Coverslip 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.

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 sample in the first syringe is quickly delivered into a watchglass or onto a slide. After the technologist verifies the presence of white-gray marrow particles in the sample, push smears and/or coverslip smears from this unanticoagulated sample are made immediately. All films should be rapidly air dried. The appearance of fat as irregular holes in the films also give the assurance that marrow and not just blood has been obtained. This type of smear is referred to as a direct smear and is usually used to evaluate morphology. Although some evaluation of cellularity and M:E ratio is possible, particle smears or biopsy sections provide a more accurate representation of these factors.

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.

Viability is a measure of the percentage of cells that are alive. Since motile cells are inherently viable a viability assessment may not be necessary when motility is high. Most laboratories set a minimum motility after which a viability will also be performed. To assess viability, place a drop of semen on a slide. Add an equal volume of a vital stain such as trypan blue. Cover with a coverslip. Allow color to develop for several minutes, but not more than 5 minutes. Count 100 cells (both motile and non-motile cells) on each of 2 slides. During the count differentiate between the white cells (living) and blue cells (non-living).

For assessing count, motility, viability and other cellular components your laboratory will require a microscope with 10x oculars and phase contrast objectives up to 20x. You will also need hand counters.For assessing morphology you will need bright field objectives of 40x and/or 100x (oil immersion).You will also need counting chambers, glass slides and coverslips and a method for staining sperm for morphology assessment.

Mix urine specimen well, and transfer 10-15 ml of urine to conical centrifuge tube. Centrifuge at 1500 rpm for 10 minutes. Decant supernatant, and resuspend sediment in 0.5-1.0ml of residual specimen. Place a drop of concentrated sediment on a glass slide and coverslip.

In this field both red blood cells and yeast are present. Since red blood cells are readily hemolyzed by dilute acetic acid, a drop is allowed to flow under the edge of the coverslip. The acetic acid lyses the red blood cells, leaving only the remaining yeast.

Coverslip defects may be mistaken for elements in the microscopic exam.