buttons containing the tissues are picked out of the beaker of wax one at a time. The button is opened, the identifying label is noted and kept. A suitable mould is chosen and filled with the molten wax. With a pair of blunt forceps that has been slightly heated over the bunsen flame, the tissue is placed in the mould of molten wax with the surface to be cut facing downwards. It is important that this is carefully oriented so that the plane of sectioning will be correct, e.g. biopsies of skin must be embedded so that the plane of the surface is vertical to the bottom of the mould; and wall of a cyst must be embedded edge down. It is usual to press down the tissue and hold it for a few seconds until it is held by the cooling wax. Immediately, one end of the identifying label is immersed in the wax. The forceps are flamed frequently to prevent wax and tissue sticking to it. The setting of the wax can be hastened if, as soon as a film has formed over the surface of the wax, the complete unit is lowered into a bowl of cold water or placed in the refrigerator. This rapid cooling also minimizes crystallisation of the wax which may occur if allowed to cool and set slowly at room temperature. 

Embedding for cytology Cytological specimens are sometimes required to be sectioned. In such a case, the processing and embedding can be carried out in a glass tubing which has one end sealed with a cork. The specimen is centrifuged between each stage of processing. The small pieces of tissue will collect at the bottom of the tubing and this facilitates sectioning. On the solidifying of wax, the cork is removed and the block pushed out of the tubing.

Note Many wax substitutes are available in the market.

Manual Processing Schedules

Many factors are taken into consideration in setting up the processing schedules. The type of fixative used will determine the point of entry to the processing schedules. The size of the tissue block is an important factor. For example, a block of only 1-2 mm in size will need shorter time than one of 3-5 mm. Room temperature will affect the rate of penetration of the various reagents. Heat generally speeds up, and cold slows down the process. Each individual laboratory should determine its own standard processing schedules. Table 1 shows a routine manual processing schedule for 3-5 mm size blocks. A rapid manual processing schedule for blocks less than 3 mm in size is shown in Table 2. The time required in each processing reagent is greatly reduced when tissues are suspended and continuously agitated. This is achieved with the automatic processing machines.

Table 1 Routine manual process (blocks 3-5 mm thick)

1. Fixation

2. Wash in water if necessary

3. 70% alcohol-during day, 3-8 hours

4. 90% alcohol-overnight, 16 hours

5. Absolute alcohol I, two hours

6. Absolute alcohol II, three hours

7. Absolute alcohol III, three hours :

8. Toulene or chloroform-overnight, 16 hours

9. Three changes of paraffin was, 3 x 1 hours

10. Embed in fresh wax.

Table 2 Rapid manual process (blocks not thicker than 3 mm)

1. Fix in Carnoy's fluid 30-60 minutes

2. Absolute alcohol 1, 30 minutes

3. Absolute alcohol II, 30 minutes

4. Absolute alcohol III, 30 minutes

5. Xylene or toluene, 15-30 minutes (until clear)

6. Three changes of paraffin wax, 3 x 20 minutes, in vacuum oven

7. Embed.

Note: Dehydration and clearing may be carried out in the wax oven using well stoppered containers

Automatic Tissue Processing

The automatic tissue processors (Fig. 4.1) greatly reduce the time and labour needed to process tissues. Therefore, they allow a more rapid investigation to be carried out. The reduction in processing time is due to the fact that the tissues are constantly agitated, and therefore, the transfer of tissues from reagent to reagent is performed at shorter intervals. The use of the automatic machines permits the transfers to take place even during shut down hours of the laboratory. All models feature a mechanism of suspension and agitation and automatic transfer from solution to solution at pre-set times. Most of them have twelve stations, the last two being wax baths.

Dehydration, clearing and preparation for embedding are accomplished with the automatic tissue processors.

 Most of these machines work on the principle of a central, rotating spindle that carries a bucket or basket containing the tissue blocks suspended from the outer end of a horizontal radial arm. A clockwork device which is electrically operated, rotates a notched clock-face card on the circumference of which a spring loaded tooth' or hook moves. Notches are cut to suit the desired time schedule. When the tooth moves into one of the notches, a motor is automatically activated to lift the central spindle which, by means of a fixed helical screw, automatically rotates as it rises so that the bucket arm is swung over the next beaker of solution. The spindle is then lowered, placing the bucket in the solution. 

