Use of haemocytometer to quantify concentration of cells' suspensions

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Published: 2021-12-17 DOI: 10.17504/protocols.io.b2yvqfw6

Hemocytometer

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Abstract

This protocol presents the procedures to estimate the concentration of cells' suspensions using an hemocytometer

Steps

Wash the hemocytometer with a washer bottle and dry it using a paper towel

Cover the hemocytometer with the coverslip and place it on a flat surface

Vortex briefly the cell suspension whose concentration will be calculated

Using a micropipette collect 10-100 µl of the previously agitated suspension

Place the micropipette tip on the hemocytometer edge near the coverslip

Empty the spore suspension slowly into the chamber of the hemocytometer

Place the hemocytometer in the microscope's mechanical stage and secure it using the stage clip

Turn on the microscope and focus on the hemocytometer

Look out for the first square to be counted

10.

Count the number of cells on each square

Note

If the cells are touching the upper or the left lines of the square, cells are to be counted, but if they are touching the lower or right line they should not be counted

11.

Count all the squares following a zig-zag

Citation

12.

Write down the number of cells on each square

Note

The lower limit for accurate counting of cells in a hemocytometer is usually considered to be 2.5 x 105/ml. For cell numbers greater than 2.5 x 106/ml, it is generally recommended that the sample be diluted.

Note

Count the number of cells on at least five squares. Ideally, repeat the procedure twice for higher accuracy

13.

Estimation of the concentration

Note

According to the cell's size cells can be counted on squares 1, 2 or 3

13.1.

If cells were counted on squares of size 1

Concentration (cells/ml)= Number of counted cells*10,000/Number of squares counted

Note

If the cells' suspension has been diluted the formula should be adjusted to: Concentration (cells/ml)= Number of counted cells10,000/Number of squares counteddilution

13.2.

If cells were counted on squares of size 2

Concentration (cells/ml)= Number of counted cells*160,000/Number of squares counted

Note

If the cells' suspension has been diluted the formula should be adjusted to:Concentration (cells/ml)= Number of counted cells160,000/Number of squares counteddilution

13.3.

If cells were counted on squares of size 3

Concentration (cells/ml)= Number of counted cells*250,000/Number of squares counted

Note

This square size is typically used to count Plasmodiophora brassicae resting spores

Note

If the cells' suspension has been diluted the formula should be adjusted to:Concentration (cells/ml)= Number of counted cells250,000/Number of squares counteddilution

Use of haemocytometer to quantify concentration of cells&#39; suspensions

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Abstract

Steps

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Use of haemocytometer to quantify concentration of cells' suspensions