Differential counting of living and dead cells of bacteria

Abstract

By means of the procedure here described it is possible to determine with considerable accuracy the number of bacterial cells in a suspension and at the same time to determine the size and form of the cells and to differentiate the living and dead cells. The method makes use of the principle of the Breed and Brew 1 method of counting bacteria in milk, and the negative staining method of Benians. 2

A measured quantity of bacterial suspension is mixed thoroughly with an equal quantity of 2 per cent. aqueous Congo red solution; the mixture is allowed to stand ten minutes. After again shaking the mixture, 0.01 c.c. is removed by means of a capillary pipette of that capacity and discharged on to a clean slide which has been clamped to the table over a piece of white paper on which a 2 cm. square has been ruled. By means of a stiff wire the drop of liquid is spread as evenly as possible over this area. After it has become thoroughly dry the slide is immersed a moment in a 1 per cent. solution of hydrochloric acid in 95 per cent. alcohol. This turns the dye blue and also fixes the film. If covered with a layer of cedar oil the slides will keep indefinitely, but if exposed to the air they fade considerably.

Cells which were alive at the time of staining are unstained and appear as white spots on a blue ground. While the cells themselves may shrink considerably after fixation and drying, a number of comparative measurements have shown that the clear space in the film faithfully reproduces the size and form of the living wet cells. With favorable material flagella may be demonstrated by this stain.