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Counting Cells

This document provides instructions for counting cells using a Neubauer chamber. The Neubauer chamber contains a grid with squares of a known area (0.0025 mm2) that aid in calculating cell concentrations from cell counts. Cells are counted within delineated squares under 10x magnification and the number of cells is then used to calculate the number of cells per microliter and milliliter based on the known volumes associated with the grid squares and chamber depth. An example calculation is shown where cells counted in several squares are averaged and converted to an estimated 18,250 cells per microliter for the given sample.

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Athanasios S. Arampatzis
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336 views3 pages

Counting Cells

This document provides instructions for counting cells using a Neubauer chamber. The Neubauer chamber contains a grid with squares of a known area (0.0025 mm2) that aid in calculating cell concentrations from cell counts. Cells are counted within delineated squares under 10x magnification and the number of cells is then used to calculate the number of cells per microliter and milliliter based on the known volumes associated with the grid squares and chamber depth. An example calculation is shown where cells counted in several squares are averaged and converted to an estimated 18,250 cells per microliter for the given sample.

Uploaded by

Athanasios S. Arampatzis
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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How to count cells with a Neubauer chamber

Rafael Martinez 14072011

This example is using a Neubauer chamber Bürker, 0.100 mm depth, 0.0025 mm 2. The numbers
written on the chamber mean that the space between the chamber and the cover slip is 0.100 mm and
that the smallest square on the grid has an area of 0.0025 mm2.

Clean the Neubauer chamber and the cover slip with 70% EtOH.Load 10µl of sample on the loading
groove. The sample will diffused by capillarity.

With the microscope, using a 4x objective, identify the nine main squares of the counting chamber
delimited by three lines each as shown in the following image:

Now change to 10x objective and focus one of the 9 main squares. The counting is performed in the
area delimited by three lines. Cells that touch the upper and left border are counted (black color) whilst
cells that touch the right and lower border are not counted (red color).
Perform the procedure in four main squares. The ideal number of cells must be between 30 and 70. If
the number is too high consider to make a dilution.

Calculation:

The smallest square has an area of 0.0025 mm2 therefore each main square has an area of 0.04 mm2
(0.0025 x 16 = 0.04).

The depth of the chamber is 0.100 mm (space between the glass slide and cover slip) then the volume is
calculated as:

(0.04) x (0.100) = 0.004 mm3 = 0.004 µl

To calculate the amount of cells in 1µl a rule of three is applied. If we have X amount of cells in
0.004µl, how many cells are in 1µl?:

# cells 0.004 µl

? 1µl

Then:
Cells in 1µl = (number of cells in a main square)(1µl) /0.004

Cells in 1 ml = (cells in 1µl) x (1000)


Example:

Number of cells in square 1 = 75; square 2 = 70; square 3 = 72; square 4 = 75. Average = 73.
If there are 73 cells in 0.004 µl, how many will be in 1 µl?

73 0.004 µl

? 1µl

Then:
cells in 1 µl = (73)(1)/0.004

Shorter:
cells in 1 µl = 73/0.004

Answer:
cells in 1 µl = 18250

Cells in 1 ml:
cells in 1 ml = (18250)(1000)

cells in 1 ml = 18 250 000

Sources:

http://www.lo-laboroptik.de/englisch/info/info.html
http://evolution.unibas.ch/ebert/lab/counting.htm#a4

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