Fixation 



SMEAR PREPARATIONS FROM FLUID MATERIAL 



71 



for spreading has a great deal against it. 

 There is a risk tliat the edge of the upper 

 slide will scratch the lower, and though 

 these scratches are not apparent in smears 

 which are to be examined under an oil im- 

 mersion objective, they are objectionable 

 in a dry slide. It is also difficult to secure a 

 sUde which is entirely flat and which will 

 thus make a layer of uniform tliickness, 

 for the tliickness of the laj^er obviously 

 depends on the degree of contact between 

 the upper sHde and the lower. The writer 

 has recently been using a thin sheet of 

 transparent plastic (methyl methacrylate) 

 in place of glass and has obtained very 

 much better results. To prepare such a 

 sheet for use one cuts, or saws, a 3" X 1" 

 rectangle from it and then polishes one of 

 the edges by rubbing it briskly backward 

 and forward on a sharpening stone or on a 

 ^ piece of fine sandpaper. This turns up a 

 feather edge on both sides of the edge 

 which has been flattened. This is removed 

 by taking the shp and holding it at just 

 about the same angle at which it will be 

 used for preparing the smear and giving it 

 one or two quick strokes on the finest 

 sandpaper. The use of a soft material like 

 this not only insures that there will be no 

 scratches on the slide, but also guarantees 

 that the edge used for smearing will always 

 remain in contact with the slide. After two 

 or three hundred smears have been made 

 the piece of plastic may be thrown away 

 and a new one taken. 



The method described is the standard 

 procedure for producing thin smears. 

 These are necessary for those fluids, such 

 as vertebrate blood or mammalian seminal 

 fluid, which contain very large numbers of 

 objects which must be separated as widely 

 as possible if they are satisfactorily to be 

 studied. 



There are a number of fluids, however, 

 from which thick smears must be made 

 either because, as in the case of inverte- 

 brate blood, they contain relatively few 

 cells or because, as in the case of malarial 

 diagnostic smears, one is seeking for a 

 parasite which is relatively sparsely dis- 

 tributed through the material. These thick 

 smears are made with the aid of a loop of 

 wire held in a needle-holder of the type 

 found in bacteriological laboratories. This 



loop is dipped into the fluid to be ex- 

 amined, and used to spread it with a 

 rotary motion in tlie center of the shde. 

 This is very similar to the preparation of 

 smears of bacterial material which is de- 

 scribed in some detail in Chapter 21. 



Fixing Smears 



Smears may be fixed by drying, by 

 alcohol, or in one of the conventional fixa- 

 tives. When a smear is to be fixed by dry- 

 ing it is, as soon as it has been made, 

 waved in the air and then set on one side 

 for subsequent treatment. This procedure 

 is excellent in the case of objects such as 

 bacteria or erythrocytes, which do not 

 change their shape after drying, or for 

 materials such as white blood corpuscles, 

 which it is not desired to preserve in their 

 normal shape. No other object can, how- 

 ever, be considered satisfactory unless it 

 has been fixed, and the simplest method 

 of doing this is to pass the smear, just as it 

 is drjdng, through a jet of steam. This 

 technique has been described in Chapter 6 

 for mounting amebas and need not be re- 

 peated here. 



All other smears should be fixed before 

 they are dried and it is something of a 

 problem to fix them without removing the 

 material from the shde. It is obvious that 

 if the material is freshly smeared onto a 

 glass shde and then dropped into a fixative 

 of some kind, it will be washed off. The 

 logical solution to the problem is to use a 

 fixative in a vapor phase, and nothing is 

 better for this purpose than osmic acid. 

 To use this material, a couple of glass rods 

 are placed in a petri dish sufficiently far 

 apart to permit the sUde to rest on them 

 without the smear touching them. A drop 

 or two of a solution of osmic acid, usually 

 of 2 % strength is put on the bottom of the 

 petri dish and the cover replaced. It must 

 be emphasized that osmic acid fixes the 

 mucous membrane of the nose and throat 

 just as readily as it does a smear and every 

 precaution must be taken to avoid inhal- 

 ing the vapors. As soon as the smear is 

 made, and before it has time to dry, it is 

 placed face down across the two glass rods 

 so that it is exposed to the vapor but not 

 to the liquid. The cover is then replaced on 

 the petri dish, and the slide left in place for 



