Question

Describe the principle of Trypan-Blue and cell counting formulas.

Answer 1

ans -

The Trypan Blue dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye.Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color .Add 0.1 mL of trypan blue stock solution to 0.1 mL of cells. Load a hemacytometer and examine immediately under a microscope at low magnification. Count the number of blue staining cells and the number of total cells. Cell viability should be at least 95% for healthy log-phase cultures.

The no. of cell can be calculated by divide the live cell count by the total cell count to calculate the percentage.