Measurement Of Bacterial Growth Rates Research Articles

High-throughput Method for Novel Medium Development for Culture of Anaerobic Gut Bacteria.

Gut microbiota play important roles in the health of their host and detailed investigation of these organisms requires in vitro culture. Culturing strictly anaerobic bacteria can be a challenge as the gut environment they inhabit is nutritionally complex. Use of complex media containing nutritionally rich but undefined gut fluid reduces the accuracy of physiological and metabolomic studies. Here we present a high-throughput protocol for comparing growth rates of fastidiously anaerobic bacteria on different media. These protocols can be used to develop a solid medium made up of commercially sourced ingredients, providing replicable growth conditions for previously uncultured anaerobic bacteria. As many fastidious bacteria grow poorly in a liquid broth, these protocols measure bacterial growth rate on solid media. These protocols speed up and simplify the growth rate measurement process by using a multiwell format and equations in place of physical McFarland standards to calculate approximate cell density. Bacterial strains belonging to the families Erysipelotrichaceae and Lachnospiraceae (phylum Firmicutes) isolated from the hindgut of Kyphosus sydneyanus were used to demonstrate the efficacy of these protocols. Bacterial growth rates were compared between a nutritionally rich medium with gut fluid versus a novel replicable medium with mannitol. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of solid YCFA growth medium Basic Protocol 2: Collection of fish gut samples and plating to single isolates Basic Protocol 3: Genetic identification of single isolates with colony PCR and 16S rRNA gene sequencing Basic Protocol 4: Measurement of bacterial growth rates on solid media.

High temperatures enhance the microbial genetic potential to recycle C and N from necromass in high‐mountain soils

Climate change is strongly affecting high-mountain soils and warming in particular is associated with pronounced changes in microbe-mediated C and N cycling, affecting plant-soil interactions and greenhouse gas balances and therefore feedbacks to global warming. We used shotgun metagenomics to assess changes in microbial community structures, as well as changes in microbial C- and N-cycling potential and stress response genes and we linked these data with changes in soil C and N pools and temperature-dependent measurements of bacterial growth rates. We did so by incubating high-elevation soil from the Swiss Alps at 4°C, 15°C, 25°C, or 35°C for 1month. We found no shift with increasing temperature in the C-substrate-degrader community towards taxa more capable of degrading recalcitrant organic matter. Conversely, at 35°C, we found an increase in genes associated with the degradation and modification of microbial cell walls, together with high bacterial growth rates. Together, these findings suggest that the rapidly growing high-temperature community is fueled by necromass from heat-sensitive taxa. This interpretation was further supported by a shift in the microbial N-cycling potential towards N mineralization and assimilation under higher temperatures, along with reduced potential for conversions among inorganic N forms. Microbial stress-response genes reacted inconsistently to increasing temperature, suggesting that the high-temperature community was not severely stressed by these conditions. Rather, soil microbes were able to acclimate by changing the thermal properties of membranes and cell walls as indicated by an increase in genes involved in membrane and cell wall modifications as well as a shift in the optimum temperature for bacterial growth towards the treatment temperature. Overall, our results suggest that high temperatures, as they may occur with heat waves under global warming, promote a highly active microbial community capable of rapid mineralization of microbial necromass, which may transiently amplify warming effects.

Rapid nondestructive measurement of bacterial cultures with 3D interferometric imaging

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline and is a staple bioanalytical method for efforts ranging from research to standard clinical diagnostic tests. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we demonstrate white-light interferometry (WLI) as a practical tool for accelerated and improved measurement of bacterial cultures. High resolution WLI surface profile imaging was used for nondestructive characterization and counting of bacterial colonies on agar before they became visible to the naked eye. The three-dimensional (3D) morphology of Gram-negative (Pseudomonas fluorescens) and Gram-positive (Bacillus thuringiensis) bacterial species were monitored with WLI over time by collecting surface profiles of colonies on agar plates with high vertical resolution (3–5 nanometers) and large field of view (3–5 mm). This unique combination of sensitive vertical resolution and large field of view uniquely provided by WLI enables measurement of colony morphologies and nondestructive monitoring of hundreds of microcolonies. Individual bacteria were imaged within the first few hours after plating and colonies were accurately counted with results comparing favorably to counts made by traditional methods that require much longer wait times. Nondestructive imaging was used to track single cells multiplying into small colonies and the volume changes over time in these colonies were used to measure their growth rates. Based on the results herein, bioimaging with WLI was demonstrated as a novel rapid bacterial culture assay with several advantageous capabilities. Fast nondestructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates and colony morphology with this method may be beneficial in research and clinical applications where current methods are either too slow or are destructive.

