A Microscopic Method of Distinguishing Dead from Living Bacterial Cells

Abstract

This paper is concerned with the use of neutral red in distinguishing, microscopically, between dead and living cells. The technique of microcultures was used to study the relation between the viability of a cell and its ability to stain in whole or in part. Cells of Escherichia coli, Schizosaccharomyces pombe and a yeast isolated from ale can be considered dead whenever the cytoplasm proper is tinged, even slightly, with stain. Yeast cells with stained vacuoles and unstained cytoplasm are weakened, sick cells that may or may not recover. In a culture, there is a small number of cells that are not stained and look healthy, but are unable to multiply. Other cells do not stain because their autolysis has progressed too far; these look disorganized, have a low refractive index and are easy to recognize. Their number is usually small. When comparisons are made between numbers of viable cells in cultures of E. coli as obtained microscopically, using neutral red as an indicator of viability, and numbers of viable cells as given by plating, a close agreement between the 2 is found when groups, instead of single cells, are taken into consideration. The concentration of neutral red varies with the organisms. In the case of E. coli, a concn. of 0.005% was adopted. This concn. is harmless even to the youngest and most sensitive cells, and the organism grows readily in broth containing neutral red in that concn.