Object:
To determine the relative proportions (differential count) of white blood corpuscles in the human blood.
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Requirements:
Sterilized needle, clean slides; Leishman’s stain or Wrights stain and microscope.
Procedure:
Prick the finger with a sterilized needle; wipe away the first drop of blood. Take the second drop on the end of a clean slide; place the slide on a smooth surface holding it steady with the left hand.
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Hold a second slide or drawing slide at 45° just in front of the drop of the blood, draw this slide in such a way that the blood spreads along its edge.
Push the second or drawing slide forward 45° on the first slide without exciting any pressure, a film of blood will be made on the first slide. Dry the blood film in air, but do not heat it.
Stain the blood film with Leishman’s stain or Wright’s stain. Wait and after one minute carefully adds distilled water to the stain, the water should be twice the volume of the stain used.
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Mix the water and stain well by sucking the mixture in and out of a pipette. After a time a greenish metallic scum forms on the surface of the mixture.
Allow the diluted stain to aid from 7 to 10 minutes. Drain of the stain and wash film for 10 seconds with distilled water.
The film should be rose pink in colour, if it is too purple, then wash again with distilled water. Dry the slide and examine it by the microscope for white blood corpuscles.
First examine the stained film under low power of microscope to get an idea of leucocytes.
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Then examine under high power or oil immersion lens after thinly smearing the stained film with oil.
In a longitudinal strip of the film count the various kinds of white blood corpuscles from top to the bottom.
Count 100 or 200 white corpuscles noting on a paper each type of white blood corpuscles encountered and calculate the percentage.
Calculation:
The following formula can be adopted to calculate the percentage of different types of white blood corpuscles:
Number of type cells / Total number of white blood corpouscles * 100
Total number of white blood corpuscles The normal proportion of white blood corpuscles is as follows:
Neutrophils. 50—70 per cent or 3000—6000 per cu mm
Eosinophils. 14„ 150—300„
Basophils….. 05—1 „ 0—100
Lymphocytes. 20—40„ 1500—2700„
Monocytes….. 2—8, 300- 600„
Precautions:
It is absolutely essential for differential count of white blood corpuscles that the film of blood is well spread with no clogging of corpuscles.