THE TREATMENT OF THE MATERIAL.
INTRODUCTION. The material obtained by autopsy, surgical operation, curettage, excision, spontaneous discharge, animal experimentation, etc., may be examined microscopically in the fresh state, or prepared for microscopic examination by methods of fixation, hardening and imbedding. The methods necessary for such histologic studies of pathologic material are given in the following pages, arranged as far as possible in their logical order. Only those methods are given that, in the light of the writer’s experience, yield the best results, from the standpoints of economy of time, labor, and expense, and perfection of result. The number of histologic methods contained in the literature is so great that it is out of the question for the student or practical worker to try out all of them. To give all of these methods would create confusion. I have attempted to avoid this by giving in full detail only those which in our laboratory experience have yielded the best results. So many methods represent but slight variations of some original method, and in the great majority of cases these variations add so little or nothing of value to the original method that in such cases the latter alone is given in full, with references only to the variations of the method. The individual equation plays such a large part in the judgment of laboratory methods that allowance has been made for this when certain variations or alterations in original methods have been strongly recommended by expert laboratory workers.
The purpose of the microscopic examination is the revealment of pathologic changes too small to be recognized by the naked eye, and the securing of a diagnosis that cannot be made macroscopically, as well as the confirmation of diagnoses based upon the gross appearances. Aside from these more immediate practical considerations, the microscopic examination of tissues is concerned with the solution of etiologic and pathologic problems, and the extension of our knowledge of disease. The aim of pathologic technique is the fixation of tissues for microscopic examination in such a manner that all of the morphologic and chemical elements and constituents of the tissue are perfectly preserved, so that with differential staining methods they are all brought out with sufficient contrast to be readily and correctly identified. In a certain number of methods this ideal is attained, and to Weigert, more than to any other worker in the field of pathologic technique, are we indebted for such ideal methods.
The choice of methods will depend upon the source and nature of the material, the object of the examination, the time-element and the degree of responsibility involved. The cellular elements of all pathologic fluids, secretions and excretions should be examined in the fresh state as well as in fixed preparations. For the demonstration of various chemical and morphologic features that are lost or altered by processes of fixation and imbedding, and when a rapid diagnosis is required, the examination of the material in its fresh state or by the freezing method is indicated. When the freezing method cannot be employed because of the changes in cells and tissue produced by it, when very thin or serial sections are desired, when a rapid diagnosis is not required, and when a very careful and minute study is desired, with the application of various staining methods, then the material should be fixed, hardened and imbedded and cut upon a microtome. Whatever method is chosen, it must be borne in mind, particularly in practical diagnostic work, that the portions chosen for microscopic examination must represent the characteristic anatomic structures of the tissue or organ, that living tissue be included, that the pathologic condition be represented both in its fully-developed state and at the transition-border between it and the healthy tissue, and that when sections are cut the block or tissue must be so oriented as to give the most comprehensive view of the tissue and the pathologic process. To accomplish this fully it is often necessary to make a number of blocks representing different areas of the material, and to cut these in different planes.
CHAPTER XVII.
THE LABORATORY OUTFIT.
For practical diagnostic work or for pathologic research various instruments and utensils are necessary, although the expense of fitting up a working pathologic laboratory is not as great as it is often thought to be. The most expensive item, as well as the most important, is the microscope. This should be of the best make, and should be carefully selected and tested before the final purchase. As a rule the German makes, Zeiss, Leitz, and others, are to be preferred to the American instruments, in spite of the higher cost due to the duty imposed. I have found the German microscopes uniformly good and standing the wear and tear of a teaching laboratory much better than the American-made stands. I have never seen a poor Zeiss or Leitz objective, but cannot say the same thing of other makes. On the other hand, I have seen some American objectives that were as good as any German ones, but there are not many such. If one is going to buy an American microscope it should be bought on the same principle that one would buy a violin or a piano, wholly on its individual merits; and these can be ascertained only by having the instrument carefully examined and tested by an expert. Most laboratory workers will agree that the Zeiss instruments are the best; they are also the most expensive. For all practical purposes a Leitz stand costing ninety to one hundred dollars is quite good enough. A medium-sized continental stand, with rack and pinion and micrometer screw for coarse and fine adjustment, a triple nose-piece with dust-protector, Abbé condenser, iris diaphragm, plane and concave mirrors, three objectives (low, high and 1/12 oil-immersion), and two eye-pieces, a low and a high, form a complete outfit that answers all practical requirements. The new type of stand with curved arm and large stage, permitting the examination of all parts of a Petri dish or glass plate, and with the mechanism of the fine adjustment protected from any strain when the instrument is lifted by the arm is especially recommended. I have also found the black-finish very practical. The entire outfit need not be purchased at once; the stand with its accessories and a low power may first be purchased, and the higher-power objectives obtained later. One of the first luxuries is a movable adjustable stage. A very good and relatively cheap one is made by the Spencer Lens Co., of Buffalo. If a Zeiss stand is purchased the objectives A, D, and 1/12 oil-immersion, and oculars 2 and 4 best meet the requirements. Of the Zeiss apochromatic series, the objectives 16.0, 8.0, 4.0, and oil-immersion 2.0 mm., apert. 1.30, and oculars 4, 6 and 8 are most serviceable. The apochromatic objectives and the compensation-oculars are expensive, and need not be used for ordinary work, but are indispensable for photographic work. The Leitz objectives, 3, 6, or 7, and 1/12 oil-immersion, with oculars 2 and 4, and the equivalent objectives of the Spencer Lens Co. or Bausch and Lomb will answer all ordinary needs. For the purpose of microscopic measurements an ocular micrometer is necessary. This may be obtained as a separate eye-piece, or as a round piece of glass with measured divisions marked upon it that may be put into an ocular. The value of the scale must be determined for every lens and tube-length by estimating the number of its parts covering one part of a stage-micrometer marked in hundredths of a millimetre.
Fig. 50.—A satisfactory outfit for the working laboratory. Continental stand, medium-sized, with large stage, three objectives, etc.
An instrument of any one of the above-mentioned makes, carefully selected and tested, should last its owner a life-time if proper care is taken of it. It should receive the same careful attention accorded a good violin or piano. It should be protected from dust, action of chemicals, heat, sunlight, and rough usage. When carried it should be supported in such a way that its weight is not thrown upon the thread of the adjusting screws. The adjustment, draw-tube and iris diaphragm should be carefully oiled at intervals, using the least possible amount of the best microtome oil. It is not necessary here to enter into the construction and theory of the microscope, as this knowledge has usually been obtained before the pathologic laboratory is reached. Experience has shown me, however, that it is always necessary to remind students, even those experienced for some time in the use of the microscope, of certain fundamental principles in the adaptation of microscopic technique to pathologic work. The following rules are of value:—
1. Use a low-power objective for all work except for the study of bacteria, microparasites and finer cell-structures. The aim should be to obtain as much of a bird’s-eye view of the “geography” of the section as possible. Contrast plays a very important part in pathologic diagnosis; and it is lost in high-power work, so far as the relations of cells and tissue-elements and pathologic products are concerned. The student almost invariably enters the pathologic laboratory with a fixed “high-power habit,” and he is usually greatly surprised to learn how much he misses with the high-power and how much he can see with the low-power. A motto used many years in my laboratory, “Low-power objective and high-power cortex,” is of greater educational value than may appear at first sight. A slide should be examined first with the naked-eye, as it is held against a window or light; then it should be examined under the low power. Rarely will it be necessary to use a high-power except for the purposes mentioned above.