Cell Viability and Cell Counting
introduction
Cell viability refers to the assessment of the number of healthy cells in a sample. This evaluation is a vital indicator for understanding the mechanisms of gene, protein, and pathway functions involved in cell survival or death after exposure to toxic agents. Cell viability is typically determined by measuring the cells' ability to exclude vital dyes such as DAPI. A fraction of the cells that exclude DAPI or show normal nuclear morphology have already lost mitochondrial function and are thus irreversibly committed to death.
Next, we will examine the dyes commonly used in cell viability staining.
Cell Viability Staining
The dyes used in viability staining differentiate between live and dead bacterial cells in a sample. The differentiation of these fluorescent dyes is based on the integrity of the cell's plasma membrane. A red and a green dye are added to the sample. The green dye permeates all cells (both live and dead), while the red dye, which contains propidium iodide, only penetrates cells whose membranes are no longer intact (and are thus dead). Therefore, cells that retain the green dye are alive, while cells that take up the red dye are dead.
Fluorescent Staining Using DAPI
DAPI is a common dye for counting bacteria or cells. It is a non-permeable DNA dye that is largely excluded by the plasma membranes of live cells. However, it can enter damaged membranes, where it interacts with the cell's DNA. The dye's full name is (4′, 6-diamidino-2-phenylindole). DAPI is a fluorescent dye that stains nucleic acids and reacts relatively weakly with inert and non-biological materials. Therefore, it can be useful in distinguishing between living and non-living components in a sample. Cells stained with DAPI appear blue under a fluorescence microscope. However, it is important to note that this dye is non-specific, meaning it stains all cells and does not differentiate between live and dead cells or different species or organisms.
Fluorescent Antibodies for Viability Staining
Fluorescent antibodies can be used to identify or track a specific organism within a complex or diverse microbial population. Specific antibodies are developed in the laboratory to bind with particular organisms. The creation of these antibodies is often time-consuming and costly, but they can be an important tool in cell viability staining.
Green Fluorescent Protein (GFP)
This type of staining involves introducing a gene encoding a green fluorescent protein (GFP) into the genome of a bacterium through genetic engineering. When the sample is observed under ultraviolet microscopy, cells labeled with GFP will fluoresce green. However, GFP cannot be used in the study of natural populations because it must be integrated into the genome. Nonetheless, GFP-labeled cells can be introduced into an environment and monitored over time as the GFP is passed on to offspring.
Limitations of Viability Staining
While viability staining is useful in the laboratory for gaining insight into the total number of bacterial cells in an environment, it has several limitations. Some key limitations include:
Observing small cells can be challenging even with a microscope. These cells may be overlooked or hidden behind non-living particles in a sample.
Quantifying a large number of cells may be difficult and may require dilution of the sample.
Counting cell aggregates or cells attached to substrates can be challenging, as individual cells cannot be distinguished from each other in a cell mass.
In natural samples stained for viability, distinguishing between dead cells and non-living material can be difficult.
These dyes cannot differentiate between species, thus they cannot measure species diversity in a mixed sample.
Cell Counting
Cell counting refers to counting or measuring the number of cells, usually performed using a hemocytometer (Neubauer chamber) or a cell counter. This method is used in the biological sciences, including medical diagnostics and treatments. Cell counting is a subset of flow cytometry and has applications in research and clinical laboratories.
To plate cells into multi-well plates, a process known as seeding, it is necessary first to count the cells of interest. Additionally, to calculate the percentage of viability, counting is required, which will be discussed in this article.
The following method provides general instructions on how to use a Neubauer chamber:
First, the attached cells should be trypsinized as done in cell passage, and after centrifugation, the supernatant should be discarded. The cell pellet should be resuspended in one milliliter of complete culture medium.
Clean the Neubauer chamber with alcohol and place a coverslip on it.
Take 10 microliters of the cell suspension (pipette well to ensure uniformity) and gently transfer it to the space between the coverslip and the chamber. Allow it to fill the entire area under the coverslip through capillary action.
Place the Neubauer chamber under an inverted microscope. Each side of the chamber contains 9 large squares, and the method for counting cells within these squares may vary. Generally, it is done by counting 4 corner squares, ensuring to count them in order so as not to forget which ones have been counted. For cells that are located on the edges of the squares, a conventional rule is applied: only count the upper and left edges (if starting from the upper left square). Count the cells carefully.
After counting the squares, take the average of the numbers, and then calculate the total number of cells as follows.
Calculation of Cell Concentration
Cell concentration is calculated as follows:
Cell Concentration = Average Number of Live Cells per Square × 10^4
Another method involves the use of trypan blue. This method is employed when we need to perform a precise viability calculation or when the cell count is high. The following method allows you to accurately determine cellular viability.
Cell Viability is equal to the number of live cells divided by the total number of counted cells. (If cells completely absorb trypan blue, they are considered dead.)
Steps to Perform the Procedure:
Prepare a 0.4% trypan blue solution in isotonic saline buffer at pH 7.2 to 7.3.
Place 10 microliters of the cell suspension into a new Falcon tube and add 10 microliters of 0.4% trypan blue to it. Gently pipette to mix thoroughly.
Using a pipette, transfer 10 microliters of the cell suspension mixed with trypan blue to the space between the coverslip and the slide, allowing it to fill the entire area underneath via capillary action.
Place the Neubauer chamber under an inverted microscope. Each side of the chamber consists of 9 large squares, and the method for counting cells within these squares may vary. We typically count the 4 corner squares, ensuring to count them in order to avoid forgetting which ones have been counted. For cells located on the edges of the squares, a convention is established: only count the upper and left edges (if starting from the upper left square). Count the cells carefully.
Live cells will have a colored membrane due to trypan blue, while dead cells will either be fragmented or have blue staining penetrating their cytoplasm.
Count the number of blue-stained cells and the total number of cells, and calculate as follows:
Diluition Factor = 2
Cell Concentration = Average Number of Cells per Square × Dilution Factor × 10^4
Percentage of Live Cells = (Number of Live Cells / Total Number of Cells) × 100
Consequently, using these methods, we can calculate both cell counting and cellular viability.