Vol. 45, No. 5 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1983, P. 1651-1658 0099-2240/83/051651-08$02.00/0 Copyright C 1983, American Society for Microbiology Comparison Between Direct Methods for Determination of Microbial Cell Volume: Electron Microscopy and Electronic Particle Sizing E. MONTESINOS,* I. ESTEVE, AND R. GUERRERO Department of Microbiology and Institute for Fundamental Biology, Autonomous University of Barcelona, Bellaterra (Barcelona), Spain Received 2 November 1982/Accepted 3 March 1983 Size frequency distributions of different phototrophic and heterotrophic micro- organisms were determined by means of scanning and transmission electron microscopy and electronic particle sizing. Statistically significant differences existed among the three techniques used in this study. Cells processed for electron microscopy showed lower mean cellular volumes than those processed for electronic particle sizing, reflecting a shrinkage by factors ranging from 1.1 to 6.2 (mean, 2.3). Processing of cells for scanning electron microscopy caused higher shrinkage than processing for transmission electron microscopy. Shrinkage was dependent neither on the size nor on the cell wall type of the microorganism. When processed for scanning electron microscopy, phototrophic bacteria were strongly shrunken, whereas heterotrophic microorganisms were less affected. A direct relationship existed among phototrophic bacteria between percentage of shrinkage and specific pigment content. This was probably a consequence of the pigment extraction by organic solvents during the dehydration process, previous to the critical point drying, necessary to examine the specimens under the scanning electron microscope. To understand the role of planktonic bacteria in natural ecosystems, an estimate of their bio- mass is of fundamental importance. Biochemical methods have been proposed to assess biomass (13, 23), but these indirect techniques have many interferences and require standardization, and results are difficult to interpret when mixed populations of algae and other eucaryotic micro- organisms coexist with bacteria. Planktonic bacterial biomass has been calcu- lated from direct microscopic examination of total numbers and average cell volume. The latter has been mainly measured by means of microscopic methods, including phase contrast (16, 18), epifluorescence (9, 10), and transmis- sion (23) and scanning electron microscopy (TEM and SEM, respectively) (4, 10, 15, 25). Although microscopy is time-consuming, much information can be obtained because it permits an assessment of the relative proportion of different microorganisms on the basis of their size and morphology. This advantage is of espe- cial relevance when studying natural communi- ties of phototrophic bacteria, which are fre- quently composed of coexisting purple and green sulfur bacteria, nonphototrophic microor- ganisms, and algae and zooplankton (3, 11), which can be separately quantified by means of the microscope. SEM is one of the most widely used methods for determination of bacterial biomass because of its high resolution and has been recently adapted to digitizer-microcomputer systems (14), thus reducing the time consumed in the analysis. But SEM may seriously underestimate cell size and, as a consequence, biomass, be- cause the drying steps during specimen prepara- tion can cause significant shrinkage in both eucaryotic tissues (5, 12) and bacterial cells (10, 21). There is considerable disagreement about this point among microscopists, and there is a need for more definitive work to clarify it. The objective of the present study was to compare the apparent size frequency distribu- tions of different microorganisms in pure cul- tures and field samples by using SEM, TEM, and electronic particle sizing with the Coulter counter (CC). We were mainly interested in the physiological ecology of phototrophic bacteria. Therefore, the study includes phototrophic bac- teria as an important part. MATERIAS AND METHODS Microorganisms and growth conditions. Phototroph- ic bacteria were cultivated in Pfennig medium (2) supplemented with sodium acetate (0.1%). Chroma- tium minus, Chromatium vinosum, Thiocapsa roseo- persicina, and Rhodopseudomonas capsulata were isolated from field samples of Lakes Ciso and Vilar 1651 on August 31, 2015 by guest http://aem.asm.org/ Downloaded from