THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 36, No. 3, pp. 235-239 September, 1961 Copyright © 1961 by The Williams & Wilkins Co.

Printed in U.S.A.

THE REFRIGERATED MICROTOME (CRYOSTAT) IN A HORIZONTAL DEEP FREEZE

BERNARD KLIONSKY, M.D. Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas

The applications and advantages of the cryostat are receiving increasing recognition for the preparation of fresh frozen sections for histochemistry, fluorescence microscopy, and surgical or autopsy pathology.3' 6"9

With the cryostat, it is possible to prepare thin unwrinkled sections of single or multiple pieces of fresh tissue. Tissues that are very difficult to cut by means of the conventional technics for frozen section may be processed easily. This paper deals with an advance in the general principles of the frozen section and of the cryostat technic, consisting of an inclined mounting of a microtome in an open-top deep freeze. The resultant simpli­fication of operation and drastic reduction in price may help to make such equipment more readily available and usable.

REVIEW OF THE PRINCIPLES OF

FROZEN SECTIONS

The basic principle of the frozen section is the freezing of water in tissues in order to provide sufficient rigidity for cutting. The addition of a cold knife offers the advantage that the zone of tissue melting during cutting is restricted to the micro-layers on the surfaces of the section.10 A major portion of the section remains frozen during and after cutting. The use of the cold knife, therefore, permits the use of the Adamstone-Taylor technic.1 Enclosure of the microtome and the knife in a deep freeze permits the choice of an appropriate and stable temperature near the optimum for cutting tissue, which then neither

Received, January 23, 1961; accepted for publi­cation May 31.

Dr. Klionsky is Assistant Professor of Pathol­ogy-

This investigation was supported in part by grant H-2922 from the National Heart Institute, Public Health Service, Department of Health, Education, and Welfare.

overfreezes nor thaws during extended periods of sectioning. Condensation of water on the knife and on the microtome is controlled more easily with an enclosed microtome than with one exposed to humid air. The refrigerated microtome also permits the use of a plastic plate that prevents curling and rolling of the sections by forcing them between the plate and the knife blade. The sections lie flat on the knife and may be picked up directly on a warm slide or cover slip to which they will adhere. An antiroll device can be obtained with the Cambridge microtome. An adaptation of this design to the Minot and to the Spencer microtomes was made in this laboratory and a photograph and diagram of con­struction are presented. With ingenuity and appropriate variation in the length of the front axle piece and of the side mounts, an identical device could be adapted to any microtome (Figs. 1 and 2).

A detailed description of the principles and use of the antiroll device and of the methods of freezing, sectioning, fixing, and staining cryostat tissue is available.6'7 '9

The strong merits and relatively slight disadvantages of the cryostats commercially available at the time this investigation was begun have been discussed.7 Such cryostats suffered from the disadvantage that access to the interior was provided through a vertical door or window. Upon opening such a door, there was ready opportunity for the heavier cold air to pour from, and for warm, moist, room air to enter the chamber. This contributed to variations of the interior temperature and to excessive condensation of moisture on the microtome and knife. Several ingenious external con­trols or glove-fitted portholes helped to minimize the size of the vertical door and the length of time that the cabinet must be left open. Although these devices worked very satisfactorily, they contributed to

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236 KLIONSKY Vol. 36

FRONT VIEW

PLATE ONLY

SIDE VIEW COMPLETE ASSEMBLY

~///////\\ *J.8

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SECURING SCREW VERTICAL

ADJUSTMENT

TOP VIEW COMPLETE ASSEMBLY

3

'-> n=3ia Securing | O ^ screw for

'—R?—i adjustment

A NT I- ROLL PLA TE FOR MICROTOME

All components are brass except 4.5cm square plast­ic plate.

1:1 cm scale throughout

Equivalents for Drills

cm

.9 5

.47

.32

Inches 3 / 8 3/|6

' ' 8

FRONT VIEW-AXLE PIECE ONLY All screws were 8 32.

