The images below were obtained in an experiment to enumerate leukocytes in a blood sample using Neubauer counting chamber.

The following solution was prepared and added to the Neubauer chamber and viewed under a compound microscope:

100 microlitres of acetic acid (acidic reagent to haemolyze RBCs) + 50 microlitre of Leishman's stain + 1-2 drops of blood.

Are these granular transparent circular objects in the images the leukocytes? If yes, do these include the smaller ones too? I am unable to decide which ones to take into account.

Moreover is this count normal (for instance, I counted around 250 (excluding the very small ones and applying the margin rule) in the top left box of the grid (Image 3))?

Bottom right (40X)

Top right (40X)

Top left (40X)

Bottom left (40X)

Full grid

  • $\begingroup$ the amount of blood should be taken quantitatively ,not without measuring, so one or two drops of blood will not give correct calculations . $\endgroup$ – JM97 Feb 28 '17 at 16:44

First I'd make sure all the RBC were gone just by visually inspecting. What should remain are your WBC and platelets at this point, and platelets are tiny so you can probably count anything between 5µm and 25µm. So for me that would include basically everything I can see minus background or debris.

So the problem now is with quantifying your count. You should know what the volume of your blood is, and here's why: What you're doing with a hemocytometer is counting #cells in a given volume, but you also diluted your whole blood with 150µL before you counted. So for example if a drop is ~60µL, and you diluted it with 150µL your dilution factor is 3.5, and it's important because your cell density equation is enter image description here

For reference, you typically end up with 4-10 million leukocytes per mL of blood, however.

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