Pipet Information and Courses from MediaLab, Inc.

Important safety precautions must be observed when handling cerebrospinal fluid. The following guidelines apply:Semi-automatic micropipettes and disposable plastic chambers are the safest option for CSF testing. Many laboratories still use the hemacytometer with disposable pipets.If disposable materials are not used, soak contaminated reusable pipets, hemacytometer and coverslip in 70% alcohol or Wexide.All disposable items should be placed in a biohazard container for appropriate disposal.Wash hands thoroughly when the examination is completed.Spinal fluids which are to be discarded must be placed in biohazard containers for appropriate disposal.Careful attention to specimen processing and handling will help ensure that accurate results are obtained.

Calibrated automatic pipets should be used on specimens that require a dilution. Mouth pipetting must of course always be avoided. Automated cell counters may not be the best choice for cell counts on CSF because of the variation in background counts. A high background count could cause a false increase in normal or slightly elevated count. The dilution required is based on the appearance of the sample. AppearanceDilution RatioVolume of SampleVolume of Diluentslightly hazy1:1030 ul270 ulhazy1:2030 ul570 ulslightly cloudy1:10030 ul2970 ulslightly bloody1:10030 ul2970 ulcloudy1:20030 ul5970 ulbloody1:20030 ul5970 ulturbid1:100000.1 ml of a 1:100 dilution9.9 ml

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.

The following is a list of materials needed for semen analysis. Laboratories will differ slightly in the equipment used. Use of this equipment will be described further in the later pages of this course. Materials needed include:graduated test tube or serological pipets with safety bulb to measure volumepH paper in neutral to basic range (e.g. 7.2-8.8)counting chamber and/or automated counting machineglass slides and coverslips for wet mount if motility and sperm count are to be assessed separatelyhand counterif dilution is donediluting fluid calibrated automatic pipetspositive pressure pipets and glass boreslight microscope with phase contrast objectives for sperm count and bright field objectives for morphology assessmentglass slides and fixative for morphology slidesset-up for performing Papanicolaou or other morphology stainingEvery laboratory should also have a copy of the "WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction", published on behalf of the WHO by Cambridge University Press. The fourth edition was published in 1999.

If the specimen is more viscous than normal, it may be difficult to dilute it or to load it onto counting chambers in the undiluted condition. In this rare situation the semen may need to be manipulated to reduce the viscosity before a count is done. One method to do this is to repeatedly pipet the specimen up and down with an equal volume of culture medium. Care must be taken to avoid foaming. Other methods include enzyme digestion, for example with bromelain at a concentration of 1 gm / liter, or addition of a small amount of emulsifier, such as Alevare or chymotrypsin. Any manipulation of this type must be recorded on the report sheet. Calculation of the number of sperm per milliliter will also have to be corrected for any dilution.