Dec 16, 2021
Version 2
Use of haemocytometer to quantify concentration of cells' suspensions V.2
- 1University of Alberta
- Plant pathology
Protocol Citation: abotero 2021. Use of haemocytometer to quantify concentration of cells' suspensions. protocols.io https://dx.doi.org/10.17504/protocols.io.b2yvqfw6Version created by abotero
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 16, 2021
Last Modified: December 16, 2021
Protocol Integer ID: 56053
Keywords: Hemocytometer
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Abstract
This protocol presents the procedures to estimate the concentration of cells' suspensions using an hemocytometer
Guidelines
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.
Materials
Hemocytometer
Microscope
Vortex (optional)
Micropipette 10-100µl
Pipette tips
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
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
Count all the squares following a zig-zag
Expected result
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
Estimation of the concentration
Note
According to the cell's size cells can be counted on squares 1, 2 or 3
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 cells*10,000/Number of squares counted*dilution
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 cells*160,000/Number of squares counted*dilution
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 cells*250,000/Number of squares counted*dilution