The examination of the natural (unstained) blood is of greatest importance to the clinician, though the making and examination of dry preparations are also very valuable, especially when the microscope is not at hand at the bedside.
The examination of unstained preparations is made as follows: A very thin cover glass and a slide are cleansed with water and alcohol and thoroughly dried. The tip of the finger or lobe of the ear is washed with soap and water, dried, and pricked with a needle or lancet. The first drop is wiped away with a linen cloth, and a second very small drop allowed to issue forth. The summit of this drop is touched with the middle of the cover glass held in forceps, and the cover glass is at once laid on the slide, where the blood drop quickly spreads out between the two. It is not advisable to assist the spreading by pressure, since the mechanical injury may bring about changes in the shape of the corpuscles. The best results are obtained with the thinnest cover glasses; in fact, with thick ones labor is in vain. If, as is usual, several preparations are made one after another, the drop of blood remaining must be wiped away each time and a fresh one pressed out for every new preparation. The proper size of the drop is learned by experience, though it may be said that tyros in blood examinations always employ too large drops. The preparation is successful only when the red blood corpuscles are isolated one from the other and present to the observer their flat surface unaltered. Rouleau formation, thornapple appearance, etc., make this examination more difficult or even impossible.
For the microscopic examination a good immersion lens is useful; in fact, indispensable if the details of the hematozoa are to be studied, though it is possible, with a good dry system (400 to 500 diameters), to see the different forms, especially if the observer has a certain amount of experience. It is well known that Laveran made his discovery with a low power and found even the smallest forms.
The complete study of the preparation may require considerable time. In severe cases it is not rare to see a number of parasites in every field when the examination is at once successful, but in mild cases, and occasionally in pretty severe ones, an entire preparation may be gone over before a parasite is encountered. For these cases a movable stage is very convenient. The time in the course of the fever at which the examination is undertaken plays an important role, since the parasites are usually found most numerous in the peripheral blood from several hours before the paroxysm to its acme, though they may be absent. On the other hand, when the temperature has again fallen is an equally good time to make the examination.
If there is no microscope at hand to examine immediately the natural blood, or if there is any other reason for making the examination later, dry preparations are made and studied after staining.
These dry preparations are made in two ways-either by placing on the cover glass containing the drop of blood a second cover glass, allowing the blood to spread out between them, and sliding them apart, or by drawing the edge of a cover glass through the drop on the finger tip and drawing this, inclined at an angle, over a second and a third. Equally good preparations are obtained by spreading the blood on a slide with the shaft of a straight needle. The drops must naturally be small, for the layer of blood should be so thin that the individual corpuscles are isolated from one another and present their flat surface to the observer. The cover glasses or slides are dried as rapidly as possible by waving to and fro. After they are thoroughly dried, the preparations are fixed in equal parts of alcohol and ether or simply absolute alcohol for five minutes. Their further manipulation is dependent on the stain to be used.
For clinical purposes the best stain is the Romanow sky-Ziemann stain or any of its modifications. [It is made in the following way:
Medicinal methylene blue.................... 1.0 part
Sodium carbonate........................... 0.5 "
Keep in an incubator or in the sun until the solution, originally blue, becomes a deep purple. This change takes place in a few days. Solution B:
Eosin A G brand or B A brand or pure eosin for blood work............................... 1 part
For staining, dilute each of these solutions 20 times with water. Mix equal parts of each solution and pour on the slide. Stain for five to twenty minutes. The nuclei of the leukocytes, the platelets, and the nuclei of the parasites are stained a deep "red." This stain is readily made, never fails, and makes the detection of parasites an easy matter. It, moreover, gives characteristic staining effects, "stippling" of the red cell in the case of the simple tertian parasites, and also, though under what exact conditions is not certainly known, in the case of the malignant tertian parasite, though the stippling is in this case of a different character. A variety of other stains, but none so satisfactory as this, may be used, such as Loffler's alkaline methylene blue and methylene blue and eosin.-ED.]
In using Loffler's methylene blue (30 c.c. of a concentrated alcoholic methylene blue solution + 100 c.c. of a 1 : 10,000 caustic potash solution) the preparation is, after fixation in alcohol and ether, dried between filter papers, placed for about a minute in the staining solution, washed in water, dried and mounted in xylol Canada balsam. The red blood corpuscles remain unstained. The parasites and the nuclei of the leukocytes (and the nuclei of erythroblasts, if there are any) are stained blue.