In this article we will discuss about counting of cells in hemocytometer about why Is Cell Counting Important? and its procedure with example.

## What is Cell Counting?

A growth curve can be created to analyse the growth characteristics of a specific cell type or cell line, from which a population doubling time, lag time, and saturation density can be calculated. The lag phase, which is the time it takes for the cell to recover from subculture, attach, and spread; the log phase, in which the cell number begins to increase exponentially; and the plateau phase, in which the culture becomes confluent and the growth rate slows or stops, are all shown on a growth curve.

An increase in cell quantity is also a common way of determining how hormones, nutrients, and other factors affect a certain cell type. Because it is so broadly defined and impacted by many various factors, such as mitogens, changes in nutrition levels, transport, membrane integrity, attachment factors, and so on, growth, or the increase in total cell number over time, is an excellent indicator of a biological response.

Changing attachment, reducing or lengthening the lag phase, hanging plating efficiency (or survival at subculture), changing the death rate, changing the pace of development through the cell cycle, or changing the plateau density are all examples of factors that might affect cell quantity. A growth curve can be used to distinguish between these impacts.

## Why Counting the Cells?

The most straightforward way to measure cell number and thus cell proliferation is to use a hemocytometer. To reliably and directly quantify and standardize experimental settings, cells can be counted before, during, and after setting up an experiment.

Furthermore, using a dye like trypan blue while performing hemocytometer counts provides the investigator with a quantitative benchmark for the viability of the cells by performing a differential count of cells that do not take up the dye vs those that do in cell count using hemocytometer.

The hemocytometer is clearly the least expensive and time-consuming way for counting cells, but it can produce data that is as accurate as any other approach and can examine both total and viable cell count using hemocytometer. This makes cell count using hemocytometer an excellent choice for student labs or laboratories where cell counts aren’t done frequently.

## Needs for Cell Counting

- Trypsinized cell cultures
- Neubauer hemacytometer
- Coverslip
- Tally counter
- 0.4% trypan blue in PBS
- Pasteur pipettes
- Light Microscope

**Figure 1: **Counting of cells in hemocytometer.

## Cell Counting Procedure

- Use 0.4 percent trypan blue to make a 1:5 dilution of the cell suspension. If required, dilute further with PBS and carefully resuspend with a Pasteur pipette.
- Place a drop of the Pasteur pipette suspension at the edge of the “V” form on the hemacytometer chamber and cover it with the coverslip. Repeat on the opposite side of the chamber (Figure 2).
- Place the chamber on the microscope’s stage and use modest (4x) power to concentrate on the etched lines of the chamber.
- The hemocytometer is made up of 25 smaller squares grouped into nine 1-mm squares. One 1-mm square has a volume of 0.1 mm
^{3}. - Focus on one of the 25 smaller squares surrounded on both sides by three parallel lines with a 10X objective (Figure 1). Count all the cells that aren’t dyed for a viable cell number.
- Count the blue cells and those that aren’t dyed separately from the overall cell number, viable cell number, and percent viability.
- Make a point of counting cells that are on the top and right, rather than the bottom and left, to avoid counting cells that are on the border.
- At least 100 cells/mm
^{2}must be counted. If the square has fewer than 100 cells, count one or more squares and repeat for the other side of the chamber. - Multiply the number of cells counted in a 1 mm square (or the average of whichever many squares you counted) by 10
^{4}to get the number of cells/ml.

**Figure 2: **Counting cells using a hemocytometer and trypan blue. Viable cells contain intact cell membranes and do not uptake trypan blue, appearing bright/clear in the hemocytometer. Dead cells have damaged cell membranes and uptake trypan blue, appearing blue in the hemocytometer. Cell viability can be estimated by taking the ratio of live/dead cells.

## Note

It is important not to overfill or under fill, but rather to allow the drop to be drawn over the surface by capillary action.

## Cell Counting Examples

viable cells/mL

If the cell counts for each of the 16 squares were 50, 40, 45, 52, the average cell count using hemocytometer would be:

(50 + 40 + 45 +52) ÷ 4 = 46.75

75 x 10,000 (10^{4}) = 467,500

467,500 x 5 = 2,337,500 live cells/mL in original cell suspension.

To calculate viability

Live cell count: 2,337,500 cells/mL

Dead cell count: 50,000 cells/mL

2,337,500 + 50,000 = 2,387,500 cells

2,337,500 ÷2,387,500 = 97.9% viability