Hemacytometer Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Hemacytometer and links to relevant pages within the course.
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| A 1:100 dilution was made on a slightly cloudy CSF. The cells were slightly overlapping each other on the hemacytometer. Which of the following dilutions should be made? | View Page |
| Safety Precautions 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.
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| Examining CSF with the Hemacytometer 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. | View Page |
| More on Undiluted Specimens In an undiluted specimen, count and differentiate red cells and white cells at the same time. You can count red cells on a hand counter and use the differential counter for white cells.
If you cannot differentiate white cells from red cells in the undiluted specimen, a plain capillary tube may be filled with crystal violet acetic acid diluent which is subsequently expelled from the tube. A very thin coating of the diluent will remain on the inside of the tube. CSF is drawn halfway up into the tube, which is then rocked back and forth to mix. The hemacytometer is then filled with the fluid containing stained white blood cells and lysed red cells.
If cells are numerous and overlapping and it is necessary to focus through several planes in order to see all of the cells, a dilution must be made.
When macroscopic appearance is turbid, milky or bloody, a significant dilution is usually necessary. | View Page |
| Examining a Diluted Specimen Examining a diluted CSF specimen involves the following steps:
Mix the CSF sample manually 6 - 10 times or place it on a mechanical mixer for 5 - 10 minutes.Use a calibrated automatic pipet and place the appropriate volume of sample and diluent in a tube. Mix the diluted sample well.Use a Pasteur pipet and fill both sides of the hemacytometer. Allow the cells to settle for 5 minutes in a moist environment.Count cells in the four corner squares and the center square on both sides of the chamber. The number of cells counted times the dilution factor is then equal to the number of cells per microliter.
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| How many large hemacytometer squares contain a total volume of 1 microliter of fluid? | View Page |
| A 1:10 dilution is made on a CSF sample. A total of 10 squares on both sides of the hemacytometer are counted and a total of 150 cells are recorded. What is the count per microliter? | View Page |
| Examining CSF with the Hemacytometer (continued) White cells are less refractile and appear somewhat granular in appearance. In general, white cells will be larger than red cells. The segmented nucleus in neutrophils can be seen on high power. Lymphocytes and monocytes may be more difficult to differentiate in an undiluted, unstained specimen.Cells are counted in the four corner squares and the center square on both sides of the hemacytometer. The number of cells counted equals the number of cells/microliter.The ruled area of one side of a hemacytometer is shown on the right, with routine counting squares for red and white cell counts. Each large square is 1 mm wide by 0.1 mm in depth. The area for counting an undiluted specimen is 10 square millimeters, or 5 large squares on each side.
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| Safety Precautions Safety precautions should be observed when handling seminal fluid. The following guidelines should be followed:If non-disposable items are used, soak contaminated items(e.g.hemacytometers and coverslips) in 70% alcohol or Wexide®.All disposable items should be placed in a biohazard bag for autoclaving.Gloves must be worn and hands thoroughly washed when the examination is completed.Seminal fluids that are to be discarded should be placed in biohazard bags for autoclaving. | View Page |
| Calculating sperm count on a hemacytometer The formula for calculating the sperm count when 5 small squares within the large center square are counted is:
Number of sperm counted in 25 squares on each of 2 sides x dilution factor/volume x 1000 = sperm/ml.
Example: 100 sperm are counted in the five small squares of one side of the hemacytometer, 110 sperm are counted in 5 small squares of the other. The dilution is 1:20.
Number of sperm in 25 squares on 2 sides = 210 x 5 = 1050
Sperm/ml = 1050 x 20 (dilution factor) divided by 0.2 mm3 x 1000 = 105 million sperm/ml. | View Page |
| Diluting a specimen for counting on a hemacytometer Following liquefaction (20-30 minutes), mix the sample manually by swirling the container several times. Thorough mixing is essential for accurate counting. Calibrated automatic pipettes are used to prepare a dilution. Because of the viscosity of semen, the semen should be added to the diluent using a positive pressure pipettor.
The dilution often used for routine sperm counts is 1:20 but the actual dilution factor will vary depending on the total sperm count. For high concentration specimens a greater dilution will be necessary. For low concentrations an undiluted or minimally diluted specimen may be required. The appropriate dilution is determined by estimating the concentration needed to do a count of at least 100 cells per side of the loaded hemacytometer.
The diluent that may be used for sperm counts on a hemacytometer can be as follows: 5 gm of sodium bicarbonate in 100 ml of distilled water, plus 1ml of formalin (neutral). | View Page |
| Other counting chambers Some professionals believe that sperm counts done by hemacytometer are not accurate because of the need to dilute the viscous semen prior to counting. There are several other counting methods available to assess sperm concentration.The advantages of the following methods are: the specimen does not have to be diluted motile and non-motile sperm can both be counted avoiding the need for wet mount evaluation of motile cells. Note that counting moving sperm can be difficult and takes significant practice to avoid error. For each of these methods accurate counts are best obtained when at least 100 sperm per replicate are counted. Makler (Zygotek Systems, Inc.). An undiluted sample is placed on the chamber and covered with the coverglass. Ten squares on the grid contain 0.000001ml. CellVu (Millennium Sciences, Inc). Two sides of a special slide are loaded with a drop of undiluted semen. Coverslips with special grids are placed on top of the sperm according to manufacturer's directions. Sperm on both sides are counted. MicroCell (Conception Technologies) has two chambers on a single, disposable slide. A special eyepiece with a grid is needed for counting. | View Page |
| Loading and counting using a hemacytometer Fill both sides of the hemacytometer. Focus on the large center square with the 20X objective. The counting area consists of five small squares in the large center square. The squares usually counted are the four corner squares and the center square, all of which are marked R. A minimum of 100 sperm should be counted in the five small “R” squares. If the number of sperm is low then 10 squares or all squares may be counted to obtain the 100 per side.
Count both sides of the hemacytometer and take the average of the two counts to calculate the actual count per ml. | View Page |
| Neubauer hemacytometer The picture on the right shows the counting chamber of the Neubauer hemacytometer. This counting method is used to count many types of cells. To use this chamber for counting sperm the specimen will usually need to be diluted. Proper loading of the hemacytometer is also important for accurate sperm counts to be obtained. | View Page |