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

2022 ◽  
Vol 12 ◽  
Author(s):  
Jörg S. Deutzmann ◽  
Grace Callander ◽  
Wenyu Gu ◽  
Albert L. Müller ◽  
Alexandra L. McCully ◽  
...  

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.

2019 ◽  
Author(s):  
David C. Vuono ◽  
Bruce Lipp ◽  
Carl Staub ◽  
Evan Loney ◽  
Joseph J. Grzymski

AbstractBatch cultures are a low maintenance and routine culturing method used in anaerobic microbiology. Automated tools that measure growth curves from anaerobic microorganisms grown in traditional laboratory glassware, such as Balch-type tubes, are not commercially available. Here we present a new MicrobiAl Growth Intervalometer (MAGI) that captures microbial growth curves through photo conductivity of the medium using a diffused light pattern of specified frequency, rather than photo-attenuation of collimated light used in traditional systems, and is configured with an offset photodetector/emitter to minimize direct impingement of light from the source to improve the resolution of the solution’s density. MAGI is operated by software-driven automation and offers investigators a low noise/high gain instrument with capabilities for remote visualization and data acquisition. MAGI is a low maintenance, low cost, and robust platform primarily for anaerobic cultivation and growth monitoring. We demonstrate the utility of this device by first showing that growth rates and generation times inEscherichia coliK-12 are reproducible to previously published results. We then tested MAGI to measure growth curves of an environmental organism,Intrasporangium calvum, under various media compositions. Our results demonstrate that MAGI is a versatile platform to measure growth curves in media under various redox conditions (microaerobic and anaerobic), complex mediums such as Luria-Bertani (LB) broth and minimal media, and for resolving diauxic growth curves whenI. calvumis grown on a disaccharide. Lastly, we demonstrate that the device can resolve growth curves for μM concentrations of resources that yield low biomass. This research advances the tools available to microbiologist aiming to monitor growth curves in a variety of disciplines, such as environmental microbiology, clinical microbiology, and food sciences.


2007 ◽  
Vol 1064 ◽  
Author(s):  
Somesree GhoshMitra ◽  
Tong Cai ◽  
Santaneel Ghosh ◽  
Arup Neogi ◽  
Zhibing Hu ◽  
...  

ABSTRACTQuantum dots (QDs) are now used extensively for labeling in biomedical research due to their unique photoluminescence behavior, involving size-tunable emission color, a narrow and symmetric emission profile and a broad excitation range [1]. Uncoated QDs made of CdTe core are toxic to cells because of release of Cd2+ ions into the cellular environment. This problem can be partially solved by encapsulating QDs with polymers, like poly(N-isopropylacrylamide) (PNIPAM) or poly(ethylene glycol) (PEG). Based on biological compatibility, fast response as well as pH, temperature and magnetic field dependent swelling properties, hydrogel nanospheres has become carriers of drugs, fluorescence labels, magnetic particles for hyperthermia applications and particles that have strong optical absorption profiles for optical excitation. The toxicity of uncoated QDs are known; however, there have been a very limited number of studies specially designed to assess thoroughly the toxicity of nanosphere encapsulated QDs against QD density and dosing level.In this work, we present preliminary studies of biological effects of a novel QD based nanomaterial system on Escherichia coli (E. coli) bacteria. Cadmium chalcogenide QDs provide the most attractive fluorescence labels in comparison with routine dyes or metal complexes. Nanospheres on the other hand are the most commonly used carriers of fluorescence labels for fluorescence detection. The integration of fluorescent QDs in nanospheres therefore provides a new generation of fluorescence markers for biological assays. Hydrogels based on PNIPAM is a well known thermoresponsive polymer that undergoes a volume phase transition across the low critical solution (LCST) [2]. Therefore, the inherent temperature-sensitive swelling properties of PNIPAM offer the potentiality to control QD density within the nanospheres. In the present work, E. coli growth was monitored as E. coli served as a representation of how cells might respond in the presence of hydrogel encapsulated QDs in their growth environment. The present work describes the successful encapsulation of CdTe QDs in PNIPAM gel network. Microgel encapsulated QDs were synthesized by first preparing PNIPAM microspheres with cystaminebisacrylamide as a crosslinker and CdTe QDs capped with a stabilizer. The CdTe QDs were bonded into PNIPAM microgels through the replacement of CdTe's stabilizer inside PNIPAM microspheres. Growth curves were generated for E. coli growing in 20 mL of LB media containing hydrogel encapsulated QD nanospheres (400 nm diameter) at relatively higher (0.5mg/mL) and lower (0.01mg/mL) concentration of solution. From the growth curves, there was no evidence at lower concentration (0.01mg/mL) that the hydrogel encapsulated QDs prevent the microbial cells from growing but at higher concentration (0.5mg/mL), microbial growth was inhibited. Transmission Electron Microscopy (TEM) was used to characterize QD size and density inside the hydrogel nanospheres. Scanning Electron Microscopy (SEM) was used to observe size and morphology of the hydrogel particles. Further investigation is going on cell growth response at different QD density and to evaluate the limiting hydrogel concentration for different QD densities.


