Microtomy: Microscopical study of sections of animal & plant material

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1 ICENI MICROSCOPY STUDY GROUP Microtomy: Microscopical study of sections of animal & plant material Introduction. Many living things are extraordinarily beautiful. The microscopist has a privileged view and this may be extended and enhanced by looking inside; this can be done by making very thin sections. This is microtomy. Microtomy may also be used to extend our knowledge of how living things are put together and so allow deductions to be made about how they work. This paper is concerned only with light microscopy. So that sections can be examined under a microscope, light must pass though them so sections need to be very thin indeed. Thicknesses of 8-10 µm are appropriate for biological sections and µm botanical sections. [For geological specimens, 30 µm is commonly used.] Before living things are sectioned, they must first be fixed (killed) and then preserved. FAA, Formalin Acetic Acid, is usually used for plant material, Bouin s fluid for animal. It must penetrate the tissues and some subjects, seeds for instance, are designed to exclude any penetration so may need piercing to allow the liquid access. Specimens may be held in FAA indefinitely but zoological specimens will harden if left too long in Bouin s fluid after fixation these must be thoroughly rinsed in 70% alc. and then stored in this medium or, better still, a mixture of 70% alc. plus some glycerine. After FAA, transfer to 70% alcohol, then to 90%, then to absolute alcohol and, after hours in xylene, finally to a further bath of absolute alcohol. Embedding. To allow the specimen to be very thinly sliced, it must be first embedded. [The simplest support can be provided using a carrot. This is first cut into a cylinder to fit closely into the cutting instrument or microtome. The carrot is then split longitudinally and a small V taken out of both flat faces to accommodate the specimen.] More usually, specimens are embedded in wax. A suitable embedding wax would be solid up to its melting point and liquid thereafter. For this the wax must be homogeneous. Some, such as candle wax, a petroleum derivative, are mixtures so their constituents melt at different temperatures. A range of microtomists waxes are available, each with a different melting point. The choice of wax will be determined by the conditions of its use. Higher melting points may be appropriate in hot countries or centrally heated rooms. The Iceni Microscopy Study Group (IMSG) has chosen one with a melting point of 56 C. As with a carrot, wax plugs are prepared to fit the microtome. For clean cuts, they must be a good fit. A good way to obtain them is: Select a plastic tube or bottle to act as a former; it should have the same diameter as the desired wax plug. Drill holes to accommodate this former in a piece of scrap soft wood. Make foil cups by using the former to push pieces of cooking foil into the holes. Remove the cups, perhaps by blowing into them, and fill them with granules of the embedding wax. Put cups with granules into a hot cupboard or onto a hotplate so the wax melts. Store the wax plugs in their foil cups until required. The specimen must be really well supported against the cutting blade. It will only be firmly supported if the wax has penetrated the specimen. To achieve this, specimens are soaked in molten wax for 24 hours. To avoid cooking the specimens, the wax should be held only a few degrees above its melting point. The following day, take a pair of forceps warmed to the wax temperature and use them to rearrange the specimen so that when it is sliced we ought to see what is wanted. The foil cup of wax and specimen is then removed from the heat so, while holding the specimen in place, the wax sets. Ernie Ives recommends storing plugs in 10% glycerine. 1

2 Microtomes. The Cambridge Rocking Microtome A rotary microtome of older construction Microtome (C. Reichert, Vienna, Pictures from Wikipedia While sections can be cut by hand using a razor, it is difficult to achieve constant thicknesses. Mechanical microtomes allow cuts of a predetermined thickness to be made and to be repeated. Such machines come in a wide variety of shapes and sizes. All have extremely sharp blades that must be frequently (and correctly) sharpened. These blades are dangerous! When very thin sections are cut using the best microtomes, the heat generated by each cut warms the trailing edge of each newly cut section just enough to stick it to the next, producing a ribbon of sections. This allows several sections to be mounted side by side so that, conveniently, progressive views of the internal structures may be seen all on one slide. The IMSG has sledge, rotary, bench and hand microtomes. Sharpening the blade. Blades come in various profiles such as doubleconcave, planar-concave, wedge and chisel. 2