FIG. 1. Scale diagram for construction of an antiroll device. With in­genuity in the mounting and with appropriate variation of the length of the axle piece, this can be adapted to any microtome. A plastic plate, 2.5 cm. in width, is adequate for most specimens and is easier to adjust than the wider one which is illustrated.

increased cost and to inconvenience in operation of these cryostats.

Most of these problems can be eliminated by placing the microtome in a refrigerated box with an open top2 (Fig. 3). This presents a horizontal interface between the warm and cold air. Access to the interior can be accomplished without significant loss of cold air or entrance of warm, moist air. The top of such a unit can be left open

almost indefinitely without condensation on the objects stored within or without significant alteration of interior temperature (the widespread use of such boxes in grocery stores throughout the country is witness to their effectiveness).

With the open top, it is no longer necessary to utilize the external control and glove devices that were designed to limit sharply the length of time the vertical door was

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Sept. 1961 T H E HORIZONTAL CRYOSTAT 237

FIG. 2 (left). Artist's drawing demonstrates assembly and attachment of antiroll device to knife-carriage assembly of microtome.

FIG. 3 (right). The horizontal freezer is illustrated with external control for a rotary microtome. The front section of the plastic lid is open.

open (Figs. 4 and 5). It now is possible for anyone to build his own simple cryostat provided that he has a microtome and a small, inexpensive, horizontal freezer* cap­able of maintaining a temperature of from — 13 C. to — 20 C. at the cutting edge of the knife. A variety of rust-proof microtomes are available f and may be preferable in

* A suitable but inexpensive freezer can be ob­tained from the Master-Bilt Refrigeration Manu­facturing Co., 4209 Folson Avenue, St. Louis 10, Missouri. For an additional charge the company will complete installation of your microtome in the freezer.

f The National Instrument Laboratories, Inc., 828 Evarts Street, N. E., Washington 18, D. C , will provide a porcelainized (Cambridge rocking-arm) microtome, complete with knife, antiroll device, and necessary hardware to provide for the external operation of the microtome.

The International Minot microtome, model 3310, is available complete with antiroll device from the International Equipment Co., Boston, Massachusetts. An aluminum freezing block is also available.

The Lipshaw rotary microtome, model 50 AB, is available from the Lipshaw Manufacturing Co., 7446 Central Avenue, Detroit 10, Michigan.

that they require less time for maintenance. Standard, rotary, paraffin microtomes that are not rust-proof have been used suc­cessfully, however. With proper main­tenance, these can be used satisfactorily over long periods of time.4

For the convenience of the operator, it seemed preferable to use the microtome in other than its standard horizontal position. Placing the microtome at an angle with the front end elevated made the knife, tissue, and chuck more accessible to the control of the operator. Several microtomes were tested at elevations ranging from horizontal to 60 degrees of vertical tilt. They operated satisfactorily at all levels. Final considera­tions of the proper angle are governed by 2 factors: (1) all portions of the microtome must be below the frost line of the freezer; and (2) the final position of the chuck and knife should allow the operator to have the assistance of gravity in cutting the sections. Practically, an angle of 20 to 30 degrees was most convenient. With the aid of gravity it is much easier to obtain sections that are not compressed and wrinkled. I t

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238 KLIONSKY Vol. 86

is perhaps unfortunate that for many years conventional freezing microtomes have required pathologists to defy gravity and cut their fragile frozen sections uphill. For the further convenience of the operator it is recommended, although not necessary, that the actual cutting mechanism of the micro­tome be controlled from outside the cabinet. This requires that the angled platform support and position of the microtome be so chosen that the center of the microtome drive shaft is placed properly in relation to the refrigeration coils in the side walls of the freezer. The hole that must be drilled in the side wall of the freezer must avoid the coils (an error in drilling can convert a freezer to scrap metal). The margins of the hole should be sealed with a collar of material such as Synthane* to prevent access of moisture to the insulation and to provide a bearing surface for the extension shaft that couples the microtome to the external wheel. If the microtome is used at an angle, care must be taken to replace the wheel in the proper position on the shaft to insure proper counterbalancing of the instrument.

