Fig. 52.—Type of freezing-microtome, for the use of ether or ethyl-chloride. Cathcart model.
Fig. 53.—Carbonic-acid freezing-microtome. Becker model.
Fig. 54.—Bardeen freezing-microtome attached to carbonic-acid gas-cylinder.
The carbonic acid outfit consists of a microtome arranged so that it can be connected with a cylinder of compressed carbonic acid gas, as shown in the Aschoff-Becker or Bardeen models. (Figs 53 and 54.) The cylinders or drums containing a charge of 15-30 lbs. of the liquid carbonic-acid are furnished by the trade at reasonable rates. It is not necessary to buy the drums, as they are replaced by full ones as needed. They are provided with valves and can be fastened in an upright position or laid flat upon a table, in any way convenient for the attachment to the microtome. Connection is made between the valve of the cylinder and the object-holder of the microtome by means of strong rubber tubing which should be securely wired at both ends, or by a flexible metallic tube. The latter is preferable, as the rubber-tubing often bursts, or is so stretched by the pressure of the gas that it must be frequently replaced. Object-holders with flexible metallic tube attachment for use in an ordinary microtome are supplied by Bausch and Lomb and other firms. My personal experience makes me prefer the Bardeen freezing microtome (see Fig. [54]) to all others. It is cheap and can be easily attached to the valve of the gas-cylinder, which is fastened horizontally to the table-top, as it has a screw-thread fitted to the uniform thread of the drum-valve. The object-holder can be raised or lowered between the glass-tracks on which the knife runs, so that sections of a definite thickness can be obtained. The most satisfactory knife is that of the type of a plane-bit set in a wooden handle. (See Fig. [55].) It must be well honed and stropped. The tissue, which should not be more than 5 mm. thick, is placed upon the object-holder in a drop of water, albumin fixative, or saturated solution of gum-arabic, and pressed firmly against the plate as the gas is turned on slowly and evenly. Freezing is usually accomplished in one-half to one minute. The best results are obtained by turning the gas on for about 15-20 seconds, then turning it off for several seconds; the tissue will continue to freeze; if not hard enough the gas is turned on again for 15 seconds and then turned off. If necessary this may be repeated until the tissue is properly frozen. The interruption of the gas-flow prevents over-freezing and thereby lessens the amount of change in the cells. When sufficiently frozen the gas is then turned off; the knife, with bevel-edge down, is held in the right hand with its handle between the palm and the ball of the thumb with the back of the hand uppermost; and the edge of the knife set at an angle of 45° to the tracks, along which it is rapidly pushed back and forth, shaving the sections from the frozen tissue as the latter is pushed up above the level of the tracks by means of the micrometer screw turned with the left hand. The screw should have been adjusted at the proper height before freezing, so that no time is lost in getting the object-holder up to the height for cutting. By holding the elbow and fore-arm closely against the body and pushing from the elbow with wrist-joint fixed the shaving of sections can be accomplished very quickly, so that with one freezing several hundred sections can be obtained. The microtome-screw should not be turned by the left hand until the knife on its return has cleared the tissue. A little practice in co-ordinating movements is necessary for expert work. The sections should be allowed to collect upon the knife-blade until a large number have been cut; they are then swept off the blade by the finger into cold freshly-boiled physiologic salt-solution. Sections of tissue that have been previously fixed can be put into 60 per cent alcohol, where the sections will unroll and straighten out perfectly flat. When the method of freezing by alternately turning on and off the gas, as given above, is followed, and the amount of freezing in the interval noted, there is no danger of over-freezing, and the breaking and crumbling of sections, with the production of marked artefacts, is avoided. When over-freezing has occurred the block may be partly thawed out by the finger. But over-freezing, as well as repeated freezing and thawing, may cause so much damage to fresh tissue that a diagnosis cannot be obtained from the frozen sections. After thawing out in the physiologic salt-solution the sections of fresh tissue obtained by freezing are treated according to the methods given in the next section of this chapter.
Fig. 55.—Knife for Bardeen freezing-microtome.
It must be emphasized here that the process of freezing is an active one, and alters the relation of cell-structures. With many fresh tissues the changes resulting from the freezing are so great that no diagnosis can be made. It seems necessary here to warn against the routine employment of the rapid method of freezing and staining fresh tissues in the diagnosis of material obtained by surgical operation. It has become a fad with some surgeons to make a pathologic diagnosis by the freezing method while the patient is on the table. Consequently, as the result of diagnoses made by the rapid freezing and staining method, many mistakes are made, even by supposed experts in this line. Particularly in the diagnosis of sarcoma is it easy to make mistakes because of the altered aspect of the cells caused by freezing. Normal lymphnodes, tonsils and inflammatory infiltrations may look like spindle-cell sarcoma in the sections prepared by the rapid freezing and staining method; and the exact nature of many other pathologic conditions cannot be accurately determined from such sections. On the other hand, a certain number of pathologic conditions can always be recognized in sections obtained in this way, and this fact justifies the employment of the method when properly controlled. In all cases in which the pathologic condition is not clearly evident in sections obtained by the rapid freezing and staining method no diagnosis should be given. In such cases the tissue should be fixed and then cut upon the freezing-microtome, or imbedded in celloidin or paraffin and then cut. Even when the patient is upon the table the tissue removed can be put into a 10 per cent formol solution for a few minutes and then frozen directly in gum without washing out the formalin. The longer the time that can be used for this short preliminary fixation the better the sections will be and the less the production of artefacts by the freezing. The process of fixation can be hastened by warming the fixing-fluid. I advise this short fixation before freezing in all cases of operative diagnostic work when the diagnosis is wanted as soon as possible. For all other work with the freezing microtome, when the question of time is not so important, fixation with formol for 12-24 hours should be carried out. This combination of formol-fixation and the freezing method permits the early diagnosis of autopsy and operation material, makes possible the demonstration of fat and other substances altered or dissolved out by the imbedding methods, and is a convenient way of selecting tissues, requiring more complicated staining processes. (See also Page [239].) The further treatment of sections of fresh tissue obtained by freezing will be found in the second section of this chapter.