The bucket is continuously agitated by a short repetitive up and down motion of the entire assembly head or the arm. The last two beakers are made of metal and contain the molten wax which is kept at a constant temperature by thermostats located within the walls of the beakers.

Most of the automatic tissue processors are designed to operate a 24-hour schedule, although

some are made to operate a one hour schedule for fast processing, or a 7-day schedule for slow processing. 

There is a mechanism that allows the delay of actual starting time for 24 hours. The machines also do incorporate a number of safety devices. A cut out device automatically comes into operation when the processing cycle is completed with the tissue bucket in the second wax bath until it is removed by hand. Another safety device operates when the wax in the first wax bath has solidified. In this case the transfer mechanism will transfer the tissue containers straight to the second wax bath. Also in the event of a power failure or a fuse being blown, a battery operated alarm bell located in a strategic position in the laboratory, goes off.

The perforated tissue containers (cassettes) are made of stainless metal or plastic material. The 

plastics have almost completely superseded the metal ones. They are particularly useful when processing tissues fixed in corrosive agents. 

They are made to interlock whereas the metal containers have close fitting lids and all are designed with one, two, four or six compartments or divisions. Special baskets are available for processing fragmentary tissues such as curettings. The individual tissue containers are loaded into a large carrier basket which is suspended on the central spindle. The reagent containers should be kept filled with correct strength of fluids to marked levels and checked to ensure that they are firmly positioned. The wax bath should be checked daily to ensure that the current is flowing.

Note

Small pieces of curretings may first be wrapped in filter paper or gauze and then placed in the perforated tissue containers. This should be done in the absence of the special containers for such tissues.

Automatic Machine Processing Schedules

Processing with the automatic tissue processor will very much depend on the type of tissues, nature of the work and individual performance of the machine. Table 4.3 is a model routine automatic processing schedule for 3-5 mm thick blocks. Table. 4.4 is a 48 hour schedule for 3-5 mm thick blocks. Most of the machines have a delayed start device whereby the week-day cycle can be used at week-ends.

Table 4.3 Routine automatic process (blocks 3-5 mm thick)

1.70% alcohol                        3 hrs

2. 90 % alcohol hangers         3 hrs

3. Absolute alcohol I              1hr

4. Absolute alcohol II             1hr

5. Absolute alcohol III            2hr

6. Absolute alcohol IV            2hr

7. Touene i                              1 1/2

8. Touene II                             2 1/2 9. Wax bathIII                         3 hours

10. Wax bath II                        3hrs

Total                                    22hours

Table 4.4 48-hour automatic process (Block 3-5 mm thick)
1. 10% formal-saline 12.	4hrs
2. 10 % formal-saline II	4hrs
3. 70 % alcohol	4hrs
4. 90 % alcohol	4hrs
5. Absolute alcohol	4hrs
6. Absolute alcohol II	4hrs
7. Absolute alcohol III	4hrs
8. Absolute alcohol IV	4hrs
10. Chloroform II	4hrs
11. Wax bath I	4hrs
12 Wax bath II	4hrs
Total	48 hrs

Note :The latest models of automatic tissue processors are vastly modified and computerised.

Vacuum Impregnation 

It is very often necessary to facilitate rapid penetration by the wax. To achieve this, the vacuum impregnation technique is employed. The technique is based on the production of negative pressure inside the embedding oven. This negative pressure bastens the extrusion of air bubbles and of the clearing agent from the tissue block, thus resulting in the rapid penetration of the wax.

This technique is desirable for:

(i) Urgent biopsies 

(ii) Dense tissues 

(iii) Lung tissues 

(iv) very fatty tissues

The vacuum impregnation oven (Fig.4.2) commonly in use consists of a flat bottomed brass vacuum chamber with a heavy glass lid resting on a thick rubber washer which provides an air-tight junction. The vacuum chamber is immersed in a thermostatically controlled water jacket. Air is allowed into the chamber when the oven is undernegative pressure by means of a valve fitted on one side of the chamber. The interior is connected to the vacuum pump by a small tube which is on the opposite side of the chamber.

