Study Notes on Microtome Sections

The below mentioned article provides study notes on Microtome Sections:- 1. Section-Cutting 2. Mounting of Sections 3. Stretching of Sections 4. Drying of Sections 5. Staining the Sections 6. Staining the Sections 7. Schedule of Staining the Section.

Microtome Sections # Section-Cutting:

Rotary microtomes are commonly used for section-cutting. Move the block holding the socket backward as far as possible with the help of the backward movement handle, which usually lies at the far side of the microtome. Adjust the thickness of sections.

Ordinarily, flower buds are cut at 14 µ and root tips at 12 µ thickness. Cut out one block from the large one cast previously. Trim it with a scalpel and/or blade, holding it gently with the left hand and obtain a perfect square or rectangle with the material at the centre. Place the block-holder vertically in a small tube. See that there is sufficient paraffin on it.

Now hold the trimmed block gently with the thumb and forefinger of the left hand, moderately heat a scalpel, place it flatly on the block-holder, and rest the block on the scalpel and then remove the scalpel. Prick the four sides of the block where it has joined the holder with a hot needle. Submerge the block in cold water for a few seconds. Trim further, if necessary.

Now fix the block-holder in the block-holder socket with the help of its screws. Fix the razor in its socket. The block and razor should lie parallel to each other. The razor tip should be slightly tilted inwardly (about 8° angle). Bring the block and the razor as close as possible without touching each other. Now cut sections by moving the front wheel in a clockwise direction.

The ribbon of sections will slip over the razor. Support it with a needle or brush held by the left hand. When the material is exhausted, dislodge the ribbon with a brush and place it carefully on a half foolscap piece of blotting paper or, preferably, on a large, glossy and somewhat stiff paper.

Precautions:

Microtome section cutting is a critical job and requires considerable practice to obtain perfection.

However, the following precautions should be noted:

1. The block-holding socket is moved backward as far as possible because, if the microtome gets stuck midway while section cutting, the ribbon will be disturbed. Resetting it may cause the loss of a portion of the material with the result that the entire material is not present in sections.

2. Thickness of sections depends upon the size of the cells. The idea is to get sections one cell in thickness.

3. Trimming the block is a very critical step. If it is trimmed into odd shapes the ribbon will not be straight and there may be difficulty in joining of the sections to form the ribbon. The excess paraffin should be trimmed gradually, there may be excess paraffin on one side but it should not be cut in one stroke.

The four sides should be trimmed in such a way that the material finally lies at the centre. Longitudinal sections are normally made in case of flower buds, and transverse sections in case of root tips. Obviously, trimming should be done accordingly. Another defect often noted in trimming is that the top of the block becomes narrow and the base progressively broader.

This should be avoided. The block often needs a second trimming after mounting on the block-holder, as it sometimes bends due to pressure while mounting. For this, all the excess paraffin should not be trimmed away initially. After final trimming, the block should be of such a size that at least four ribbons can be accommodated on the slide.

4. If sufficient paraffin is not present on the block-holder, add some and melt it with a hot scalpel. This is necessary to ensure proper fixing of the block and, at the same time, reduce the chance of the metallic holder rubbing against the razor. While fixing the block, the scalpel method should be followed.

As the paraffin on the holder and that of the block melt simultaneously it ensures perfect fixing. Pricking with a hot needle makes fixing doubly sure. It is all the more necessary because often the block gets detached while section cutting. Dipping the fixed block in cold water is also done for proper fixing.

5. Note carefully before section cutting that the block and the razor are perfectly parallel to each other; otherwise the ribbon will not be straight. Very often the ribbon becomes curved or dentate. This is due to faulty trimming or not keeping the block parallel to the razor (Fig. 3.4).

6. The razor is placed at a slightly inward angle to avoid rubbing of the block against the lower side of the razor. The lower side of the razor is quite thick; so if it is placed vertically, with every stroke the block will be pressed against this thick side of the razor and sections will not come out.

7. As the block strikes the razor a section is produced and the friction against the razor produces a little heat energy. When sections are cut in quick succession this energy is sufficient to join the very thin sections end to end to produce a ribbon. This is why paraffin of suitable melting point has to be taken while casting blocks. If the paraffin is too hard the heat energy produced by friction is not enough to join the sections.

