Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models

Abstract

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).