The oven first of all has to be assembled in the following manner: 

1. Attach a venturi water pump to a cold water tap on the mains supply. 

2. Connect the vacuum pump to a trap bottle with pressure tubing, 

3. Attach the bottle to a glass Y-piece with pressure tubing. 

4. Fit one end of the Y-piece to a manometerand the other end to a vacuum oven. A glass stopcock is finally inserted in the pressure tubing between the Y-piece and the trapbottle. 

The oven having been properly assembled, impregnation is carried out thus:

1. To a container of molten wax, transfer the cleared tissue and place it in the vacuum oven. 

2. Press the heavy glass lid firmly into position. 

3. Shut the valve and evacuate the chamber with the venturi pump to give a negative pressure of 400-500 mm on the manometer. 

4. Close the stopcock between the Y-piece and the trap bottle and shut off the pump (the water pump must never be turned off while the stopcock is still open in order to prevent suck back of water). 

5. At the end of the impregnation time, gradually open the screw valve to allow the pressure to return to normal. 

6. Embed the tissue. 

Most recent automatic tissue processors have in built vacuum impregnation facility. These new

machines are computerised so that they can be programmed for various functions and schedules.

Celloidin and Low Viscosity Nitrocellulose (LVN)

The celloidin and the low viscosity nitro-cellulose are both forms of cellulose nitrate, of which the LVN is of less viscosity. The LVN, because of its low viscosity, penetrates more readily and may be used in higher concentrations than celloidin. It gives a much harder block and allows thinner sections to be cut.

Fixed and dehydrated tissues are impregnated with increasing concentrations of celloidin or LVN dissolved in a mixture of absolute alcohol and ether. Clearing agents or antemedia are not utilised and no heat is required in the process. As a result, distortion to the tissues is minimal.

Impregnation

Following complete dehydration, 

1. Transfer the tissue to a mixture of equal parts of absolute alcohol and ether. This is to speed up the impregnation process. 

2. Transfer the tissue to 2% solution of celloidin or 8% solution of LVN for 4-7 days. 

3. Transfer the tissue to 4% solution of celloidin or 16% solution of LVN for 4-7 days. 

4. Transfer the tissue to 8% solution of celloidin or 24% solution of LVN for 2-3 days.

Embedding

1. Fill the special embedding mould upto the half way mark with 8% celloidin or 24%. LVN; place the tissue in position with surface to be cut uppermost. 

2. Fill the mould with more of the embedding solution. 

3. Place the mould in a desiccator containing ether vapour.

4. When all the air bubbles have been removed by the ether vapour, place the tissue with the surface to be cut facing downwards in the mould. This will prevent any air bubble from being trapped beneath the tissue.

5. Remove the mould to a second desiccators containing chloroform vapour to harden the celloidin or LVN to the desired consistency. The desired consistency is determined when the thumb pressed on the block leaves no impression.

6. Transfer the block from the mould into pure chloroform. The block slowly sinks to the bottom of the solution.

7. Finally transfer the block to 75% alcohol until required for cutting.

A similar substance to celloidin and LVN that is widely used is the necoloidine. It is supplied as a solution of 8% of pyroxyline in ether alcohol. It is thickened (by evaporation) to 16% for use.

Advantages of Celloidin and LVN 

1. There is a considerable minimisation of shrinkage of the tissues mainly due to the absence of heat in the process. 

2. Both celloidin and LVN improve cutting qualities of large block of dense tissues as a result of the reduced hardening and plasticity of the medium. 

3. They give superior cohesion to tissue layers of different consistency. 

4. They are very useful for the preparation of sections of brain where thick sections are often required. The shrinkage and distortion often encountered with paraffin wax is eliminated. 

It is also necessary to mention the disadvantages of celloidin and LVN: 

1. Processing time is long and tissues such as brain can take up to three weeks to process. 

2. Sections thinner than 10 um are difficult to cut: 

3. Serial sections are difficult to produce because celloidin and LVN are non-ribboning media.

4. Because blocks are stored in 75% alcohol and must not be allowed to dry, storage is a

problem. 

5. Celloidin and LVN are highly explosive if allowed to dry and so they must be used with care.

GELATIN EMBEDDING

Gelatin is a chemically modified form of collagen, a protein that is insoluble and which is distinguished by its high content of glycerin and hydroxyproline. The collagen used in gelatin is identified as a form of organic matter in the bones of cattle and from the white connective tissue fibres in the skin of cattle and pigs.

