Wash the finger well and allow a few minutes to elapse for the circulation to become normal. Prick the finger lightly with a blood-lancet, regulating the depth of the puncture so that the blood will not flow without gentle pressure. Quickly dip a clean glass rod into a vessel containing diluting and fixing fluid, and place two or three good-sized drops upon the finger over the puncture. Then exert gentle pressure above the puncture so that a small drop of blood will exude into the fluid. Mix the two by passing the rod lightly several times over the surface of the blended drop. (Some workers first place a drop of the fluid upon the finger and then make the puncture through it, this necessitating less care as to depth of the puncture.) Now transfer a drop of the diluted blood from the finger to a watch-glass which contains two or three drops of the fluid, and mix well. From this, transfer a drop to the counting slide of the Thoma-Zeiss hemocytometer, and cover. An ordinary thin cover will answer for this purpose, and is preferable because it allows the use of a higher power objective. Allow the slide to stand for at least five minutes, and then with a one-sixth or higher objective count the plaques and the red corpuscles in a definite number of squares. At least 100 plaques must be counted. The number of red corpuscles per cubic millimeter of blood having been previously ascertained in the usual manner ([p. 152]), the number of plaques can easily be calculated by the following equation:
r:p :: R:P; and P = p x R/r.
r represents the number of red corpuscles in any given number of squares; p, the number of plaques in the same squares; R, the total number of red corpuscles per c.mm. of blood; and P, the number of plaques per c.mm.
Beginners are apt to take too much blood and not to dilute it enough. Best results are attained when there are only one or two plaques in a small square. With insufficient dilution, the platelets are more or less obscured by the red cells.
The following diluting and fixing fluid is recommended:
| Formalin | 10 c.c. |
| 1 per cent. aqueous solution sodium chlorid (Color with methyl-violet if desired.) | 150 c.c. |
This fluid is cheap and easily prepared, and keeps indefinitely. It fixes the plaques quickly without clumping, and does not clump nor decolorize the reds. It has a low specific gravity, and hence allows the plaques to settle upon the ruled area along with the reds. Fluids of high specific gravity cause the plaques to float so that they do not appear in the same plane with the reds and the ruled lines.
VI. STUDY OF STAINED BLOOD
A. MAKING AND STAINING BLOOD-FILMS
1. Spreading the Film.—Thin, even films are essential to accurate and pleasant work. They more than compensate for the time spent in learning to make them. There are certain requisites for success with any method: (a) The slides and covers must be perfectly clean; thorough washing with soap and water and rubbing with alcohol will usually suffice; (b) the drop of blood must not be too large; (c) the work must be done quickly, before coagulation begins.