Preparing Fresh Tissues for the Microscope

Abstract

While engaged in general pathologic work I shared the common distrust of frozen sections of fresh tissues for microscopic diagnosis. On taking charge recently of the laboratories of the Drs. Mayo, surgeons, I carefully tested the various methods hitherto published and found them either too slow for results while the patient waits under the anesthetic or else giving poorly differentiated cell detail. After considerable experimentation the following technic was discovered, and for the last six months it has given uniformly excellent preparations:1. Bits of fresh tissue not more than 2 × 10 × 10 mm. are frozen in dextrin solution and cut in sections of from 10 to 15 microns thick.2. The sections are removed from the knife with the tip of the finger and allowed to thaw thereon.3. The sections are unrolled with camel’s-hair brushes in 1 per cent. NaCl solution.4. The sections are stained from 10 to 20 seconds in neutral Unna's polychrome methylene blue.5. They are washed out in 1 per cent. NaCl solution.6. They are mounted in Brun's glucose medium.The microtome which I use is the Spencer automatic with a CO 2 attachment in which vulcanite is substituted for brass in the wall of the freezing chamber, thus insulating the freezing plate. Thawing the section on the finger prevents to a great extent the formation of bubbles. The well-made camel’s-hair brushes used by artists are much more useful for handling tissues than those usually furnished by laboratory supply houses. A heavy, shallow watch glass over a black surface is the best receptacle in which to unroll sections. Sections are best handled in the stain folded over a lifter made of a small glass rod drawn out and bent at convenient angle. The section is kept constantly moving while it is in the stain. The stain is contained in a minute cup to facilitate the rapid recovery of the section should it slip from the lifter. Washing out is done in several ounces of salt solution in a white porcelain dish and is continued only while the stain comes away freely. Brun's glucose medium (which is made by mixing distilled water 140 c.c., glucose 40 c.c., and glycerin 10 c.c., then adding camphorated spirit 10 c.c. and filtering), is held in an oval dish of porcelain (an “undecorated match safe”) of such a size that a three-inch slide will rest in a slanting position, with one end in the bottom of the dish and the other on its edge. The section is spread out on the slide while it is in this position. The slide is then carefully withdrawn from the dish, the excess fluid removed, a coverslip dropped over the section and the specimen is ready for the microscope.The whole process can be gone through in one and a half minutes from the time the tissue is placed on the freezing plate of the microtome until the stained specimen is on the stage of the microscope. The resulting coloring is uniformly good with the tissue elements sharply contrasted in red, purple and dark blue.A diagnosis may be made from such preparations in a large percentage of surgical cases in which a diagnosis is possible by a study of sections of the same thickness cut from fixed tissues and stained with hematoxylin and eosin.See PDF for full text of the original JAMA article.