Growth Rates Made Easy

In the 1960s-1980s, determination of bacterial growth rates was an important tool in microbial genetics, biochemistry, molecular biology, and microbial physiology. The exciting technical developments of the 1990s and the 2000s eclipsed that tool; as a result, many investigators today lack experience with growth rate measurements. Recently, investigators in a number of areas have started to use measurements of bacterial growth rates for a variety of purposes. Those measurements have been greatly facilitated by the availability of microwell plate readers that permit the simultaneous measurements on up to 384 different cultures. Only the exponential (logarithmic) portions of the resulting growth curves are useful for determining growth rates, and manual determination of that portion and calculation of growth rates can be tedious for high-throughput purposes. Here, we introduce the program GrowthRates that uses plate reader output files to automatically determine the exponential portion of the curve and to automatically calculate the growth rate, the maximum culture density, and the duration of the growth lag phase. GrowthRates is freely available for Macintosh, Windows, and Linux. We discuss the effects of culture volume, the classical bacterial growth curve, and the differences between determinations in rich media and minimal (mineral salts) media. This protocol covers calibration of the plate reader, growth of culture inocula for both rich and minimal media, and experimental setup. As a guide to reliability, we report typical day-to-day variation in growth rates and variation within experiments with respect to position of wells within the plates.

Modelling of bacterial growth with shifts in temperature using automated methods with Listeria monocytogenes and Pseudomonas aeruginosa as examples

Time to detection (TTD) measurements using turbidometry allow a straightforward method for the measurement of bacterial growth rates under isothermal conditions. Growth rate measurements were carried out for Listeria monocytogenes at 25, 30 and 37°C and for Pseudomonas aeruginosa over the temperature range 25 to 45°C. The classical three-parameter logistic model was rearranged to provide the theoretical foundation for the observed TTD. A model was subsequently developed for the analysis of TTD data from non-isothermal studies based on the Malthusian approximation of the logistic model. The model was able to predict the TTD for cultures of L. monocytogenes or P. aeruginosa undergoing simple temperature shunts (e.g. 25 to 37°C and vice versa), and for a multiple temperature shunt for L. monocytogenes (25–37–25–37°C and 37–25–37–25°C) over a period of 24h. In no case did a temperature shunt induce a lag.

A Rapid, Direct Measurement of Bacterial Growth Rate in Anaerobic Wastewater Treatment

Reliable design and operation of biological wastewater treatment systems demand robust models of biological degradation processes. However, methods to directly measure key bacterial growth kinetics have not been readily available. Those methods that are available rely on the classic measurement of aerobic respiration using oxygen uptake take rates. This paper shows how the thymidine assay can be used as a rapid and direct measurement of bacterial specific growth rates (mu) in situ for an anaerobic treatment process, independent of aerobic respiration. A filtration-based assay is applied and evaluated a dispersed-phase high-rate anaerobic treatment process, with results obtained in less than an hour. The chemical oxygen demand (COD) biomass in the reactor was 0.52 kg COD m(-3) and the specific growth rate of these anaerobic bacteria was 0.8 +/- 0.2 d(-1). It took the bacterial populations 21.6 hours to double. This is an important advancement from existing methods that use aerobic respiration as a pseudo measurement of bacterial specific growth rates. The method allows rapid and direct measures of microbial growth rates for anaerobic treatment processes.

Environmental stresses can alleviate the average deleterious effect of mutations.

Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness. Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organism's ability to tolerate deleterious mutations. Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations. We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities. Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses. In contrast to the perception that stress always reduces the organism's ability to tolerate mutations, our measurements identified stresses that do the opposite - that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations. Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations. We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations. The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions.

Comparison of various measures of microbial growth kinetics in suspended and biofilm cultures during biodegradation of naphthalene.

