347

A Twin-knife Microtome Attachment

By J. F. SLOANE AND J. E. HARRIS(From the Department of Zoology, University of Bristol)

SUMMARY

A microtome knife carrier, holding two knives, has been designed to produce al-ternate sections as two separate ribbons. The position of the knives can be alteredto give sections of the same or of different thickness in the two ribbons. The applica-tion of this device in various histochemical and counting techniques is-briefly indicated.

INTRODUCTION

THE knife carrier of a normal rotary microtome has been replaced byone which takes two standard microtome knives, set so that their edges

come within the limits of free travel of the block-holder. The tipper knife isfixed; the lower is so held that it can be moved forward towards the block-holder while its edge remains parallel to the edge of the upper knife. In thisway the two edges lie in separate, though parallel, vertical planes. With thisattachment, and a suitable setting of the feed cam of the microtome, it ispossible to cut two sections at every stroke, the first section being cut by theupper knife and the second by the lower (fig. i and fig. 2A).

In the model now in use the free travel of the block-holder is approximately35 mm. and the distance between the knife-edges 20 mm. This allows thecentre of the block to rise 7-5 mm. above the upper edge and fall 7-5 mm.below the lower edge, giving satisfactory clearance for a block of 10 mm.depth. The knives have a depth of 20 mm. and are flat backed. Each knife-rest is inclined at an angle of 70 from the vertical, and the lower is set back0-25 mm. (horizontally) from the upper to allow for minor variations in thedepths of the knives. The upper knife is held against its knife-rest by normalclamping screws, and the lower by strong spring plungers which press theknife forward and downward against the knife-rest (Fig. 2B).

Two large adjusting screws are fitted so that they meet the flat back ofthe lower knife. By screwing these forward and upward the knife is raisedon the knife-rest against the downward pressure of the spring plungers. Thus,because of the inclination of the knife-rest, the cutting edge of this lowerknife is advanced beyond that of the upper.

METHOD OF OPERATING

To cut twin ribbons the feed-cam of the microtome is set at the sum ofthe two thicknesses of section required and the lower knife is adjusted bytrial and error until two sections of the correct thickness are successfully[Quarterly Journal of Microscopical Science, Vol. 92, part 3, pp. 347-50, September 1951.]

348 Shane and Harris—A Twin-knife Microtome Attachment

ribboned. For any given rotation of the adjusting screws, the amount bywhich the lower knife is advanced varies with the depth of the blade andwith the thickness of the back. It is, therefore, impracticable to calibratethese screws. If two sets of sections of 10/x thickness are needed, the followingprocedure is adopted.

FIG. 1. Photograph of microtome, showing twin-knife attachment in use. Two similar ribbonsof uniform thickness are being cut. The block-holder is seen immediately above the upper

knife.

A wax block, without any tissue embedded in it, is made for trial purposesand 15 to 20 sections of 10 JX thickness are cut with each knife, separately. Ifthe ribbons are of the same length, the knives are considered to be equallysharp. If not, the knife causing the greater compression is re-sharpened andtested again. A slightly better edge is required than would be needed forordinary section-cutting.

When the edges are considered satisfactory a few sections are left attachedto each to indicate the correct part of the blade to use and both blades areset in position. The feed cam is turned to 20 p. and the lower knife adjusteduntil two ribbons form. If one ribbon is noticeably more compressed thanthe other, the sections in it are too thin and the bottom blade is readjusteduntil sections in each ribbon are of the same size. If a test of the accuracy

Shane and Harris—A Twin-knife Microtome Attachment 349

of this method is required, or if it is necessary to cut ribbons of differentthickness, section-thickness can be measured (1) by altering the feed-camsetting until the upper knife just fails to cut (the new setting giving the thick-ness of the bottom section, that of the top being obtained by difference),(2) by removing and weighing a definite number of sections from each ribbon,or (3) by direct measurement of the section-thickness on the microscope.

If ribbons curve in opposite directions, the blades are not parallel andwedge-shaped sections are being cut. Curvature is, of course, the result of

spring - ^plunger ^

\adjusting —

'screw

clamping screw1

~ knife^ rest

kniferest

FIG. 2A FIG. 2B

FIG. 2.A. General view of the twin-knife carrier as fitted to a standard rotary microtome.B. End-elevation of the upper part of the knife carrier to show method of mounting the knives.

greater compression of the thin side of the ribbon. In this case the lowerblade is readjusted so that the thickness of the sections is reduced at theoutside of the curve and increased at the inside.

When both ribbons are straight the trial block is removed and the blockto be cut is attached in the same position on the block-holder. It is advisableto use the smallest possible block to reduce the effect of inaccuracies in theknife-edges to a minimum. If the knife-edges become gummed with paraffinto the slightest degree, they are cleaned with a paint brush moistened withxylene, and all traces of xylene removed with methylated spirit before furthercutting.

In order to identify corresponding sections in the two ribbons the end ofthe block is pricked with a needle. The first sections in each ribbon showingthe needle hole will correspond.

It is often convenient to mount both ribbons separately on thin sheets ofmica; mounted sections are then treated by the required histological or histo-chemical techniques. At the end of the process the two sheets are cut intostrips; strips containing corresponding sections are then mounted side byside on a single microscope slide.

350 Shane and Harris—A Twin-knife Microtome Attachment

USES OF THE ATTACHMENT

Three possible uses are listed below; others will no doubt suggest them-selves to those interested in particular problems.

1. The use for which the instrument was originally designed—to providetwo similar sets of sections, for histochemical tests, or for comparisonof different methods of staining, &c. An example of this has alreadybeen described. Abercrombie and Causey (1950), using phosphorus-32to identify transplanted tissues in chick embryos, obtained autoradio-graphs from one set of sections and normal histological preparationsfrom the other.

2. The production of two sets of sections of different thicknesses. Anobvious use is in such problems as cell or mitotic counts. The averagecount on a series of identical sections will exceed the true count persection-volume by reason of the duplication in adjacent sections of asingle cell or nucleus which has been more or less exactly halved in thecutting. The difference in count, however, between two sections ofdifferent thickness will give a true estimate of the number of cells inthe volume difference between the two sections. (We are indebted toDr. F. J. Ebling for pointing out this possibility.)

3. The production of wedge-shaped sections. This is readily—sometimestoo readily—accomplished. Such sections can be employed experimen-tally to determine the best thickness for certain histological techniques(e.g. silver staining of nerve-fibres, where the intensity of impregnationoften varies considerably with the section-thickness). The 'thin edge ofthe wedge' might be of use for electron microscope studies, and a uni-form series of wedge sections has obvious possibilities for certaincounting problems similar to (2) above.

We wish to thank Dr. H. J. Thomas, with whose help the first model ofthe twin-knife holder was made, and we take this opportunity, too, of acknow-ledging the very great help rendered by Willcocks (Clevedon), Ltd., ofClevedon, Somerset, in the development of the apparatus.

REFERENCEABERCROMBIE, M., and CAUSEY, G., 1950. Nature (Lond.), 116, 229.