1. Week 4: Hemocytometry Hemocytometer RBC and WBC Thoma pipet Unopette
Dilution problem
Microscopy

2. Red Cell and White Cell Counts
RBC problem: anemia, polycythemia
WBC problem: leukocytosis, leukopenia
RBC reference ranges
Male x 106/L (x 1012/L)
Female x 106/L (x 1012/L)
WBC reference range
Both x 103/L (x 109/L)

3. Diluting Fluid For RBC For WBC Physiologic saline (0.8% NaCl)
Hayem’s solution
Reese-Ecker
For WBC
3% Acetic acid
0.1 N HCl

4. Thoma Pipet RBC Aspirate to 0.5 or 1.0 Dilute to 101 DF = 200 or 100
WBC
Aspirate to 0.5 or 1.0
Dilute to 11
DF = 20 or 10
Discard volume of one (1) in the stem

6. Improved Neubauer Hemocytometer

7. Hemocytometer Chamber
Depth = 0.1 mm
Grids
RBC use 5 small squares in the center large square
WBC use 4 corner large squares
Unopette for WBC use all 9 large squares

8. Hemocytometer
Chamber

9. Counting Rule
Do not count cells touching
Bottom line
Right line

12. RBC Example… Aspirate blood to 0.5, then dilute to 101 DF = 200
Counted 500 erythrocytes in 5 small squares
RBC = rbc x 25 Sm Sq x DF (200)
5 Sm Sq mm3
= 5.00 x 106/mm3 (L)

14. WBC Example… Aspirate blood to 0.5, then dilute to 11 DF = 20
Counted 160 leukocytes in 4 large squares
WBC = 160 wbc x 1 Lg Sq x DF (20)
4 Lg Sq mm3
= 8.0 x 103/mm3 (L)

15. Unopette Self-contained reservoir and 20 mL pipet
Ammonium oxalate diluent
DF = 100
Count leukocytes in all 9 large squares
Special formula to facilitate calculation

16. Unopette

17. Unopette WBC Example… Counted 70 leukocytes in 9 large squares
Add 10% of count
Multiply by DF
WBC = (70 wbc + 7 wbc) x DF (100)
mm3
= 7.7 x 103/mm3 = 7.7 x 109/L

18. Compound Microscope Ocular (adjustable binocular) Mechanical stage
Condenser
Objectives (parfocal)

19. Care of Microscopes Carry with care Protect long objective lens
Clean and disinfect stage
Wipe oil off OIL
Save lamp life