2021 ◽  
Author(s):  
Milanka Radulovic ◽  
◽  
Svetlana Mitrovski

Peat is a natural substrate for growth of microorganisms because it is rich in compounds that microorganisms can use as sources of carbon, nitrogen and growth factors. Peat originating from Vlasina lake in Eastern Serbia is especially rich in organic matter. The content of humic substances (humic acid, fulvic acid and humine) is almost twice that found in other peat-rich regions of similar origin and geochemical age. Humic and fluvic acids are known to promote microbial growth. In this work, humic and fulvic acids were first extracted from Vlasina lake peat and then added to minimal medium (synthetic, low ionic strength medium). The humic substances were added separately and combined in a 1:1 ratio by mass to study their individual and combined effect on microbial growth of Escherichia coli ATCC 25922 (Gr–), Staphyloccocus aureus (Gr+) i Aureobasidium pullulans, strain CH-1. The microbial growth was measured microspectrophotometrically over a 24-hour period and growth curves were obtained for a range of acid concentrations between 25 µg cm-3 and 100 µg cm-3. It was found that both humic and fulvic acids promote the growth of all three microorganisms by up to a maximum of 40%-80% the extent of which varied with the concentration of the acid and the identity of the microorganism. In general, humic acid was found to result in higher microbial growth (at highest concentrations, up to ~80% for all three microbial species).


2020 ◽  
Vol 35 (3) ◽  
pp. 252-265 ◽  
Author(s):  
V. Nguyen ◽  
J. De Beenhouwer ◽  
J. G. Sanctorum ◽  
S. Van Wassenbergh ◽  
S. Bazrafkan ◽  
...  

2020 ◽  
Vol 8 (5) ◽  
pp. 740
Author(s):  
Anna M. Alessi ◽  
Bing Tao ◽  
Wei Zhang ◽  
Yue Zhang ◽  
Sonia Heaven ◽  
...  

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems.


2019 ◽  
Vol 10 ◽  
Author(s):  
David C. Vuono ◽  
Bruce Lipp ◽  
Carl Staub ◽  
Evan Loney ◽  
Zoë R. Harrold ◽  
...  

2007 ◽  
Vol 244 (3) ◽  
pp. 511-517 ◽  
Author(s):  
George T. Yates ◽  
Thomas Smotzer

2019 ◽  
Vol 116 (29) ◽  
pp. 14698-14707 ◽  
Author(s):  
Yoav Ram ◽  
Eynat Dellus-Gur ◽  
Maayan Bibi ◽  
Kedar Karkare ◽  
Uri Obolski ◽  
...  

Determining the fitness of specific microbial genotypes has extensive application in microbial genetics, evolution, and biotechnology. While estimates from growth curves are simple and allow high throughput, they are inaccurate and do not account for interactions between costs and benefits accruing over different parts of a growth cycle. For this reason, pairwise competition experiments are the current “gold standard” for accurate estimation of fitness. However, competition experiments require distinct markers, making them difficult to perform between isolates derived from a common ancestor or between isolates of nonmodel organisms. In addition, competition experiments require that competing strains be grown in the same environment, so they cannot be used to infer the fitness consequence of different environmental perturbations on the same genotype. Finally, competition experiments typically consider only the end-points of a period of competition so that they do not readily provide information on the growth differences that underlie competitive ability. Here, we describe a computational approach for predicting density-dependent microbial growth in a mixed culture utilizing data from monoculture and mixed-culture growth curves. We validate this approach using 2 different experiments withEscherichia coliand demonstrate its application for estimating relative fitness. Our approach provides an effective way to predict growth and infer relative fitness in mixed cultures.


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