This research focused on measurement of bacterial growth rates and activities in suspended cultures and biofilms using Pseudomonas putida (ATCC 17484) for biodegradation of naphthalene. A comparison was made of the effect of three biomass measures: optical density, total cell protein, and living cell number on the calculated rate of naphthalene disappearance. Living cell number was determined by optimizing a fluorescent staining technique and using epifluorescence microscopy. All three techniques gave similar results in terms of the specific growth rates, and in all three there was a lag time between the disappearance of naphthalene and the appearance of biomass. This was most likely a consequence of the production of intermediate products detected in the chromatograms. Inclusion of a one-hour lag time in the integrated Monod expression considerably improved the agreement between experimental and calculated values. However, further improvements to an understanding of the overall rate processes may require more detailed kinetics of the individual biochemical pathways.

Measurement of bacterial growth rates in soil

Measurements of the average growth rates of soil bacteria from a soil under three treatments; a conventional high input corn soybean rotation, a reversion to a native successional community (4 years) and a never ploughed grassland, were made by two methods. The specific rates of synthesis of bacterial DNA were determined from the specific activities of the DNA precursor, deoxythymidine triphosphate (dTTP), and purified bacterial community DNA after 3H thymidine incorporation. These rates were compared with the specific respiration rates of the soil microbiota determined from the initial rate of C mineralization and the microbial biomass C. We show how these two measurements can be used to estimate values for microbial growth yield and maintenance coefficients in soil. Both methods ranked the soil treatments in the same order. The reversion treatment had the highest thymidine incorporation and specific respiration rates and the corn soybean rotation the lowest. The grassland soil had the highest microbial biomass but an intermediate growth rate. Based on thymidine incorporation the doubling time of the bacterial biomass at 25°C was 160 days in soil from the corn soybean rotation and 107 days in the reversion treatment. Fractionation of the 3H-labelled DNA according to its %guanine+cytosine content showed growth of soil bacteria to be widespread among bacterial genera and not confined to a few active species. The soil bacterial growth rates found inre are much lower than those found in some other studies using thymidine incorporation but are in agreement with annual C budgets for the soils.

Measurement of bacterial growth rates on the rhizoplane using 3H-thymidine incorporation into DNA

Bacterial growth rates on the rhizoplane of rape seedlings grown in sand were determined using 3H-thymidine incorporation into DNA. Axenic roots incorporated thymidine into DNA, which had to be subtracted from values for roots with associated bacteria. Thymidine incorporation into rhizoplane bacterial DNA ranged between 0.6 and 1.4 pmol thymidine h−1 root−1 for 6 to 26-day-old plants. Using a conversion factor, the turnover time of bacteria was calculated to decrease from 9.2 h for 6-day-old plants to 160h for 26-day-old plants. A similar value was found for rhizosphere bacteria of plants grown for 26 days in natural soil.

Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA

Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10(5) cells g(-1) day(-1). These rates are 2-1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed.

Comparison of methods for measurement of bacterial growth rates in mixed batch cultures

Eight methods of assessing growth rate constants of bacteria were compared in batch cultures of 3-micrometers-filtered estuarine water from the Skidaway River in Ga. Mixed assemblages of bacteria were grown under four nutrient regimes of added yeast extract ranging from 0 to 100 mg/liter. Linear and exponential growth rate constants were computed from changes in cell densities, biovolumes, and ATP concentrations. Exponential growth rate constants were obtained from the frequency of dividing cells and RNA synthesis as measured by [3H]adenine uptake. Rate constants obtained during lag, exponential, and stationary growth phases depended largely on the method used. Constants calculated from changes in cell densities, frequency of dividing cells, and adenine uptake correlated most closely with each other, whereas constants calculated from changes in ATP concentrations and biovolumes correlated best with each other. Estimates of in situ bacterial productivity and growth vary depending on the method used and the assumptions made regarding the growth state of bacteria.

Bacterial growth rates and temperature optima in a stream with a fluctuating thermal regime1

Direct measurement of bacterial growth rates in a natural stream showed that the unicellular and filamentous populations had doubling times of 42–51 hr when the water temperature was 0–5°C, 8.4–10.8 hr when water temperature was predominantly 11–16°C, and 2.8–6.0 hr when the water temperature was 16.5–21.0°C. The populations were not optimally adapted to the prevailing stream water temperature, but rather to temperatures 5–20°C higher.