If it is too soft the frictional energy makes the sections further soft and they become crumpled. Sometimes warming the block and the holder with slightly warm water or cooling with cold water helps to eliminate these defects. If the sections fail to form a ribbon reduce the thickness of the sections and, if it crumples, increase the thickness of the sections by a few microns. Sometimes this also helps in obtaining a ribbon.

8. For flower buds and L.S. of root tips cut the entire material into sections. While making T. S. of root tips, cut sections to fill one slide; divisions are not found beyond this region.

9. Never touch the edge of the razor with a needle or scalpel. Dislodge the ribbon at the end of section cutting with a brush. Friction with metal may cause minute dentation or folding of the edge of the razor. Once this happens, the razor cannot be used again without sharpening or polishing it.

Microtome razors are very costly. Remove the grease from the surface of the razor before using it and, after section cutting, clean its surface and apply grease. The microtome also needs cleaning and oiling from time to time for smooth running.

Microtome Sections # Mounting of Sections:

Cut the ribbon into 1½” (4 cm) long strips. Put a small drop of Meyer’s fixative (white of egg 50 ml, Glycerol 50 ml; Sodium salicylate 1 gm.) on a clean grease-free slide. Rub it gently with the little finger all over the slide and drain off the excess fixative, if any. Flood the slide with a thin film of water with the help of a dropper. Dip the tip of a scalpel in cold water and touch the ribbon with it.

The ribbon will adhere to it. Free the other end of the ribbon with a needle or scalpel, if necessary. Shift the ribbon and place it on the slide, keeping the glossy surface of the ribbon below. In this way transfer as many ribbons to the slide as it can accommodate (Fig. 3.5), maintaining the continuity of the ribbon.

Microtome Sections # Stretching of Sections:

Transfer the slide on to a hot plate kept at 37-43°C, on a thin film of water. Wait for a few seconds. The ribbons will automatically stretch on being warmed. Take two needles and further stretch the ribbons by pulling in opposite directions. Dip the needles in cold water to keep cool.

Microtome Sections # Drying of Sections:

Take out the slide from the hot plate. Hold it with the thumb and forefinger of the left hand without touching the ribbons. With the help of a needle push the ribbons to one side of the slide and tilt the slide after placing the needle below them to drain off the excess water.

Adjust the ribbons with the needle in such a way that on one side of the slide about 2 cm open space and on the opposite side about 2-3 mm open space are left. Wipe off the under-surface of the slide and keep it on the hot plate for 3 hours to overnight for drying. The slides can now be stored indefinitely.

Precautions:

1. The size of the slide is 3″ × 1″ (75 × 25 mm) and that of a microtome cover-glass 2″ × 1″(approx.) (50 × 25 mm). Hence, if the ribbon is cut into 1½” (37 mm) long pieces, after stretching they will be 2″ (50 mm) long (approx.) to fill the cover glass completely. If, however, the ribbon is slightly crumpled, it will stretch more — so the initial pieces should be made smaller.

2. Meyer’s fixative is rubbed with the little finger, because it is the least used finger and, consequently, the cleanest. The slide is flooded with water to keep the ribbons floating, so that they can stretch when warmed.

3. The ribbons are transferred to the slide maintaining their continuity. In case of root-tips the ribbon-pieces are mounted as soon as the material comes in the ribbon. In case of flower buds the initial and final sections are discarded and only the middle portion of the ribbon is taken, because this is where the anther sections containing the spore mother cells are present. In both cases, usually only one slide is made.

4. The lower surface of the ribbon which remains in contact with the razor is glossy. While mounting, this surface should come in contact with the slide for better adherence to it.

5. Full stretching of the ribbon is of utmost importance. As the ribbon stretches, the sections also stretch, eliminating the possibility of minute folds remaining in the sections. Stretching has to be done under controlled temperature.

So a thermostatically controlled hot plate is most suitable for stretching. Direct heating over a burner should never be made, for it may melt away all the paraffin and the sections may get displaced and distorted due to over-heating. Heating over an alcohol flame may, however, be made with utmost care.

6. The ribbons are mounted on a thin film of water on the slide to keep them floating, so that they can stretch freely. A thin film of water is kept in-between the slide and the hot plate surface for intimate contact between the slide and the hot plate both of which are solid. This ensures uniform heating of the ribbons.

