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Microscope Blood Work and Red Blood Cell Counting. Equipment such as haemacytometes for counting.
Microscope Blood Work
Haemacytometers The haemacytometer is an apparatus for counting the blood corpuscles, and consists of a counting chamber, two mixing pipettes, and suitable optically plane cover glasses. The blood is first diluted with a solution known as " Toisson's" solution, for the red or white, or with a solution of acetic acid when a count of white cells only is being made. In counting red corpuscles a dilution of 1-200 is generally used, but in certain cases 1-100 may be employed. The blood is drawn into the pipette up to the mark 0*5 in the case of 1-200 dilutions, and up to the mark 1 for 1-100 dilutions. The pipette is then immediately placed in the diluting fluid, which is drawn up to the mark 101 above the bulb. Both ends of the pipette are then closed with the fingers, and the pipette shaken to ensure an even mixing, the glass bead in the bulb facilitating this. For white corpuscles, a dilution of 1-10 is employed and the other pipette is used. For filling the counting chamber, a drop of the mixture is blown out of the pipette, after allowing several drops to go waste, into the centre of the counting chamber. The cover glass is then placed over the cell. The drop of blood must not be allowed to overflow the platform into the groove which surrounds it, and the cover glass must be in perfect contact with the object slide, and all must be scrupulously clean. The counting chamber consists of a plate of glass with an annular groove ground upon it. The circular portion inside the groove is ground and polished to a distance of 1 mm. Below the level of the plate of glass.
In the Thoma haemacytometer this portion is ruled with a diamond into squares l/400th of a square mm. Each in area. It will therefore be seen that the amount of liquid resting upon each square has a cubic capacity of l/4,000th of a cubic mm. The liquid which has been placed in the counting chamber is allowed to settle, and the corpuscles will therefore be in contact with the bottom of the cell. It will be found that it is a simple matter to count the corpuscles contained in each square. The usual method is to count, say, 100 squares, and it must be noted that in dealing with those actually on the lines, only those on two sides of the square should be counted, and this rule should be applied throughout. The number of corpuscles in 1 cubic mm. Of undiluted blood is then obtained by multiplying together the rate of dilution, the number of corpuscles counted, the volume of each square (l/4,000th of a cubic mm.), and dividing by the number of squares counted. The above is the general method of counting the red corpuscles ; but in the case of the white corpuscles, as there are a very much smaller number of these, the method generally employed is to count the total number of the whole ruled area of the counting chamber, which is 1 sq. mm. Cell Counting Chambers There are other forms of counting chambers, such as the Biirker, Fiichs-Rosenthal, Breuer, and Zapperts ; the method of employment in all these is the same, but the ruling and also the counting are different in each case. The use of a mechanical stage greatly assists the counting. A simpler form of haemacytometer can be used which depends for its action on Mr. Rheinberg's beautiful process of making graticules and glass scales. A glass plate is photographed with squares in the pattern of a chess-board, so that alternate squares are tinted, although they are transparent. This plate is dropped into an eyepiece between the lenses, and by means of a stage micrometer the drawtube can be varied until a definite number of squares are equal to *1 of a millimetre in the micrometer. The chess-board glass plates are supplied with squares either 1/4, 1/2, 1, or 2 mm. In size. They are made to cover the whole field of view, or as a small block of squares in the centre of the field. The latter are to be preferred for blood counts. The only other requirement is a 3 X 1-inch slip with a metal ring cemented to it which is 1 mm. Thick, into which the blood is placed covered with an ordinary cover glass. Suppose a 1/6-inch object glass is being used, a 1-mm. Chess-board plate dropped into the eyepiece can be made by drawing out the drawtube to the required position according to the eyepiece and object glass employed, of such an apparent size that nine squares, three each way, correspond to '1 mm., and the count of nine squares will give the number in a cubic tenth of a millimetre. If a 1/2-mm. Chess-board plate be used, then thirty-six squares, six each way, correspond to a cubic millimetre. The most convenient size to select will depend upon the class of object to be counted and the object glass that is used. Due to the alternate squares being tinted, a count can be made with much less eye-strain than with the ordinary haemacytometer, and this method is preferred by some apart from the question of the cost of the apparatus.
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Microscope Technique 
