scholarly journals Environmental and physiological factors affecting high-throughput measurements of bacterial growth

2020 ◽  
Author(s):  
Esha Atolia ◽  
Spencer Cesar ◽  
Heidi A. Arjes ◽  
Manohary Rajendram ◽  
Handuo Shi ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Escherichia Coli ◽  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Physiological State ◽  
Maximal Growth ◽  
Initial Cell ◽  
Maximal Growth Rate

AbstractBacterial growth in nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multi-well plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.Abstract ImportanceHow starved bacteria adapt to and multiply in replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allows for accurate growth-rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic-acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

scholarly journals Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Esha Atolia ◽  
Spencer Cesar ◽  
Heidi A. Arjes ◽  
Manohary Rajendram ◽  
Handuo Shi ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Physiological State ◽  
Maximal Growth ◽  
Content Type ◽  
Initial Cell ◽  
Maximal Growth Rate

ABSTRACT Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

scholarly journals AN ACRIFLAVINE SENSITIVITY MUTATION WHICH AFFECTS TO THE MAXIMAL GROWTH RATE OF ESCHERICHIA COLI

1975 ◽  
Vol 50 (4) ◽  
pp. 319-328
Author(s):  
HAKOBU NAKAMURA ◽  
ATSUSHI SUGANUMA ◽  
JOSEPH GREENBERG
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Maximal Growth ◽  
Maximal Growth Rate

scholarly journals Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

1999 ◽  
Vol 65 (6) ◽  
pp. 2631-2635 ◽  
Author(s):  
Sonja Isken ◽  
Antoine Derks ◽  
Petra F. G. Wolffs ◽  
Jan A. M. de Bont
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Organic Solvents ◽  
Critical Concentration ◽  
Bacterial Membrane ◽  
Maximal Growth ◽  
Wild Type ◽  
Active Efflux ◽  
Maximal Growth Rate ◽  
Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals Loss of Function inEscherichia coliExposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Sarah Forbes ◽  
Nicola Morgan ◽  
Gavin J. Humphreys ◽  
Alejandro Amézquita ◽  
Hitesh Mistry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Biofilm Formation ◽  
Benzalkonium Chloride ◽  
Loss Of Function ◽  
Free Medium ◽  
Content Type ◽  
Expression Of Genes ◽  
Repeated Passage

ABSTRACTAssessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues,Escherichia coliMG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P< 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure ofE. colito BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility.IMPORTANCEExposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in theEscherichia colitranscriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

Osteosarcoma Developed in the Period of Maximal Growth Rate Have Inferior Prognosis

2008 ◽  
Vol 30 (6) ◽  
pp. 419-424 ◽  
Author(s):  
Jun Ah Lee ◽  
Min Suk Kim ◽  
Dong Ho Kim ◽  
Jung Sub Lim ◽  
Kyung Duk Park ◽  
...  
Keyword(s):  
Growth Rate ◽  
Maximal Growth ◽  
Maximal Growth Rate

Estimating Bacterial Growth Parameters by Means of Detection Times

1999 ◽  
Vol 65 (2) ◽  
pp. 732-736 ◽  
Author(s):  
József Baranyi ◽  
Carmen Pin
Keyword(s):  
Growth Rate ◽  
Specific Growth Rate ◽  
Bacterial Growth ◽  
Specific Growth ◽  
Growth Parameters ◽  
Physiological State ◽  
Cell Number ◽  
Maximum Specific Growth Rate ◽  
Specific Rate ◽  
The Individual

ABSTRACT We developed a new numerical method to estimate bacterial growth parameters by means of detection times generated by different initial counts. The observed detection times are subjected to a transformation involving the (unknown) maximum specific growth rate and the (known) ratios between the different inoculum sizes and the constant detectable level of counts. We present an analysis of variance (ANOVA) protocol based on a theoretical result according to which, if the specific rate used for the transformation is correct, the transformed values are scattered around the same mean irrespective of the original inoculum sizes. That mean, termed the physiological state of the inoculum,α̂, and the maximum specific growth rate, μ, can be estimated by minimizing the variance ratio of the ANOVA procedure. The lag time of the population can be calculated as λ = −ln α̂/μ; i.e. the lag is inversely proportional to the maximum specific growth rate and depends on the initial physiological state of the population. The more accurately the cell number at the detection level is known, the better the estimate for the variance of the lag times of the individual cells.

scholarly journals Effect of Salt Stress on Mutation and Genetic Architecture for Fitness Components in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (10) ◽  
pp. 3831-3842
Author(s):  
Christopher Kozela ◽  
Mark O. Johnston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Salt Stress ◽  
Mutation Rate ◽  
Genetic Architecture ◽  
Maximal Growth ◽  
Fitness Components ◽  
Environmental Variance ◽  
Maximal Growth Rate ◽  
Mutational Variance

Mutations shape genetic architecture and thus influence the evolvability, adaptation and diversification of populations. Mutations may have different and even opposite effects on separate fitness components, and their rate of origin, distribution of effects and variance-covariance structure may depend on environmental quality. We performed an approximately 1,500-generation mutation-accumulation (MA) study in diploids of the yeast Saccharomyces cerevisiae in stressful (high-salt) and normal environments (50 lines each) to investigate the rate of input of mutational variation (Vm) as well as the mutation rate and distribution of effects on diploid and haploid fitness components, assayed in the normal environment. All four fitness components in both MA treatments exhibited statistically significant mutational variance and mutational heritability. Compared to normal-MA, salt stress increased the mutational variance in growth rate by more than sevenfold in haploids derived from the MA lines. This increase was not detected in diploid growth rate, suggesting masking of mutations in the heterozygous state. The genetic architecture arising from mutation (M-matrix) differed between normal and salt conditions. Salt stress also increased environmental variance in three fitness components, consistent with a reduction in canalization. Maximum-likelihood analysis indicated that stress increased the genomic mutation rate by approximately twofold for maximal growth rate and sporulation rate in diploids and for viability in haploids, and by tenfold for maximal growth rate in haploids, but large confidence intervals precluded distinguishing these values between MA environments. We discuss correlations between fitness components in diploids and haploids and compare the correlations between the two MA environmental treatments.

Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation

2001 ◽  
Vol 47 (4) ◽  
pp. 290-293 ◽  
Author(s):  
T Wauters ◽  
D Iserentant ◽  
H Verachtert
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Growth Rate ◽  
Tannic Acid ◽  
Unsaturated Fatty Acids ◽  
Acid Resistance ◽  
Maximal Growth ◽  
Wild Type ◽  
Iron Deprivation ◽  
Maximal Growth Rate

Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae. Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S. cerevisiae. Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants. While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions. Also, the wild type grown anaerobically was not affected by iron-limited conditions. Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants. During iron limitation, the wild type showed a reduced oxygen uptake rate. Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol. Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.Key words: Saccharomyces cerevisiae, tannic acid resistance, iron deprivation, cytoplasmic petite mutant.

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