7. If the ribbons are found to be curved after mounting, touch the two inner edges of the curve with two needles and pull them upwards while stretching. This will make them straight.

8. The final size of the ribbons — after stretching — is limited by the length of the microtome cover glass, which is about 2″ (5 cm). At one side of the slide, 2-3 mm of empty space is left so that the ribbons or sections do not get damaged by friction with the slide box. The rest of the empty space is left at the other side of the slide for labelling.

Microtome Sections # Staining the Sections:

Microtome sections are stained in cylindrical or rectangular staining Jars or Coplin Jars provided with lids. Each jar should be filled up to about 2½” (6 cm) with the reagent so that the fingers are not dipped in the reagent while staining. A large number of jars are required and they are arranged in a row according to priority.

Transferring a slide from one jar to another should be done slowly to keep the mixing of the reagents at a minimum; but not so slowly that the slides dry up. Slides can be stained singly or in pairs, taken in a back-to-back position. When the reagents become discoloured due to repeated use, they should be changed.

Both root tips and flower buds are stained in 1% aq. crystal violet solution. As the sections are embedded in paraffin, the first step is removal of this paraffin by xylol. Then xylol is replaced by alcohol and alcohol by water. The slides should be kept in xylol and abs. alcohol for as little time as possible, because both these reagents make the sections brittle.

The slides are stained after reaching down to water and then are mordanted in KI and iodine mordant for a few seconds. Mordanting increases the stain-retaining capacity of the chromosomes. Mordanting after staining is called post-mordanting. For some materials which do not stain easily, pre-mordanting is also done in 1 % chromic acid for overnight followed by washing in running water for 3 hours.

It should be remembered that for crystal violet staining, a chromic fixative is always used. Chromium particles become impregnated on the surface of the chromosomes and these adsorb the crystal violet stain. Mordanting is followed by quick dehydration in abs. alcohol and then differentiation in clove oil.

Crystal violet is highly soluble in alcohol. So dehydration in alcohol is done as quickly as possible; otherwise all the stain will be washed out. After differentiation, clove oil is cleared by xylol and, finally, the slides are mounted in Canada balsam.

Microtome Sections # Schedule of Staining the Section:

Pass the slide with paraffin sections through the following grades: Xylol I (Down), Xylol II (Down), Xylol III (Down) — 1 hr. in each Xylol — abs. ethyl alcohol mixture (1:1) — 1 hr.; absolute ethyl alcohol, 95%, 90%, 80%, 70%, 50% and 30% alcohol — 30 mins. in each. If required, the slides can be kept for overnight in 70% alcohol. Finally, keep in water at least 15 mins.

This downgrade is a long-drawn process. When so much time is not available, as during an examination, the durations of all the treatments can be reduced to half. But for a really good permanent preparation this should be avoided.

Stain in 1% aq. crystal violet solution for 30 mins. Keep in Pot. Iodine mordant (1 gr KI and 1 gm. iodine crystals dissolved in 100 ml 80% alcohol) for 45 seconds. Pas through abs. ethyl alcohol I (UP), II (UP) and III (UP) — 2 to 3 dips in each.

At this stage a piece of blotting paper should be kept nearby and, after 2 to 3 rapid dips in each alcohol jar, the slides should be rapidly jerked against the wall of the reagent jar and then on the blotting paper.

The entire process of dehydration in alcohol must be done as quickly as possible. Differentiate in clove oil I (2-5 mins.) and observe under the low power of microscope after placing a clean dry slide under the stained slide.

If cytoplasm and nucleus cannot be differentiated, put the slide back in clove oil I and again observe under microscope. After differentiation, keep the slide in clove oil II (10-15 min.). For differentiation, as long as the clove oil is fresh, less time is required; and more and more time is required as the clove oil gets more and more used. This has to be learnt by experience.

Pass through xylolI (UP), II (UP) and III (UP) — 1 hr. in each. If required, the slides can be kept in xylol III (UP) for overnight. Mount in Canada balsam. Put 3 drops of Canada balsam on the slide immediately after taking it out of xylol III (UP). Dip the cover- glass in xylol and place it gradually on the slide. Press the cover-glass gently with a needle to bring out excess balsam and dry the slide on a hot plate.