A Novel Algorithm for the Determination of Bacterial Cell Volumes That is Unbiased by Cell Morphology

Abstract

Abstract:The determination of cell volumes and biomass offers a means of comparing the standing stocks of auto-and heterotrophic microbes of vastly different sizes for applications including the assessment of the flux of organic carbon within aquatic ecosystems. Conclusions about the importance of particular genotypes within microbial communities~e.g., of filamentous bacteria! may strongly depend on whether their contribution to total abundance or to biomass is regarded. Fluorescence microscopy and image analysis are suitable tools for determining bacterial biomass that moreover hold the potential to replace labor-intensive manual measurements by fully automated approaches. However, the current approaches to calculate bacterial cell volumes from digital images are intrinsically biased by the models that are used to approximate the morphology of the cells. Therefore, we developed a generic contour based algorithm to reconstruct the volumes of prokaryotic cells from two-dimensional representations~i.e., microscopic images! irrespective of their shape. Geometric models of commonly encountered bacterial morphotypes were used to verify the algorithm and to compare its performance with previously described approaches. The algorithm is embedded in a freely available computer program that is able to process both raw~8-bit grayscale! and thresholded~binary! images in a fully automated manner.

Abundance of Common Aerobic Anoxygenic Phototrophic Bacteria in a Coastal Aquaculture Area

“…Since the cell in the present data set were almost spherical or short rods, cell volume was calculated as a spherical shape using one-half of the cell length, r , and the following equation: V = 4/3 × π × r 3 . For calculation of cell volume as a rod, the calculation value is estimated to be 3.3 times less than that calculated as a sphere (Zeder et al, 2011). Although these methods can detect Anoxic-PB also, as mentioned above, our sampling sites and depth do not contain bottom materials brought up from bottom, and microscopic observation did not show large particles where Anoxic-PB would present.…”

Comparison of bacterial communities on limnic versus coastal marine particles reveals profound differences in colonization

“…Eight samples (i.e. stained membrane filter pieces) were processed per run with an average speed of 4.7 positions per minute, including nested autofocusing, z‐stack acquisition and extended depth of field imaging (Zeder and Pernthaler, ; Zeder et al ., ) on each position. Image acquisition of 2882 limnic and marine microbial particles (1405 and 1477 respectively) was performed on the same microscope, but running the MPISYS in a semi‐automated mode – as fields of view had to be selected manually (as detailed in Supporting Information http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12466/suppinfo).…”

Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

“…Triplicate filters from water depths of 20, 150, 400, 550, and 700 m were stained with 4=,6-diamidino-2-phenylindole (DAPI) subsequently to the CARD-FISH procedure, and the DAPI and CARD-FISH counts were done with a Zeiss AxioImager.Z2 microscope (Carl Zeiss AG, Jena, Germany) with an automated stage. Image acquisition was done using the software package AxioVision, release 7.6 (Carl Zeiss AG, Jena, Germany), comprising an automated focusing routine, an automated sample area definition using the macro MPISYS, and a manual image quality assessment (22)(23)(24). The software took pictures of the DAPI channel (350 nm) and the FISH channel (488 nm) at different fields of view along a track on the sample.…”