Low-Cost Clamp-On Photometers (ClampOD) and Tube Photometers (TubeOD) for Online Cell Density Determination.

Jörg S Deutzmann,Frauke Kracke,Alfred M Spormann,Albert L Müller,Wenyu Gu,Alexandra L Mccully,Jenna Kim Ahn,Grace Callander

doi:10.3389/fmicb.2021.790576

Abstract

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

Highlights

Determination of suspended cell density in a liquid culture is essential in virtually any field of microbiology
Turbidity measurements of a dilution series of fixed E. coli cells showed a linear relationship of ClampOD and TubeOD output [-log10(voltage)] to Optical density (OD) values determined by the commercial cuvette spectrophotometer Ultrospec2100 pro for short path lengths (e.g., Hungate tubes)
When the incident light intensity was reduced to a blank reading of about 90% of the maximum, ClampOD and TubeOD output correlated perfectly with cuvette-based OD measurements over a range from OD = 0 to about OD = 2 (Figure 3B)

Summary

Introduction

Determination of suspended cell density in a liquid culture is essential in virtually any field of microbiology. Turbidity of the sample can be measured as the decrease in light transmission through a sample when other modes of light attenuation, such as absorption by colored substances, are absent (Koch, 1968). Absorption (A) measurements are based on the Beer-Lambert law (A = ε·l·c), and are used to determine the concentration (c) of a substance of known absorptivity (ε) based on the transmitted light through a sample of length l. At low turbidity where the Beer-Lambert law is valid (e.g., OD < 0.3 for most photometers and microbial cultures at 600 nm wavelength), light attenuation is proportional to cell density and the path-length of the light through the turbid solution. The correlation of cell count per volume and OD depends greatly on these properties and calibration for each strain, medium, and growth condition (Koch, 2007; Stevenson et al, 2016) is necessary

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