MANUAL PHYSIOLOGY PRACTICAL - Repository:The Medical ...

FM/UOJ
Experiment 8
IDENTIFICATION OF LEUCOCYTES AND DIFFERENTIAL COUNT
The white cells are a group of different types of cells. It is very important to be
able to identify the cells on a stained slide for counting the number of each type of cells.
(It is presumed that the total white cell count is already done). The proportion of each
type of cell in a stained blood smear is determined; the number of each type of cell is
determined from the two values.
Instruments:
Slides, staining tray, Stop watch, and microscope with oil-immersion objective.
Reagents:
Leishman’s stain: This is a mixture of eosin, methylene blue and some derivatives
of methylene blue. The powder, containing all those in correct proportion, is dissolved in
methyl alcohol ( 150 mg per 100ml ). The stains are neutral salts of weak acids i.e. they
are buffer salts. These salts can bind and give color to different substances only when
they ionise and become active ions. This does not happen in alcohol solution. Eosin is
said to be acidic stain because the staining is due to the anion: methylene blue is basic
because the staining is due to cation.
Buffered water, pH 6.9: The buffered water is necessary to provide the medium
for ionization of the dyes for staining.
Method:
Preparation of blood smear
Blood smear is made by first taking a drop of blood on a slide and spreading to
form a thin film of blood. Take a drop of blood, from a finger prick. Near one end of a
clean and dry glass slide. Take another slide which has sharp edges (this slide will be
referred to as spreader) and place it on the middle of the first slide at an angle of 45.
Draw the spreader, keeping an angle 45 to touch the drop of blood. The blood spreads
along the edge of the spreader. Draw the spreader quickly towards the other end of the
slide. (A good smear will be fairly uniform, neither too thick nor thin, and will have no
uneven streaks or spots on it). Keep the slide until it dries completely.
Staining
Place the slide across two of the parallel supports on the staining tray so that the
slide is horizontal. Add stain by a dropper just to cover the film (about 10-12 drops) and
Blood
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