**3.1 Applications of cell viability assessment**

Cell viability assessment has a wide range of applications in biomedical research and drug development. Some key applications include:

• Evaluating the effect of drug candidates on cells: Cell viability assays can be used to determine the cytotoxic potential of new drug candidates, providing valuable information on their safety profile and aiding in the selection of promising candidates for further development.


### **3.2 Methods for determining cell viability**

There are numerous methods available for determining cell viability, each with its advantages and limitations. Some of the most commonly used methods include:

*Dilution:* A simple technique in which the number of viable cells is estimated based on their ability to proliferate in a diluted environment. The dilution method involves serially diluting a cell suspension and then assessing cell growth in each dilution. Viable cells will continue to grow and divide, whereas non-viable cells will not proliferate. By comparing cell growth in each dilution, researchers can estimate the percentage of viable cells in the original sample. Although this method is simple and relatively easy to perform, it may not be suitable for all cell types or experimental conditions and may be less sensitive than other methods.

*Surface viable count:* This method involves counting the number of viable cells on a solid surface, such as a culture dish, after exposure to the test substance. The surface viable count method is based on the ability of viable cells to grow and form colonies on a solid surface, such as a culture dish or agar plate. After incubating the cells with the test substance, the viable cells are plated onto the surface and allowed to grow for a specified period. Researchers then count the number of colonies formed, which is proportional to the number of viable cells in the sample. This method is relatively simple and can provide accurate results, but it may not be suitable for non-adherent cell types or slow-growing cells.

*Roll tube:* A technique in which viable cells are embedded in a semi-solid agar medium and incubated for a specified period, allowing for the observation of cell growth and viability. In the roll tube method, viable cells are mixed with a semi-solid agar medium and poured into a glass tube, which is then rolled to create a thin layer of agar containing the cells. The tube is incubated, allowing viable cells to grow and form visible colonies within the agar. By counting the colonies, researchers can determine the number of viable cells in the sample. This method is useful for detecting slowgrowing or fastidious cells but can be more labor-intensive and time-consuming than other methods.

*Nalidixic acid:* This method uses nalidixic acid to selectively inhibit the growth of nonviable cells, enabling the determination of viable cell counts. The nalidixic acid method involves selectively inhibiting the growth of nonviable cells by incorporating the antibiotic nalidixic acid into the culture medium. Viable cells will continue to grow in the presence of the antibiotic, whereas nonviable cells will not. By comparing cell growth in the presence and absence of nalidixic acid, researchers can estimate the percentage of viable cells in the sample. This method can be highly specific but may not be suitable for all cell types, as some cells may be resistant or sensitive to nalidixic acid.

*Fluorogenic dye:* Fluorescent dyes, such as calcein-AM or propidium iodide, can be used to stain live or dead cells, respectively, allowing for the quantification of viable cells using fluorescence microscopy or flow cytometry. Fluorogenic dyes are molecules that emit fluorescence when bound to specific cellular structures or molecules. For cell viability assessment, researchers often use two different dyes: one that selectively stains live cells and another that selectively stains dead cells. By measuring the fluorescence intensity of each dye in the sample, researchers can determine the proportion of live and dead cells. This method is highly sensitive and can provide rapid results but may be affected by factors such as dye penetration, cell autofluorescence, and photobleaching.

*Trypan Blue cell viability assay:* A widely used method that involves the use of the Trypan Blue dye, which selectively stains dead cells, allowing for the estimation of viable cell counts using a hemocytometer or automated cell counter. The Trypan Blue cell viability assay is a widely used and straightforward method for determining cell viability. The Trypan Blue dye selectively stains dead cells with compromised membrane integrity, while live cells with intact membranes remain unstained. After incubating the cells with the dye, researchers count the number of stained (dead) and unstained (viable) cells using a hemocytometer or automated cell counter. This method is relatively simple and quick, but it may not provide accurate results for certain cell types or experimental conditions, such as when cell membrane integrity is temporarily altered or when cell autofluorescence interferes with the detection of the dye.

The diverse methods available for cell viability assessment offer a range of options to evaluate the impact of various substances on cellular health. Each method has its advantages and limitations, and the choice of the most suitable method depends on factors such as the cell type, experimental conditions, and desired level of sensitivity and specificity.