Commercial cryostats based on these general principles have become available.f

SUMMARY

The open-top horizontal freezer con­taining a refrigerated microtome has all of the advantages of the conventional vertical cryostat. It offers additional advantages, in that it is more stable and easier to operate. Simplicity of design leads to economy of construction. Application of this simple principle makes the construction of a cryostat economically feasible for anyone who has an open-top deep freeze and a microtome.

* Synthane Corporation, Oaks, Pennsylvania. t Internat ional Equipment Co., Boston, Massa­

chuset ts ; Lipshaw Manufacturing Co., Detroi t , Michigan; Master-Bilt Refrigeration Co., St. Louis, Missouri.

SUMMAEIO IN INTERLINGTTA

Le cassa frigorific horizontal aperte in alto ha omne le avantages, como receptaculo de un microtomo refrigerate, que es offerite per le cryostato vertical conventional. Illo ha avantages additional in tanto que illo es plus stabile e plus facile a manipular. Le simplicitate del piano structural resulta in economia del construction. Le application de iste simple principio rende le construction de un cryostato economicamente possibile pro omne interessato qui possede un cassa frigorific aperte in alto e un microtomo.

R E F E R E N C E S

1. ADAMSTONE, F . B. , AND TAYLOR, A. B. : The rapid preparation of frozen tissue sections. Stain Technol., 23:109-116,1948.

2. CHANG, J . P . , AND H O R I , S. H. : A frozen sec­tion freeze-substitution technique and an improved cryostat . Proceedings of the Histochemical Society, 1960. J. Histo-chem., 8: 310, 1960.

3. D I E Z E L , P . B . : Uber die Brauchbarkeit des Kryostaten in Histologie und Histochemie. Mikroskopie., 13: 196-200, 1958.

4. F ITZ-WILLIAM, W. G., J O N E S , G. S., AND GOLDBERG, B . : Cryostat techniques: Meth­ods for improving conservation and sec­tioning of tissue. Stain Technol., 35: 195-204, i960.

5. H E S S , R.: Histologische Schnellschnitte im Kryostaten. Pa th . Microbiol., 23: 273-276, 1960.

6. IBANEZ, M. L., RUSSELL, W. O., CHANG, J . P . , AND SPBECE, A. J . : Cold chamber frozen sections for operating room diagnosis and routine surgical pathology. Lab. Invest. , 9: 98-109, 1960.

7. KLIONSKY, B. , AND SMITH, O. D . : Application of the refrigerated microtome in surgical pathology. Am. J. Clin. Path . , 33: 144-151, 1960.

8. LANGREDER, W., AND STRAUSS, G.: Beschleu-nigung und Verbesserung der gynakolo-gisch-histologischen Diagnostik durch den Schnellschnitt im Kryostaten. Zentralbl. Gynak., 80: 889-903,1958.

9. P E A R S E , A. G. E . : The production and use of fresh tissue sections for analytical and func­tional histochemistry. Acta Histochem., 5 (Supplement 1): 229-232, 1958.

10. THORNBUBG, W., AND M E N G E R S , P . E . : An analysis of frozen section techniques. I . Sectioning of fresh-frozen tissue. J . Histo­chem., 6: 47-52, 1957.

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1961 THE HORIZONTAL CRYOSTAT

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FIG. 4 (upper). With the front section of the plastic lid open, the operator has this view of a rotary microtome mounted at an angle of 28 degrees. Rotary microtomes of the Minot-type have been adapted in a similar manner.

FIG. 5 (lower). The extension couplings for the rocking-arm microtome are based on those designed for the Pearse cryostat and utilize its components. The entire plastic door is open, in order to reveal the interior of the freezer, with the microtome on its platform at an angle of 30 degrees.

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