week 4: hemocytometry hemocytometer rbc and wbc thoma pipet unopette dilution problem microscopy
Post on 20-Dec-2015
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Week 4: Hemocytometry
• Hemocytometer
• RBC and WBC
• Thoma pipet
• Unopette
• Dilution problem
• Microscopy
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Red Cell and White Cell Counts
• RBC problem: anemia, polycythemia
• WBC problem: leukocytosis, leukopenia
• RBC reference ranges• Male 4.50 - 6.00 x 106/L (x 1012/L)
• Female 3.70 - 5.20 x 106/L (x 1012/L)
• WBC reference range• Both 5.0 - 10.0 x 103/L (x 109/L)
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Diluting Fluid
• For RBC• Physiologic saline (0.8% NaCl)
• Hayem’s solution
• Reese-Ecker
• For WBC• 3% Acetic acid
• 0.1 N HCl
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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
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Improved Neubauer Hemocytometer
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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
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Hemocytometer
Chamber
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Counting Rule
• Do not count cells touching• Bottom line
• Right line
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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 (L)
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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 (L)
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Unopette
• Self-contained reservoir and 20 L pipet
• Ammonium oxalate diluent
• DF = 100
• Count leukocytes in all 9 large squares
• Special formula to facilitate calculation
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Unopette
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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
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Compound Microscope
• Ocular (adjustable binocular)
• Mechanical stage
• Condenser
• Objectives (parfocal)
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Care of Microscopes
• Carry with care
• Protect long objective lens
• Clean and disinfect stage
• Wipe oil off OIL
• Save lamp life