WHITE BLOOD CELL COUNT

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

This page has been accessed Counter times since 12 March 2009.
February, 1980, latest revision 3 January 1996, 31 Dec. '96, 8 Jan. '97, 8 Mar 01
 diluting
pipets
See related protocols: Blood Cell Counts, Blood Typing, Hematocrit.

This page is still under construction, for give the confusion....

White blood cells (WBC), or leukocytes, are involved in fighting infections and clearing away dead cells.  During infections, their numbers increase dramatically above the normal range of 4,800 to 10,800 WBC/cu mm.  Finding WBC content above 10,800 is suggestive of infection, or other problems (such as leukemia).

 Leukocytes are without pigment, and must be stained to be counted.  Therefore, the diluent contains crystal violet for this purpose.  It also contains acetic acid which fixes the WBC, and causes the lysis of RBC so they are not visible.  Note that a different dilution pipette is used than that in the RBC technique.

Each desk should assemble the following:

Image of desk set up for blood studies Equipment and supplies
Blood letting set up
2 hemacytometers
2 coverslips
one autolet
2 platforms (for drawing blood)
2 lancets (for drawing blood)
1 250 mL beaker for waste fluid
1 bottle WBC diluent (purple)
1 bottle RBC diluent (clear)
2 Kimwipes, soaked in 70% EtOH
2 paper towels


Blood cell counts can be performed using the hemacytometer. This is a precision instrument possesses a platform with microscopic grid scoring above which 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.

Because of the potential dangers of working with blood, we will first practice the necessary dilutions and use the hemacytometer to count yeast cells. Be certain to master these skills before you attempt to do the blood work.

First illustrate:
    1) the dilution pipets, explain their use and what the dilution factors would be
    2) the grids for WBC counts
    3) the grids for RBC counts.  Practice drawing water up in the pipet to the desired volume several times.
Practice drawing water up to the 0.5 and 1.0 volumes several times so that you are confident of your skill.  You will then dilute the suspensions of yeast, place an aliquot on the hemacytometer, and count all yeast in five designated squares. The convention is to count all cells touching left and bottom sides, ignore cells touching top and right sides.  When the five squares are counted, you add them up and multiply by the appropriate dilution factor (see below).

When finished for the day, wash out the pipettes and hemacytometer thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case.

PROTOCOL (These steps are similar to RBC, read that protocol carefully):



1. Swab the tip of a little-used finger with 70% EtOH.
2.  Lance with quick, firm jab to the side
of the pad of the finger, wipe away first blood.

3. Using dilution pipet with the WHITE mixer, draw up to the 0.5 mark.  Do not allow blood to congeal in pipette!  Proceed immediately to the next step:
4. Fill the pipet to the 11 mark with crystal violet diluent.  (See below for formula.)
5. Shake well to mix with the tip sealed with your finger.
6. Empty ~1/2 of pipet into waste container, add a small amount of the diluted blood apply to the second chamber of the hemacytometer.  It should flow in to fill the chamber.  (Do not over fill).
 
If the chamber is overfilled, quickly remove the overflow with a paper towel

7. Let the preparation sit for a minute (for cells to settle).

WBC stained
8. Examine under 100x, count the five fields indicated squares of blue-stained WBCs with a clicker (fields: top L & R, bottom L & R, center).  Include in the count  all cells touching left and bottom sides, ignore cells touching top and right sides.

Here are other pictures of
WBCs
WBCs (not yet)
WBCs

9. CLEAN UP THE EQUIPMENT:  Wash out the hemocytometer, pipettes and mouth pieces thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case.  Replace along with two pieces of hose in the case, return to the proper location in the drawer.

10. Calculate the WBCs/cmm: sum the 5 groups, multiply by 40. Enter your blood cell counts in the class data table.
These are RBC views which will have to suffice until we have some WBC views for illustration...

            

1 Diluent for white blood cells:

10 mg crystal violet
1.0 ml glacial acetic acid
q.s. to 100 mL with d H20