3 If the blades are not to be spoilt, it is important to know exactly how to sharpen each of them. Refer to instructions! A suitable grinding medium is aluminium oxide; 3µm for sharpening, 1µm for the final polish. These are mixed with 3 in 1 oil with a little paraffin. Alternatively, Silvo may be used for the final polish. Each grinding medium should have its own plate glass panel, kept for that grade alone. Glass plates need good support, perhaps on a piece of kitchen worktop. The need continually to sharpen blades can be avoided by using surgeons skin graft knife blades, suitably supported. Procedure. Before starting to cut, have ready on a hotplate a deepish (50+ mm) dish of warm water at a little below the melting point of the wax, say C. Also, prepare enough thoroughly cleaned slides with adhesive. Decide which is to be the top side of the slide and mark it; this can be done with a tiny scratch in one corner using a diamond cutter. If Haupt s Adhesive is to be used, use a little finger to add a small dab towards one end; this is then smeared across the slide using the back of the hand. Before sections are put onto the slides, tan the adhesive with a drop of 2 or 5% Formalin. Slide Cleaning. Slides and cover slips often come from the manufacturer with dust, grease and small glass shards on them so need washing before use. This is best done using an old toothbrush dipped first into a standard cleaning fluid (50/50 methylated / water mixed with a small dash of washing-up liquid), then with scouring powder or Jif. Finish by wiping with a soft cloth (such as a clean old handkerchief) until dry and free of abrasive, polish with chamois leather and put back into their vacuumed box ready for use. Push the slide into the water and move it under the section to be mounted. It may have to be encouraged with the paint brush. If the section needs moving, pulling it is less likely to damage it than pushing. Specimens often expand once on the slide so allow for this. Sometimes, sections turn over; the top surface will be dull and the bottom one shiny. Remove the slide with its section from the water and blot it very carefully with a single firm roll of a finger. A suitable blotting paper is that used to pack slides in boxes; alternatively, use stationer s blotting paper. After blotting, the paper should be carefully peeled away. Warm the slide in a flame to ensure it is absolutely flat. It may be stored indefinitely in this condition. Before the section can be stained, all wax must be removed. This is done by carefully lowering it into xylene for some 30 minutes. Coplin jars are ideal for this as they hold the slides vertically; almost as good are 5 slide transport boxes held vertically, perhaps in a rack. A second and third de-waxing in fresh xylene is advised. Haupt s Adhesive. From The Microtomist s Formulary & Guide by Peter Gray; Constable, Dissolve 2 g Gelatine in 100 cm 3 distilled water; Add 2 g Phenol (or Cedarwood oil or thymol) to prevent any fungal action. Add 15 cm 3 Glycerine and mix. Filter (e.g. through coffee filter paper). Other options are Isinglass, egg albumin (egg white) or, if nothing else is available, plain saliva. As each section, or ribbon of sections, is cut it should be picked up using a small artist s paint brush and floated, curved side down, onto the warm water surface to unroll and flatten. 3

4 Staining. To allow details to be seen, specimens are usually stained before mounting. Different stains may be taken up by different parts of the specimen and so provide pleasing contrasts. Experimentation may be necessary. Time in a stain depends upon how it is made up. Ernie Ives recommends the following staining processes for botanical material: Astra Blue and Safranin. Immerse in first stain for 30 minutes; rinse in water to remove surplus stain; drain and mop up surplus water with tissue. Immerse in second stain; rinse, drain and mop. Dip in Meths, reverse slide and dip again; drain and mop. Using a pipette, flush once with abs. isopropanol. Dip in abs. isopropanol, reverse slide and dip again. De-wax in xylene de-wax for 30 minutes. Clear in clean xylene for a few minutes. Mount in Canada Balsam or other resin mountant. Safranin and Fast Green. Immerse in first stain for 30 minutes; rinse in water to remove surplus stain; drain and mop up surplus water with tissue. Dip in Meths, reverse slide and dip again; drain and mop. Immerse in second stain; rinse, drain and mop. Using a pipette, flush once with abs. isopropanol. Dip in abs. isopropanol, reverse slide and dip again. De-wax in xylene de-wax for 30 minutes. Clear in clean xylene for a few minutes. Mount in Canada Balsam or other resin mountant. Over-staining may be corrected using Acid Alcohol in a Coplin jar for c.2 minutes; if satisfactory, wash in water. Mr Ives prefers rectangular cover-slips and uses a No 3 brush to ring them with Humbrol paint. For ordinary ringing, once the slide is dry, he uses a scalpel to clear the top of the cover-slip. This leaves a meniscus clinging to the edge of the cover-slip. For smart he uses a slide on edge to leave a slide s width overlapping the cover-slip top and edge. Further reading. 1. "Practical Microscopy". Eric Marson. (Out of print). 2. "Practical Section Cutting and Staining". E.C. Clayden. Churchill A guide to Wood Microtomy. E. Ives, "A practical handbook of British beetles", N H Joy Members of the IMSG at work on 1 st September Fast Green. This formula is Mr E Ives s modification from an old Fast Green in clove oil stain: Cellosolve 150 ml Isopropanol abs. 150 ml Terpineol 150 ml Fast Green FCF 0.25 g Mix liquids and add stain. Leave for 1 day to dissolve. Terpineol is not turpentine! This is much slower working than Fast Green in Cellosolve alcohol alone and, being weaker, gives a lighter green colour but is much easier to control. Toluidine Blue is another possibility. Jill adjusts her microtome 4

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