OCR BIOLOGY UNIT F221
1. Put a band (tourniquet) around the arm to make the vein stand out
2. Clean the area around the vein with an alcohol based solution
3. Push a sterile needle, attached to a sterile syringe into the vein
4. Pull back the plunger of the syringe to suck the blood into the syringe
5. When the necessary volume of blood has been extracted, remove the syringe and needle, loosen the tourniquet and press a small ball of cotton wool over the wound, then apply a suitable dressing (plaster).
Making a blood film
1. Place a small drop of blood near the edge of a clean microscope slide
2. Place the end of another slide (the spreader) on the sample slide
3. Hold the spreader at an angle of approx 30oC and push it along the slide, spreading the drop of blood into a smear.
4. Label the slide with the patient’s details and allow it to air dry, so the cells stick to the slide
5. Fix the slide using alcohol, this preserves the cells
6. Stain the slide using a Romanowsky stain, e.g. Wright’s or Leishman’s. The stain is poured over the slide, left for approx 2 minutes and the excess is washed off with water.
E.g. Leishman’s, makes some structures appear darker or a different colour. In a blood film the nucleus of leucocytes will be stained purple, this allows neutrophils, lymphocytes and monocytes to be identified from each other by the shape of their nuclei.
A special counting chamber designed for counting blood cells. It has a central platform with grooves either side of it. There is a tiny grid etched onto the platform, this looks a bit like graph paper. In the centre of the grid there are some triple lined squares, these measure exactly 0.2 x 0.2 mm. When you put the cover slip on top the platform is exactly 0.1mm below the cover slip. This means that when you look at one of the triple lined squares under the microscope you are looking at a volume of 0.1 x 0.2 x 0.2 = 0.004 mm3.
If we are counting erythrocytes, the sample is diluted with Dacie’s fluid; the blood is diluted 1 in 200.
Each triple lined square has a volume of 0.004 mm3, the blood was diluted 200 times and we count five triple lined squares (0.004 x 5 = 0.02 mm3).
So if the number of cells counted in the five triple lined squares is E, the number of red cells in 1 mm3 of blood is:
1 /0.02 x E X 200
= E X 10 000
If cells lie on top of the triple lines around the edge of the square, we apply the NORTHWEST RULE. If a cell lies on the middle of the triple lines on the north or west of the grid, we count it; if it is lying on the south or east of the grid, we miss it out.
A different dilution is used (1 in 20) and the four corner squares are used to count the cells.
Types of blood cell
Red blood cells (erythrocytes)
• Biconcave discs, transport oxygen and some carbon dioxide. Their shape means they have a relatively large surface area to volume ratio to speed up gas exchange. Their cytoplasm is packed with a pigment called haemoglobin, this associates reversibly with oxygen. Mature red blood cells have no nucleus; this gives them more room for haemoglobin. Erythrocytes are also very small and flexible so they can be flattened against capillary walls; this reduces the distance that gases have to diffuse across and speeds up gas exchange.
Leucocytes (white blood cells)
• Have small granules in the cytoplasm. These cells engulf microorganisms by phagocytosis.
• Have a large, darkly stained nucleus surrounded by a thin layer of clear cytoplasm. There are two kinds, B lymphocytes and T lymphocytes. B – produce antibodies; T- several functions including cell destruction.
• Largest kind of leucocyte. They have a large, bean shaped nucleus and clear cytoplasm. They...
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