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

The knife carrier of a normal rotary microtome has been replaced by one which takes two standard microtome knives, set so that their edges come within the limits of free travel of the block-holder. The upper knife is fixed; the lower is so held that it can be moved forward towards the blockholder while its edge remains parallel to the edge of the upper knife. In this way the two edges lie in separate, though parallel, vertical planes. With this attachment, and a suitable setting of the feed cam of the microtome, it is possible to cut two sections at every stroke, the first section being cut by the upper knife and the second by the lower (fig. 1 and fig. 2A).

FIG. 1.

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

FIG. 1.

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

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.

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.

In the model now in use the free travel of the block-holder is approximately 35 mm. and the distance between the knife-edges 20 mm. This allows the centre 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 kniferest is inclined at an angle of 7° from the vertical, and the lower is set back 0·25 mm. (horizontally) from the upper to allow for minor variations in the depths of the knives. The upper knife is held against its knife-rest by normal clamping screws, and the lower by strong spring plungers which press the knife forward and downward against the knife-rest (Fig. 2B).

Two large adjusting screws are fitted so that they meet the flat back of the lower knife. By screwing these forward and upward the knife is raised on 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 lower knife is advanced beyond that of the upper.

To cut twin ribbons the feed-cam of the microtome is set at the sum of the two thicknesses of section required and the lower knife is adjusted by trial and error until two sections of the correct thickness are successfully ribboned. For any given rotation of the adjusting screws, the amount by which the lower knife is advanced varies with the depth of the blade and with the thickness of the back. It is, therefore, impracticable to calibrate these screws. If two sets of sections of iothickness are needed, the following procedure is adopted.

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

When the edges are considered satisfactory a few sections are left attached to each to indicate the correct part of the blade to use and both blades are set in position. The feed cam is turned to 20 μ and the lower knife adjusted until two ribbons form. If one ribbon is noticeably more compressed than the other, the sections in it are too thin and the bottom blade is readjusted until sections in each ribbon are of the same size. If a test of the accuracy of this method is required, or if it is necessary to cut ribbons of different thickness, section-thickness can be measured (1) by altering the feed-cam setting until the upper knife just fails to cut (the new setting giving the thickness 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 and wedge-shaped sections are being cut. Curvature is, of course, the result of greater compression of the thin side of the ribbon. In this case the lower blade is readjusted so that the thickness of the sections is reduced at the outside of the curve and increased at the inside.

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

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

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

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

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

  2. The production of two sets of sections of different thicknesses. An obvious use is in such problems as cell or mitotic counts. The average count on a series of identical sections will exceed the true count per section-volume by reason of the duplication in adjacent sections of a single cell or nucleus which has been more or less exactly halved in the cutting. The difference in count, however, between two sections of different thickness will give a true estimate of the number of cells in the volume difference between the two sections. (We are indebted to Dr. F. J. Ebling for pointing out this possibility.)

  3. The production of wedge-shaped sections. This is readily—sometimes too readily—accomplished. Such sections can be employed experimentally to determine the best thickness for certain histological techniques (e.g. silver staining of nerve-fibres, where the intensity of impregnation often varies considerably with the section-thickness). The ‘thin edge of the wedge’ might be of use for electron microscope studies, and a uniform series of wedge sections has obvious possibilities for certain counting problems similar to (2) above.

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

Abercrombie
,
M.
, and
Causey
,
G.
,
1950
.
Nature (Lond
.),
116
,
229
.