HEMACYTOMETER AND DILUTING PIPET PRACTICE ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
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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, filling the chamber with the diluted cells, 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.  Mark the Upper L, Upper R, center, lower L and lower R squares for counting.
	3) the grids for RBC counts.

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 appropriate suspensions of yeast, place an aliquot on the hemacytometer, and count all yeast cells in five designated squares. The convention is to count all cells touching right and bottom sides, ignore cells touching top and left sides.  When the five squares are counted, you record and add them up and multiply by the appropriate dilution factor (see below).

As soon as you are finished with the dilutions, wash out the pipettes 3 times with soap and water, rinse well, finish with a distilled H₂O rinse, replace in case.  After you have completed the counting, wash the hemacytometer in the same manner.

PRACTICE EXERCISE FOR LEUKOCYTE COUNT:
 

	1. Suspend 50 mg dry yeast in 100 mL water.
	2.  Set up all equipment you will use in the dilution and preparation of the hemocytometer
	Familiarize yourself with the structure and function of the hemocytometer, and of the dilution pipets.
	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.
	7. Add a small amount of the diluted yeast to just fill the first chamber of the hemacytometer. It should flow in to fill the chamber by capillary action. (Do not over fill).  To improve your skill, repeat the dilution a second time and fill the second chamber.  After completing the counts of each, compare the numbers you have generated. 8. Let the preparation sit for a minute (for cells to settle).
	9. Examine under 100x, count the five fields indicated for WBC counts (counting the 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:

Follow the steps as outlined above, but use more concentrated yeast, and the diluting pipet which dilutes 1:200:

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 YOUR DESK WITH THE FOLLOWING EQUIPMENT FOR THE BLOOD COUNTS:
 

2 hemacytometers 2 coverslips 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 EtOH 2 paper towels 1 tray Critoseal putty