to immerse in the drop of blood to avoid air bubbles. If the blood level exceeds the 0.5 mark, withdraw excess blood by touching the tip to the skin surface. Do not touch it to gauze or cotton since these material absorb the fluid portion of the blood, leaving behind a much higher concentration of cells.
2. Wipe the blood from the outside of the pipette, taking care not to touch the very tip. Immerse the tip in the RBC diluting fluid and aspirate fluid exactly to the 101 mark, slightly rotate the pipette while doing so. It is best to hold the pipette in an almost vertical position to avoid formation of air bubbles in the bulb. DO NOT DELAY BETWEEN STEPS 1 AND 2. IF THE BLOOD IS NOT DILUTED PROMPTLY, IT WILL DRY IN THE PIPETTE. Start to draw diluting fluid into the pipette as soon an the tip of the pipette is immersed in fluid to avoid loss of blood cells. Wipe the excess diluting fluid from the pipette, taking care not to touch the very tip. Filter the diluting fluid regularly to remove accidentally introduced blood cells.
3. Remove the suction tube and shake the pipette vigorously for 3 minutes. DO NOT SHAKE IN THE DIRECTION OF THE LONG AXIS. (See figure 6-5.)
Figure 6-6.Hemacytometer counting chamber.
4. Discard the clear fluid (about three drops) from the stem of the pipette. The counting chamber (figure 6-6) must be loaded with fluid from the pipettes bulb.
Figure 6-7.Loading the counting chamber
5. Place the coverglass on the counting chamber, making sure both are clean and grease-free. (Fingerprints must be completely removed.) Load the counting chamber by touching the tip of the pipette against the edge of the coverglass and the surface of the counting chamber (figure 6-7). A properly loaded counting chamber should have a thin, even film of fluid under the coverglass. Allow 3 minutes for cells to settle. If fluid flows into the grooves (moats) at the edges of the chamber or if air bubbles are seen in the field, the chamber is flooded and must be cleaned with distilled water, dried with lens tissue, and reloaded. If the chamber is underloaded,