Question

List different methods of measuring cell growth and explain working principle of any two methods

Answer 1

The main methods for cell growth measurement are of 2 types direct and indirect, the direct cell counting methods include,

1. Manual cell counting

2. Automated mechanical counting

Indirect cell counting methods includes,

1. BACTEC MGIT method

2. Turbidity assay by spectrophotometry

3. Fluorescent dye method

4. EZMTT dye-based cell proliferation analysis

5. High-content analysis

6. Raman spectroscopy analysis

7. Electrical impedance technology

* Manual cell counting.

Traditionally, cell numbers are counted by taking an aliquot of a homogenous cell suspension and plating on a hemocytometer to count the numbers under a light microscope. The obtained cell number in a certain volume of the suspension is then converted into the cell concentration (cells per ml) in the stock solution. Bacteria are counted by a Petroff-Hausser bacterial counter, a Hawksley counter, and/or the plate colony formation method. The plate colony counting method often gives a lower cell number than the actual value, because it is often difficult to disperse bacteria into a single cell and to make sure that a single colony is not derived from several bacteria.

* Turbidity assay by spectrophotometry

Turbidity can be observed when the cell density reaches certain level; within a certain range, the number of cells is proportional to the turbidity of the bacterial culture. The cell turbidity is measured by a spectrophotometer or a colorimeter, and a standard curve is generated by plotting the absorbance at OD600nm and the actual cell numbers in the sample. Photoelectric turbidimetric counting is a simple, rapid, and continuous measurement suitable for high-throughput screening. However, its optical density is less sensitive, cannot differentiate between dead or live bacteria, and is greatly affected by cell size, morphology, and the color of the culture solution.

* Fluorescent dye method

Live or dead cells that cannot be differentiated by the light microscope can be counted after fluorescent labeling. SYTO series nucleic acid fluorescent dyes, etc. stain the DNA or RNA of the live or dead cells; PI nor SYTOX Green nucleic acid dyes cannot transfer into the live cells and stain the DNA of the damaged cell membranes. Both types of dyes can be used in combination to measure the ratio of live and dead cells. After labeling, the cells can be detected by a fluorescence microscopy or by a flow cytometry. The combination of fluorescent dyes and advanced instruments makes it possible to realize the “visualization” and investigate the mechanisms of action under the physiological and pathological conditions and to explain the significance of life effects, which is of great significance in the field of disease diagnosis and drug screening.