My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines. During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTap , and share my knowledge on the topic here to help as many people as possible. Once you have counted cells in each of the squares, you perform the hemocytometer calculations based on your total counts, dilution factor, initial volume and desired final density. The volume of a small square is specific to the hemocytometer. It is calculated by multiplying the width by the height which are the same — usually 1mm each by the depth usually 0.
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For an accurate determination, the total number of cells overlying one 1 mm 2 should be between 15 and If the number of cells per 1 mm 2 exceeds 50, dilute the sample and count again. If the number of cells per 1 mm 2 is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on both sides of the hemocytometer 8 x 1 mm 2 areas. Keep a separate count of viable and non-viable cells.
Reincubate the culture and adjust the volume of media according to the confluency of the cells and the appearance of the media. Include cells on top and left touching middle line. The cells touching middle line at bottom and right are not counted. Live cells appear colourless and bright refractile under phase contrast.
Dead cells stain blue and are non-refractile. Cite this Simulator:. Uniform cell suspension. Tally Counter. Cell Suspension. Inverted microscope preferably phase contrast. Place the cell suspension in a suitably-sized conical centrifuge tube. For an accurate cell count to be obtained, a uniform suspension containing single cells is necessary. Pipette the cell suspension up and down in the tube times using a pipette with a small bore 5 ml or 10 ml pipette.
For cells thawed from cryopreservation in 1ml cryopreservation medium , pipette up and down times using a one ml pipette. Prepare a dilution of the cell suspension in trypan blue : Approximately 10 microliters of cell suspension will be required to charge one chamber of the hemocytometer. In a conical microfuge tube, add 10 microliters of 0. Gently swirl finger vortex the cell suspension and remove 10 microliters of it using sterile technique.
Combine the 10 microliters of cell suspension with the 10 microliters of trypan blue in the microfuge tube. Pipette up and down several times to ensure a uniform cell suspension using the same pipette tip and allow to stand for minutes.
Load the hemocytometer : Moisten and affix cover slip to the hemocytometer. Ensure the cover slip and hemocytometer are clean and grease-free use alcohol to clean. The chamber should not be overfilled or underfilled. Determine the number of cells total and viable : View the cells under a microscope at x magnification. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid.
The square should contain 16 smaller squares. Count all the cells in the four 1 mm corner squares. If there are too many or too few cells to count, repeat the procedure, either concentrating or diluting the original suspension as appropriate. Each large square of the hemocytometer, with cover slip in place, represents a total volume of 0. Cleaning the hemocytometer :- Clean the instrument as soon as possible after use. Use protective clothing, gloves and eyewear.
Trypan blue is a mutagen. Air dry. Dispose of trypan blue contaminated articles in biohazard waste. This will not be a valid assumption unless the suspension is monodispersable and free of cell clumps.
Distribution in the hemocytometer chamber depends on the number of particles, rather than particle mass. Thus, cell clumps will distribute in the same way as single cells, distorting the final result. Clumping can be minimized by keeping the suspension in an ice bath in plastic tubes, and by using a diluents without calcium and magnesium.
Always mix thoroughly before sampling. Improper filling of chambers: In order to fill properly by capillary action, a hemocytometer chamber must be very clean and this also applies to the Micro pipette used to fill the chamber.
New pipettes may be dry-heat sterilized. If the cell suspension does not flow in and fill the chamber immediately and smoothly when the drop from the pipette is placed in the depression under the coverslip, the chamber must be dirty and should be recleaned.
Failure to adopt a convention for counting cells in contact with boundary lines or each other: In order to determine what to count and what not to count, concerning a cell on a boarder, you should develop a convention in which you do not count half of the cells that touch a boarder. For example you may decide not to count cells if they touch the bottom or right boarder or the top and left boarder.
You can decide on your own convention but whichever you choose, you MUST be consistent. One should count the cells in the four squares of both the upper and lower chambers for the most accuracy although, in many laboratories, for convenience, only the four squares of one of the two chambers are counted. Use the provided cover glasses: They are thicker than the standard 0.
The height of the fluid is standardized this way. Moving cells: Moving cells, like sperm cells, are difficult to count. The cells should immoblized first. Objective: The hemocytometer is thicker than a regular slide. If the sample is not dissolved well, the cells become crowded and will be difficult to count. If it is too dissolved, the sample size will not be enough to develop strong inferences about the concentration in the original mixture.
Commonly, a rough idea of the concentration must be known before beginning in order to guess an appropriate dilution. Analyze multiple chambers. If the difference is larger, the method of taking the sample may be unreliable. For drying the excess liquid do not use paper wipes. The capillary action that filled the chamber will then dry it out.
After using trypan blue, rinse the hemocytometer with distilled water to remove the dye and allow it to dry. It is not necessary for the tube used for the trypan blue dilution to be sterile.
However, if non-sterile tubes are used, make sure that all pipettes and pipette tips that come in contact with the cell suspension are sterile and that these do not come in contact with the cell suspension once they have been exposed to a non-sterile environment. Count rows or columns. Use the gridlines to help remember which areas' cells have already been counted.
Hemocytometer Counting of Cells.
Cell Counting with a Hemocytometer: Easy as 1, 2, 3
Many biological applications such as microbiology, cell culture, blood work and many others that use cells require that we determine cell concentration for our experiment. Cell counting is rather straightforward and requires a counting chamber called a hemocytometer, a device invented by the 19 th century French anatomist Louis-Charles Malassez to perform blood cell counts. A hemocytometer consists of a thick glass microscope slide with a grid of perpendicular lines etched in the middle. The grid has specified dimensions so that the area covered by the lines is known, which makes it possible to count the number of cells in a specific volume of solution. Before starting ensure that both the hemocytometer and its coverslip are clean by removing any dust particles with lens paper.
Cell Counting with a Hemocytometer
Counting cells using a hemocytometer