Expt.

No: 2 Date: BLOOD SMEAR AIM: Identification of cells in blood smear PRINCIPLE: The smearing and staining of the blood with stains like Giemsa, Leishmans or wrights stain results in the blue coloured heterochromatin and cytoplasmic granules which when viewed under a microscope can identify the cell types based on their morphology and staining. The stains used have dyes that are basic (methylene blue) and acidic (eosin) in nature that stain the nuclear and cytoplasmic elements respectively. The most numerous cells are the RBC. It is biconcave shape renders its center thinner (in thin smears) than the edges so the hemoglobin filling the cell stains more deeply in the perimeter. The lymphocytes are small, round, blue staining cells. Its heterochromatin nucleus is round ,dark staining and nearly fills the cell leaving only thin rim of light blue cytoplasm. The neutrophils are the largest cells in the field. Its nucleus is multilobed and dark staining The basophils lobated nucleus is often hidden by the coarse blue-purple granules of its cytoplasm.Neutrophilic granules are poor staining and often hard to distinguish in neutrophils. The nucleus of the neutrophil may have 3-5 lobes. The eosinophil has coarse red granules. Its nucleus often has 2or 3 lobes. Monocyted are about twice the size of the small lymphocyte or RBC.It nucleus is not very heterochromatic. Lymphocytes have a more heterochromtic nucleus and a sky blue cytoplasm. REAGENTS: Clean glass slides, sterile needles, alcohol swabs, Leishmans/giemsa stain.

PROCEDURE:

Clean the tip of the finger with the alcohol swabs Take a sterile needle and prick the finger Place a drop or two of blood on a glass slide With another slide make the smear dry out sufficiently. Add the stain such that it floods over the slide. After staining for a few minutes, wash the slides with water and dry. View the slide under a microscope and record your observation.

Note:

1. Use clean glass slides. If greasy, results non-uniform thickness and poor staining quality.

2. Thickness affected by the angle of spreading a. Thick smear-WBC morphology is not seen well b. Thin smear-RBC morphology is not preserved well. 3. Smears should be dried quickly to prevent distortion
4. If slides are not completely dried, water particles are seen after staining which results in

false interpretation.
5. Slides should be stained immediately after it is dried. Otherwise dried plasma will give

pale blue background. 6. Overstaining leaves stain particles on slides which may sometimes be mistaken for platelets. 7. Take note of PH of buffer: Should be around 6.8.

If pH is more, RBC satin dirty bluish green; neutrophils stain purplish instead of mauve; eosinophils stain bluish green instead of orangish red. If pH is less: RBC stains bright orange, others satin very pale.