Hemocytometer

• Hemocytometer


  Also known as a counting chamber, specialized glass slide used in laboratory settings to count and determine the concentration of cells in a liquid sample
• Hemocytometer


  • Primarily applied in hematology for blood cell counts, may also be used in counting blood and other cellular components in other bodily fluids
  • Simple, cost-effective and standardized instrument in clinical diagnostics used to obtain accurate and reproducible cell counts
  • Applications include determination of number of sperm cells in seminal fluid, and determination of WBC count in bodily fluids
• Body fluid cell count

  Cell counts are integral to body fluid analysis, provide information about presence or absence of certain conditions, such as infections or even the viability of samples
• Body fluids where cell count is primarily applied

  • Cerebrospinal fluid (CSF)
  • Seminal fluid
  • Synovial fluid
• Cell count in CSF and synovial fluid


  Cerebrospinal fluid is checked for the number of WBCs present to rule out infections
• Cell count in seminal fluid

  The number of normal spermatozoa is counted to test for infertility
• Cell counting procedure


  Should be performed immediately after specimen collection because granulocytes and even RBCs begin to lyse within one hour, and 40% of WBCs disintegrate after two hours
• Dilution of sample

  1. Gently mix the test tube containing the sample by inversion at least three times

  2. Attach an aspirator to the sucking end of a Thome WBC diluting pipette and then draw the sample up to the 0.5th mark in the stem

  3. For any excess samples in the pipette, gently remove them by dispensing the contents in a clean gauze

  4. While holding the pipette vertically, place the tip of the pipette in the tube containing 2% acetic acid and aspirate until the 11th mark

  5. Place the pipette horizontally and firmly hold the opening at the tip of the pipette using your thumb. Detach the sucking tube from the other end of the pipette and cover that opening with the index or middle finger

  6. Vigorously mix the sample in the pipette by doing a figure of 8 motion with your hand for 45 seconds
• Preparation of the hemocytometer


  1. Wipe the hemocytometer using a clean gauze pad dipped in 70% isopropyl alcohol and make sure it is dry before loading the sample

  2. Prepare a wet chamber by placing a damp gauze pad (use water to dampen the pad) inside a petri dish

  3. After mixing the sample, hold the pipette vertically with the index or middle finger covering the top of the pipette. Discard the first four drops of the mixture onto a piece of gauze

  4. Place a coverslip onto the counting chamber

  5. Gently release your index or middle finger from the pipette to allow a few drops of sample to flow

  6. Place the tip of the pipette on the edge of the ruled area of the counting chamber and gradually allow the mixture to seep under the cover glass. Fill this area exactly. Repeat this step on the other side of the counting chamber

  7. Place the charged hemocytometer inside the wet chamber and cover it. Incubate at room temperature for 10 minutes
• Hemocytometer

  • Manual or Automated
  • Use: to perform manual cell counts on body fluids including cerebrospinal fluid, synovial fluids, pleural fluid, pericardial fluid, peritoneal fluid, peritoneal dialysates, bronchoalveolar lavages, semen
• Synovial fluid and semen

  • Highly viscous body fluids = Synovial fluid
  • Fluids that fail to liquefy = Semen
  • Pretreated before counting
  • Clotted sample: INACCURATE cell counts
• 3 Main types of Hemocytometer


  • Fusch-Rosenthal
  • Nageotte
  • Neubauer (Improved)
• Main difference between hemocytometer types

  Different design of calibrated counting area
• Disadvantages of manual cell count

  • Labor intensive
  • Time consuming
  • Needs advanced technical skills
  • Poor precision
  • Subject to error
• Automated hemocytometer

  • Automated cell counting chambers: erroneous results with low cell counts
  • Normally, the number of RBCs and WBCs in body fluids are low
  • Solution: Define lower limits + Protocol for manual count with hemocytometer
• Hemocytometer parts and dimensions

  • 3x3mm counting grid subdivided into 9: 1x1mm2 squares, each further divided into 16, 100, or 400 smaller squares
  • 1 W square = 1 mm2 in area, best used for WBC count
  • 1 R square = 0.04 mm2 in area, best used for RBC count
• Number of squares counted

  • Depends on the total number of cells present, greater number of cells = greater accuracy
  • When not needed, high dilutions causes cellular structures to disintegrate
  • If extremely low cell counts: additional squares can be counted
  • If High cell counts: fewer cells can be counted
• Detecting errors in preparation

  • Dilutions: performed and analyzed in duplicate
  • Cell counts: Compare each chamber, must agree less than or equal to 20%
  • Not True Replication: Counting the same chamber twice, Comparing chamber counts using the same diluted sample
• WBC count/Total nucleated cell count


  Requested on almost all body fluids, determines the presence of infections, 4 W Squares