Pasteur Information and Courses from MediaLab, Inc.

Specimens that are clear may be counted undiluted as long as there is no overlapping of the cells. Examining an undiluted CSF involves the following steps: Mix the CSF manually 6 - 10 times or place it in a mechanical mixer for 5 minutes.Using a Pasteur pipet or Dispo® pipet, fill both sides of the hemacytometer and allow the cells to settle for 5 minutes. To prevent the fluid in the chamber from evaporating, place it in a Petri dish containing moist filter paper. A disposable chamber similar to a hemacytometer is preferred, if one is available.Focus on low power (10x) and scan for the presence of cells. If cells are located, switch to high power (40x) to determine whether the cells are leukocytes or erythrocytes. Erythrocytes will be smooth refractile discs or spheres. Some red cells may appear crenated. Keep in mind that some red cells may be folded or in a vertical position rather than flat. In this situation only a small portion of the cell will be visible.

Particle smears are also made from the unanticoagulated sample. The bone marrow particles are removed from the watchglass and placed on a coverslip. One of the following items: Pasteur pipet, capillary tube or broken end of a wooden applicator stick, may be used to transfer the particles. A second coverslip is placed over the first and the particles are crushed between the coverslips as they are pulled apart. Some practice is needed to perfect this technique. As mentioned previously, this type of preparation provides a more accurate assessment of marrow architecture and cellularity than the direct smear. Morphological detail is preserved on well made slides. The remaining sample may be added to a tube containing EDTA anticoagulant and additional smears may be made if needed.