Cell Counting Using a Haemocytometer Equipment & Reagents  Haemocytometer plus a supply of cover-slips  0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline  Tally Counter  Cell Suspension  Gilson pipettes or similar  Inverted microscope (preferably phase contrast) Procedure 1. Ensure the cover-slip and haemocytometer are clean and grease-free (use alcohol to clean). 2. Moisten (with water or exhaled breath) and affix cover-slip to the haemocytometer. 3. Look for "Newton's Rings" which indicate that the cover slip has adhered via suction to the haemocytometer. Newton's refraction rings are seen as rainbow-like rings under the cover-slip. 4. Mix equal volumes of 0.4% trypan blue stain and a well mixed cell suspension (not too vigorous) e.g. mix 100µl trypan blue stain with 100 µl cell suspension. 5. Pipette trypan blue/cell mix (approximately 10µl) at the edge of the cover-slip and allow to run under the cover slip. 6. Visualise the haemocytometer grid under the microscope, refer to figure 1 for layout of grid. Please note: 7. Trypan Blue is a "vital stain"; it is excluded from live cells. 8. Live cells appear colourless and bright (refractile) under phase contrast. 9. Dead cells stain blue and are non-refractile. 10. To aid accuracy and consistency of cell counts use counting system illustrated in figure 2. 11. Count viable (live) and dead cells in one or more large corner squares and record cell counts. 12. It is advisable to count around 40 to 70 cells to obtain an accurate cell count - therefore it may be necessary to count more than one large corner square. 13. To calculate cell concentration per ml: Average number of cells in one large square x dilution factor* x 104 *dilution factor is usually 2 (1:1 dilution with trypan blue), but may need to further dilute (or concentrate) cell suspensions. 104 = conversion factor to convert 10-4ml to 1ml Calculation of Cell Viability No. of Viable Cells Counted / Total Cells Counted (viable and dead) x 100 = % viable cells Ravi Kumudesh / slsmls.org

1. Haemocytometry
Ravi Kumudesh
MSc/BSc/Dip(MLT)
Sri Lanka Society for Medical Laboratory Science
1

2. Hemocytometry
• Hemocytometer
• RBC and WBC
• Thoma pipet
• Unopette
• Dilution problem
• Microscopy
2
Sri Lanka Society for Medical Laboratory
Science

3. Red Cell and White Cell Counts
• RBC problem: anemia, polycythemia
• WBC problem: leukocytosis, leukopenia
• RBC reference ranges
• Male 4.50 - 6.00 x 106
/µL (x 1012
/L)
• Female 3.70 - 5.20 x 106
/µL (x 1012
/L)
• WBC reference range
• Both 5.0 - 10.0 x 103
/µL (x 109
/L)
3
Sri Lanka Society for Medical Laboratory
Science

4. Diluting Fluid
• For RBC
– Physiologic saline (0.8% NaCl)
– Hayem’s solution
– Reese-Ecker
• For WBC
– 3% Acetic acid
– 0.1 N HCl
4
Sri Lanka Society for Medical Laboratory
Science

5. 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
5
Sri Lanka Society for Medical Laboratory
Science

6. 6
Sri Lanka Society for Medical Laboratory
Science

7. Improved Neubauer Hemocytometer
7
Sri Lanka Society for Medical Laboratory
Science

8. 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
Sri Lanka Society for Medical Laboratory
Science

9. Hemocytometer
Chamber
9
Sri Lanka Society for Medical Laboratory
Science

10. Counting Rule
• Do not count cells touching
– Bottom line
– Right line
10
Sri Lanka Society for Medical Laboratory
Science

11. 11
Sri Lanka Society for Medical Laboratory
Science

12. 12
Sri Lanka Society for Medical Laboratory
Science

13. RBC Example…
• Aspirate blood to 0.5, then dilute to 101
• DF = 200
• Counted 500 erythrocytes in 5 small squares
RBC = 500 rbc x 25 Sm Sq x DF (200)
5 Sm Sq 0.1 mm3
= 5.00 x 106
/mm3
(µL)
13
Sri Lanka Society for Medical Laboratory
Science

14. 14
Sri Lanka Society for Medical Laboratory
Science

15. 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 0.1 mm3
= 8.0 x 103
/mm3
(µL)
15
Sri Lanka Society for Medical Laboratory
Science

16. Unopette
• Self-contained reservoir and 20 µL pipet
• Ammonium oxalate diluent
• DF = 100
• Count leukocytes in all 9 large squares
• Special formula to facilitate calculation
16
Sri Lanka Society for Medical Laboratory
Science

17. Unopette
17
Sri Lanka Society for Medical Laboratory
Science

18. 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
Sri Lanka Society for Medical Laboratory
Science

19. Compound
Microscope
• Ocular (adjustable
binocular)
• Mechanical stage
• Condenser
• Objectives (parfocal)
19
Sri Lanka Society for Medical Laboratory
Science

20. Care of Microscopes
• Carry with care
• Protect long objective lens
• Clean and disinfect stage
• Wipe oil off OIL
• Save lamp life
20
Sri Lanka Society for Medical Laboratory
Science

21. Sri Lanka Society for Medical Laboratory
Science
21