Smears on Slides.—Of the various methods of spreading films on slides, that described by Daniels is quite satisfactory. In this the drop of blood is drawn along and not pushed along. The films are even, can be made of any desired thickness by changing the angle of the drawing slide, and there is little liability of crushing pathological cells. Take a small drop of blood on the end of a clean slide. Touch a second slide, about ½ inch from end, with the drop and as soon as the blood runs out along the line of the slide end, slide it at an angle of 45° to the other end of the horizontal slide. The blood is pulled or drawn behind the advancing edge of the advancing slide. An angle less than 45° makes a thinner film; one greater, a thicker film.
Instead of a slide a square cover-glass may be used and if the edge be smooth it makes a more satisfactory spreader than the slide.
Instead of the Daniels method I prefer to take up the drop of blood on the slide on which the smear is to be made, about ½ inch from the end. Then apply the spreader slide and so soon as the drop runs along the end of the spreader slide proceed as above described. This method is shown in Fig. 150.
Spreaders.—Of the various methods of making smears by means of cigarette paper, rubber tissue, needles, etc., the best seems to be to take a piece of capillary glass tubing and use this instead of a needle in making the film. There is one advantage about the strip of cigarette paper touched to the drop of blood and drawn out along the slide or cover-glass, and that is that it is almost impossible not to make a working preparation by this method.
Thick-film Methods
Such methods are of the greatest practical value in searching for malarial parasites when they are in very small numbers in the peripheral circulation, in finding trypanosomes, relapsing fever spirochaetes and filarial embryos. Ruge’s method so brings out the polymorphonuclears that such a technic can be used for opsonic index. Many workers prefer the Ross thick-film method in examining for malaria. In this about one-half of a drop of blood is smeared out over a surface about equal to that of a square cover-glass and allowed to dry. It is then flooded with 1/10 of 1% aqueous solution of eosin for about fifteen minutes. The preparation is then gently washed with water and then treated with a polychrome methylene-blue solution. After a few seconds this is carefully washed off and the preparation dried and examined.
James smears out an ordinary drop of blood so that it makes a circular smear about ¾ inch in diameter. This may be easily accomplished with a spatulate toothpick. When dry, treat the blood smear with alcohol containing HCl (Alcohol 50 cc., HCl 10 drops) until the haemoglobin is dissolved out. Then wash thoroughly in water for five or ten minutes. Allow to dry and then stain as ordinarily with the Wright or Giemsa stain.
Ruge’s Method.—The best thick-film method is that of Ruge. After the blood has dried well gently move the slide about in a glass containing a 2% solution of formalin to which has been added 1% of glacial acetic acid. After laking is completed, as shown by disappearance of brown color, treat the slide in the same way in a glass of tap water to remove all traces of acid. Next wash gently in distilled water and stain with dilute Giemsa (1 drop to 1 cc. of water) for twenty to thirty minutes. Wash in water and allow to dry without heat or blotting paper. Some workers prefer to stain the dried thick smear for one hour in a jar containing dilute Giemsa stain (1 to 40) without previous fixation or dehaemoglobinization. At present, I make my thick films by taking up a large loopful from the exuding drop of the puncture wound.
This is deposited at one end of the slide and from it three or four more daubs are made in succession toward the other end of the slide. These daubs are quickly smeared out before coagulation takes place in the first daub.