RED BLOOD CELL COUNT USING A HEMACYTOMETER

David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 

This page has been accessed Counter times since 30 December 2004. 
February, 1980, latest revision 3 January 1996, 31 Dec. '96, 8 Jan. '97, 4 Jan 03
 
See related protocols: Blood Cell Counts, Blood Typing, Hematocrit.

SAFETY NOTE:
Avoid infection with blood-borne pathogens such as HIV and hepatitis B, by using protective gloves when handling other peoples' blood.  Dispose of the blood-contaminated sharps in the marked container, and the rest as indicated by the instructor.

INTRODUCTION:
Sufficient numbers of red blood cells (RBC, or erythrocytes) are necessary for adequate transport of oxygen from the lungs to the peripheral tissues.  Too few RBCs constitutes a pathological condition known as anemia (lit., without blood).  According to the Merck Manual, normal values of RBC/cmm for males is 5.4 + 0.8 million, and 4.8 + 0.6 for females.  Anemic levels for adult males are below 4.5 million, for females below 4.0 million.  We will perform red and white blood cell counts on your blood in the lab using a hemocytometer and appropriately diluted blood.

Blood cell counts can be performed using the hemacytometer. This precision instrument possesses a platform with microscopic grid scoring.  Rails on either side hold up a cover slip so that a specified quantity of fluid is held. By properly diluting blood, counting all cells in specified squares, and multiplying by the proper conversion factor, the number of cells per cubic millimeter can be determined.

PRELIMINARIES:
You should have already illustrated the following when you performed the practice counts using yeast last week:
    1) the dilution pipets, with explanations of their use and what the dilution factors would be
    2) the grids for WBC counts
    3) the grids for RBC counts. Review the illustration of the five hemacytometer fields which you drew your notebook.

The red blood cell count may be performed at the same time as the white blood cell count and hematocrit, if you get blood to blow adequately.
 

EQUIPMENT SUPPLIES
2 hemacytometer kits, each with:
    1 hemacytometer
    1 coverslip
    1 WBC diluter pipet with hose and mouthpiece
    1 RBC pipet with hose and mouthpiece
1 autolet
2 lancet needless (for drawing blood)
2 platforms (for drawing blood)
microscope
squirt bottle with 70% ethanol
Kimwipes (to be soaked in EtOH)
1 bottle WBC diluent (purple)
1 bottle Ringer's Solution (a clear diluent for RBC)
1 250 mL beaker for waste fluid
paper towel

RED BLOOD CELL COUNT:
 

Set up all equipment on your desk so that you are sure to have everything at your finger tips for the procedure.
Prepare an autolet with a sterile lance and platform.
1. Swab towards the side of the tip of a little-used finger with 70% EtOH. (NOT close to the cuticle!)
2. Lance by placing the platform of the autolet against the finger tip and pressing the trigger.  Alternatively, us a lancet with quick, firm jab to the side of the pad of the finger.  Wipe away first blood.
3. Using the dilution pipet with RED mixer from hemacytometer kit, draw blood up to the 0.5 mark.  This is best done by slightly slanting the pipette down to allow blood to flow in.  (Do not allow air to be drawn into the pipet or it will not draw the correct volume of blood.  Slight suction should start it. (Make sure the hose is not kinked shut.)  Keep the pipette level once you have filled it.  Do not allow blood to congeal in pipette!  Immediately proceed to the next step:
4. Continuing to hold the pipet as horizontal as possible, draw Ringer's solution diluent up to the 101 mark.  (Dilution of 1 to 200.)
5. Seal the tip with your finger and shake well to mix.
6. Empty ~1/2 of pipet into waste container
add a small amount of the diluted blood to one chamber of the hemacytometer to just fill the chamber of the hemacytometer.  It should flow in to fill.  (Do not over fill).
7. Let the preparation sit for a minute (for cells to settle).
8. Center the grid at 100x, switch to 400x and count and record the RBCs in each of five fields (each with 16 smallest squares) with a clicker (fields: top R & L, bottom R & L, center).  Include in the count  all cells touching left and bottom sides, ignore cells touching top and right sides.
Calculate the RBCs/cmm by adding the cells in the 5 groups and multiplying by 10,000 (i.e., add four zeros).  Enter your RBCs/mm in the class data table.
9. Wash out the pipette thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case.

 Ringer's Solution, per 100 mL:

 860 mg NaCl
 30mg KCl
 35mg CaCl2

dissolve in dH2O and q.s. to 100 mL.