Gelatin as an embedding medium finds its main use as a supporting medium for frozen sections. Generally, tissue from which frozen sections are to be cut is not embedded, the freezing itself provides sufficient support for sectioning. But when frozen sections are to be prepared from tough or friable tissues, a supporting medium is required to prevent the tissue from fragmenting. This is where gelatin is very useful. After embedding, the blocks of tissue are put in formalin to harden.

One major advantage of gelatin as an embedding medium is the absence of dehydration at any time in the process thus avoiding distortion of the tissue by shrinkage. However, because gelatin cannot be removed from the tissue and since it is capable of taking up both acidic and basic dyes, it very often gives poor microscopical picture. Also, thin sections are not easily cut while ribboning of the sections is difficult.

Gelatin Embedding Method

Solution A

Gelatin	12.5 g
Distilled water	87.5ml
Phenol crystals	1.0g

Solution B

Gelatin	12.5 g
Distilled water	75 ml
Phenol crystals * Phenol crystals serve as preservative.	1gm

Solution C 

Formalin	5 ml
Distilled water	95 ml

Preparation 

Solutions A and B

1. Heat the distilled water to 37°C.

2. Dissolve the phenol in warm distilled water.

3. Add the gelatin and incubate at 37°C until the gelatin is dissolved.

4. Filter through surgical gauze.

5. Bottle and label. 

Solution C

Procedure

1. Place the tissue (not exceeding 3 mm in thickness) in solution A for at least 12 hours at 37°C.

2. Transfer to solution B for at least 12 hours at 37°C.

3. Using Leukhart's embedding mould, embed the tissue in solution B. Allow to cool and then trim.

4. Transfer the trimmed block to solution C for 18-24 hours. 5. Cut sections as for frozen section.

PLASTIC EMBEDDING

Epoxy resins such as Epson, Araldite, and Vestapol give less distortion to the tissue than the butyl and methyl methacrylates which they have displaced. The main use of plastics is in electron microscopy though modified forms of the above plastics are used in histology to produce 1 um sections. These modified ones are known under their various trade names such as JB4, Historesin, LR White and LR Gold.

There is very little distortion with plastic embedding and they permit very thin sections to be cut in histology and 60-70 nm for ultra thin sections. A well processed tissue embedded in plastic medium which is sectioned at 3um and mounted in the appropriate mounting media, e.g. glycerin, gives a life-like appearance when examined under oil immersion with x 65 objective.

The major drawback is that the processing cycle is complicated and the reagents have limited shelf life. The plastics are potentially harmful as they have been known to cause contact dermatitis while some are deemed to be carcinogenic.

TRIMMING OF BLOCKS

When the blocks have hardened, they are removed from the cold water or refrigerator. The identification number is carefully removed and the block is freed from the moulds. Excess wax is trimmed from the block so that the block forms a four sided prism. The important point is to have the opposite sides parallel in order to produce serial sections. The trimming away of excess wax prevents the blocks from cracking. At least 2 mm of wax should surround the tissue block. The small piece of paper bearing the identification number of the tissue may then be affixed to the block using a hot spatula. A suitable fibre block or wooden block may be used as a block holder and the number written on its side with grease pencil.

Attachment of the Block to the Block Holder

1. Heat a flat spatula (about 2.5 cm wide blade) in a Bunsen flame.

2. Place the bottom of the wax block on the spatula.

3. As soon as the wax block starts melting, slide it onto surface of the wooden or fibre block holder so that the sides of the block are parallel to those of the block holder.

4. Press the wax block down firmly and allow to harden. The block is now ready for cutting. 

Celloidin and LVN blocks  The celloidin or LVN blocks may be trimmed before storing in 70% alcohol or before starting the cutting of sections. Excess cellodin or LVN is trimmed off from all the sides except the cutting surface. Wooden holders which have deep serration cut into them are used. The block holder is coated with 4% celloidin or 16% LVN and the trimmed block is pressed firmly into position. The block and holder are held together by means of a thread tied around them for about 30-60 minutes. The celloidin sets firmly and the block and holder are reintroduced into 70% alcohol for 30 minutes. The cutting surface is trimmed with sharp scalpel or razor. The block is now ready for cutting.

Section cutting nxt