HEMACYTOMETER AND DILUTING PIPET PRACTICE
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
This page has been accessed times since 24 February 2003.
February, 1980, latest revision 3 January 1996, 31 Dec. '96, 8 Jan. '97, 8 Mar 01
Hemacytometer with cover slip
WBC diluting pipet (white), hose and mouthpiece
RBC diluting pipet (red), hose and mouthpiece
For WBC simulation: 50 mg dry baker's yeast + 100 mL water
For RBC simulation: 1 package baker's yeast + 100 mL water
50 mL beaker for waste fluid
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.
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.
PRACTICE EXERCISE FOR LEUKOCYTE COUNT:
1. Suspend 50 mg dry yeast in 100 mL water.
2. Perform the dilution twice, add sample of each dilution to hemacytometer chambers 1 and 2 as spelled out in steps 3-6:
3. Using dilution pipet with the WHITE mixer, draw yeast suspension up to the 0.5 mark. Dab with piece of paper towel if needed to adjust volume. Proceed immediately to the next step:
4. Fill the pipet the rest of the way to the 11 mark with crystal violet diluent1. (This constitutes a 1:20 dilution)
5. Shake well to mix with the hose end sealed with your finger.
6. Empty ~1/2 of pipet into waste container, add a small amount of the diluted yeast to the first chamber of the hemacytometer. It should flow in to fill the chamber. (Do not over fill).
7. Repeat steps 3-6 and fill the second chamber.
8. Let the preparation sit for a minute (for cells to settle).
9. Examine under 100x, count the five fields indicated squares of blue-stained yeast with a clicker (fields: top L & R, bottom L & R, center).
10. Calculate the WBCs/cmm: sum the 5 groups, multiply by 40. (Should be about 8,600 yeast cells/cmm)
RED BLOOD CELL COUNT PROCEDURE:
1. Mix a package of baking yeast with 100 mL of water, stir for 10 minutes to suspend.
2. Using the dilution pipet with RED mixer from hemacytometer kit, draw suspension yeast up to the 0.5 mark. This is best done by slightly slanting the pipette to allow the suspension to flow in. Slight suction should start it. (Make sure the hose is not kinked shut.) Keep the pipette level once you have filled it. Immediately proceed to the next step:
3. Draw Ringer's solution diluent up to the 101 mark. (Dilution of 1 to 200.)
4. Shake well to mix with the hose end sealed with your finger.
5. Empty ~ 1/2 of the pipet into the waste container, then add a small amount of the diluted blood to one chamber of the hemacytometer. It should flow in to fill. (Do not over fill).
6. Let the preparation sit for a minute (for cells to settle).
7. Center the grid at 100x, switch to 400x and count five fields of 16 smallest squares RBCs with a clicker (count these fields: top R & L, bottom R & L, center).
8. Calculate the number of yeast/cubic millimeter: sum the 5 groups, multiply by 10,000 (i.e., add four zeros).
9. How many in the entire package?
10. CLEAN UP THE EQUIPMENT: Wash out the hemacytometer, 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.
Count as for WBC count. (this suspension is less viscous than blood)
Grids for WBC and RBC counts:
1 Diluent for white blood cells:
10 mg crystal violet
1.0 ml glacial acetic acid
q.s. to 100 mL with d H20
SET UP PER DESK
hemocytometers, clean and polished
2 clean coverslips
2 WBC (white) diluting pipets with mouth piece and tubing
2 RBC (red) diluting pipets with mouth piece and tubing
1 bottle RBC diluent (clear)
1 bottle WBC diluent (purple)
2 hematocrit tubes, heparinized
2 self contained lancets
2 cotton balls
1 70% EtOH in squirt bottle
1 folded paper towel, torn in half, half per person
1 50 mL beaker for waste
1 250 mL beaker with about 50 mL dilute warm soapy water