PERIPHERAL BLOOD SMEAR (DEMONSTRATION)
PERIPHERAL BLOOD SMEAR (DEMONSTRATION)
To prepare, stain and examine an ideal blood smear.
APPARATUS: Clean, dry grease free glass slides with smooth edges, a drop bottle containing Leishman’s stain, distilled water, cedar wood oil, microscope, rectified spirit, lancet, cotton, empty wash bottle.
Leishman’s stain: It is a type of Romanowsky stain which contains an acidic dye and a basic dye. The acidic dye stains the basic components of the cell and the basic dye stains the acidic components of he cell. The different cells are differently stained. Leishman’s stain contains eosin and methylene blue in acetone free methyl alcohol. Methyl alcohol acts as a fixative. (acetone if present, will destroy the cell membrane). Methylene blue, the basic dye and eosin, the acidic dye exists as thiazine eosinate which dissociates into the component dyes, when diluted with distilled water 0.15gm Leishman’s stain powder is dissolved in 100 ml acetone free methyl alcohol, filtered and kept for 2-3 weeks for riping. Methyl blue stains the nucleus and the basophilic granules of the WBC, where as eosin stains the eosinophilic granules.
Other Romanosky stains include Wright’s stain, Giemsa’s stain and Jenner’s stain.
Preparation of peripheral smear:
Clean the slides using soap and water and dry using a fresh handkerchief. Choose a slide with smooth edges as the spreader slide. Obtain a moderately sized drop of blood by finger puncture method and place it about 1 cm from one end of the slide. Keep the slide horizontally on the table. Hold the spreader slide between the thumb and index finger, place the narrow edge of the spreader slide infront of the blood drop at an angle of 45°. Bring it slowly backwards till it touches the blood drop. Blood spreads along the edge for the spreader by capillary action. when the blood spreads to about 2/3 of the width of the slide, the spreader is pushed forwards with uniform speed and pressure. Dry the smear in the air by keeping the head end of the smear down.
Staining the smear:
The dried smear is placed horizontally on a staining rack. Pour the stain drop by drop till it covers the smear. Keep it for 1-2 minutes for fixing the smear. Then dilute the stain using distilled water, double the amount of stain. Mix gently by blowing air. A golden scum is seen to form over the diluted stain. Keep it for 7-10 minutes, when the smear gets stained, wash the smear in running tap water till the smear turns pink and the water flowing out is colourless. Take care to see that the tap water does not fall directly on the smear. Wipe the back of the slide and keep it in a vertical poisiton to drain and dry. The film is first examined under the low power. Focus a well stained area where the cells are uniformly distributed without rouleaux formation.Put two drops of cedar wood oil on the smear and examine under oil immersion objective (with plane mirror, condenser raised and iris diaphragm fully opened).lt is ideal to prepare and stain 2 or 3 peripheral blood smears simultaneously.
Criteria for a good smear:
1. The smear should not be serrated or wavy and should not contain any holes.
2. It should neither be too thick nor too thin.
3. The smear should occupy only the middle two thirds of the slide.
4.An ideally taken smearwill be tongue shaped with a head, body and tail.
5. The stained smear should be pink in colour and free from stain particles.
6. RBC5 should be pink in colour and WBC5 should taken up appropriate colours Neutrophilic granules - lilac, Eosinophilic granules - deep blue, Platelets should be pink to purplish blue.
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