KEVIEW — TKOPICAL MEDICINE, ETC. 



89 



They detail the techniquo both for smears and sections. That for smears may bo 

 quoted here as likely to prove useful : — 



1. Glass slides and cover-glasses are washed thoroughly with soap and water, then heated in the flame to get 

 rid of oily substances. 



2. A .small bit of the gray substance of Ijrain chosen for examination is cut out with a small, sharp pair of 

 .scissors and placed about one inch from the end of the slide, so as to leave enough room for a label. The cut in 

 the brain should be made at right angles to its surface and a thin slice taken, avoiding the white matter as much 

 as possible. 



3. A cover-slip placed over the piece of tissue is pressed upon it until it is spread out in a moderately thiu 

 layer, then the cover-slip is moved slowly and evenly over the slide to the end opposite the label. Only slight 

 pressure should be used in making the smear, but slightly more should be exerted on the cover-glass toward the 

 label side of the slide, thus allowing more of the nerve tissue to be carried farther down the smear and producing 

 more well-spread nerve cells. If any thick places arc left at the edge of the smear, one or two of them may be 

 spread out toward the side of the slide with the edge of the cover-glass. If the first smear does not seem to be 

 well spread out others should be made until a satisfactory one is obtained. 



4. For diagnosis work such a smear should be made from at least three different parts of gray matter of the 

 central nervous system : first, from the cortex in the region of the fissure of Rolando or in the region corresponding 

 to it (in the dog, the convolution arouud the crucial sulcus) ; second, from Amnion's horn ; third, from the 

 cerebellum. In many of the animals reported here smears were made from the gray matter of the cerebral cortex, 

 around the fissures of Rolando and Sylvius, from the olfactory bulb, Ammon's horn, cerebellum, medulla in the 

 region of the roots of the cranial nerves, spinal cord in the dorsal and lumbar regions, spinal and Gasseriau 

 ganglia, salivary glands, suprarenals, and some of the peripheral nerves. From the last four-named structures the 

 smears were not very successful, so only a few vrere made. 



5. The smears were dried in air, and subjected to one of the two following staining methods : — 



(a) Qiemsa's Solution. The smears are fixed in methyl alcohol (commercial is just as good as pure) for 

 about 5 minutes. The staining solution recommended last by Giemsa (1 drop of the .stain to every c.c. of distilled 

 water made alkaline by the previous addition of one drop of a one per cent, solution of potassium carbonate to 

 10 c.c. of the water) is poured over the slide and allowed to stand for one-half to three hours. The longer time 

 brings out the structure better, and in 24 hours well-made smears are not overstained. After the stain is poured 

 off, the smear is washed in running tap-water for one to three minutes, and dried with filter paper. If the smear 

 is thick, the " bodies " may come out a little more clearly by dipping in 50 per cent, methyl alcohol before washing 

 in water, then the washing need not be as thorough. By this method of staining, the cytoplasm of the " bodies " 

 stains blue and the central bodies and chromatoid granules stain a blue-red or azur. Generally the larger 

 " bodies " are a darker blue than the smaller, the smallest of all may be very light. The stain varies somewhat 

 according to the thickness of the smear. Some have a robin's egg blue tint, but this is after a longer fixation in 

 the methyl alcohol. In this case the red blood cells may have a greenish tint. The cytoplasm of the nerve cells 

 .stains blue also, but with a successfully made smear the cytoplasm is so spread out that the outline and structure 

 of most of the " bodies " are seen distinctly within it. The nuclei of the nerve cells are stained red with the azur, 

 the imcleoli a dull blue, the red blood cells a pink-yellow, more pink if the decolorisation be used. The " bodies " 

 have an appearance of depth, due to their slightly refractive qualities. 



For diagnostic purposes this method of staining may be shortened as follows : Methyl alcohol, five minutes, 

 equal parts of the Giemsa solution and distilled water, 10 minutes. In this way " bodies " are generally brought 

 out well enough for di.aguosis, and sometimes the structure shows distinctly. It is always well, however, to make 

 smears enough for the longer method of staining, in case the shorter one should prove unsatisfactory. 



(b) The eosin-methylene blue method recommended by Mallory. The smears are fixed in Zenker's solution 

 for one-half hour, after being rinsed in tap-water they are placed successively in 95 per cent, alcohol iodine one- 

 quarter hour, 95 per cent, alcohol one-half hour, absolute alcohol one-half hour, cosiu solution 20 minutes, rinsed in 

 tap-water, methylene-blue solution 15 minutes, and dried with filter paper. With this method of staining the 

 cytoplasm of the " bodies " is a magenta, light in the small bodies, darker in the larger; the central bodies and 

 chromatoid granules are a very dark blue, the nerve cell cytoplasm, a light blue, the nucleus a darker blue, and 

 the red blood cells a brilliant eosin pink. With more decolorisation in the alcohol the " bodies" are not such a 

 deep magenta and the difference in colour between them and the red blood cells is not so marked. 



The " bodies " and the structure are often more clearly defined with this method, and perhaps, on the whole, 

 it is better to use it for making diagnosis ; but when there are only tiny "bodies" present, or when the brain 

 tissue is old and soft, the Giemsa stain seems to be the more successful ; above all, when one wishes to study the 

 nature of the central structures and granules the Giemsa stain must be used. We therefore recommend strongly 

 the use of both methods. Even if both are used, and one has to wait for the longer method, the technique is far 

 simpler than any so far published. 



Van Dieson, working in our laboratory, suggests a staining method which differentiates the " Negri bodies " 

 more quickly than either of the two methods described above. So far, the best proportion of the stains used have 

 not been determined, but satisfactory results have been obtained from the following mixture : To 10 drops of 

 distilled water three drops of a sat. ale. sol. of rose-anilin-violet and six drolls of LcefiSer's solution of methylene 

 blue are added. The smears are fixed, while moist, in methyl alcohol for one minute. The stain is then poured 

 on, warmed until it steams, poured off, and the smear is rinsed in water and allowed to dry. 



The cytoplasm of the " bodies " is a deep and distinctive red, their inner structures are a dark blue, the nerve 

 cells are a light blue, and the blood cells a pale salmon-red. 



The staining mixture remains good for about an hour. 



Their summary and conclusions are as follows : — 



1. The smear method of examining the " Negri bodies " is superior to any other method so far published, for 

 the following reasons : (n) It is simpler, shorter and less expensive ; (b) the " Negri bodies " appear much more 

 distinct and characteristic. For this reason, and the preceding one, its value in diagnostic work is great ; (c) the 

 minute structure of the " Negri bodies " can be demonstrated more clearly ; (li) characteristic staining reactions 

 are brought out. 



Hydrophobia 



— continued 



