scholarly journals Polyhydroxyalkanoate granule accumulation makes optical density measurement an unreliable method for bacterial growth assessment inBurkholderia thailandensis

2019 ◽  
Author(s):  
Sarah Martinez ◽  
Eric Déziel
Keyword(s):  
Optical Density ◽  
Density Measurement ◽  
Viable Cell ◽  
Wildtype Strain ◽  
Cell Counts ◽  
Real Cell ◽  
Growth Evaluation ◽  
Unreliable Method ◽  
Growth Assessment ◽  
Similar Growth

AbstractOptical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration.Burkholderia thailandensiswas recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an ortholog of theCupriavidus necator phaCgene. When growing cultures of wildtype strain E264 and an isogenicphaC- mutant, we noted a difference in their OD600values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wildtype strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600values no longer representative of cell density. We show a direct correlation between OD600values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation ofB. thailandensisbecause of PHA production. This study also suggests thatB. thailandensiscould represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified on any study realized inB. thailandensis.

scholarly journals Changes in polyhydroxyalkanoate granule accumulation make optical density measurement an unreliable method for estimating bacterial growth in Burkholderia thailandensis

2020 ◽  
Vol 66 (3) ◽  
pp. 256-262 ◽  
Author(s):  
Sarah Martinez ◽  
Eric Déziel
Keyword(s):  
Optical Density ◽  
Type Strain ◽  
Wild Type Strain ◽  
Density Measurement ◽  
Viable Cell ◽  
Wild Type ◽  
Burkholderia Thailandensis ◽  
Cell Counts ◽  
Real Cell ◽  
Unreliable Method

Optical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration. Burkholderia thailandensis was recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an orthologue of the Cupriavidus necator phaC gene. When growing cultures of wild-type strain E264 and an isogenic phaC mutant, we noted a difference in their OD600 values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wild-type strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600 values no longer representative of cell density. We show a direct correlation between OD600 values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation of B. thailandensis because of PHA production. This study also suggests that B. thailandensis could represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified in any study performed with B. thailandensis.

scholarly journals Temperature Effect on Listeria Monocytogenes Planktonic Growth and Biofilm-Forming Ability

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Joana Catarina Andrade ◽  
◽  
Rita Bernardo ◽  
António Salvador Barreto ◽  
Telmo Nunes ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Temperature Effect ◽  
Optical Density ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Viable Cell ◽  
Crystal Violet Staining ◽  
Cell Counts ◽  
Viable Cells ◽  
Density Results

Listeria Monocytogenes is an important foodborne pathogen with the capacity to grow at low temperatures and the ability to form biofilms. These features are particularly significant to food business operators producing readyto-eat foods with a long refrigerated shelf-life not undergoing any listericidal treatment before consumption. Objectives: This work aims to assess the temperature effect on L. monocytogenes growth in planktonic suspension and in mono-species biofilms. Methods and results: Isothermal planktonic growth at 12o C and 37o C was assayed using viable cell counts and optical density measurements that revealed a strong positive correlation, confirming the reliability of combining both methods to estimate L. monocytogenes concentration. Experimental data were then fitted to Baranyi and Roberts primary predictive model and the estimated growth parameters confirmed that μmax at 37o C (0.375 ± 0.072 log Cfu/ ml/h) was higher than at 120 C (0.054 ± 0.001 log Cfu/ml/h), with identical L. monocytogenes final concentrations which emphasizes its ability to grow at refrigerated temperatures. Experimental results from the isothermal growth assay and ComBase Predictor growth model were similar, with slightly higher estimated μmax (37o C: 0.480 log Cfu/ml/h; 12o C: 0.068 log Cfu/ml/h) in the predictor growth model. The studied strains demonstrated biofilm-forming ability at 12o C, 20o C and 300 C after 5 days of growth. No significant differences in biofilm formation at different temperatures were detected considering viable cell counts values, but when using crystal violet staining optical density results significant differences were found, with the highest formation occurring at 30ºC. A positive strong correlation was found between viable cell counts and crystal violet staining optical density results. In fact, both methods complement each other, because while viable cell counts measures viable cells, crystal violet staining optical density considers total biomass (viable and non-viable cells and extracellular matrix components). Nevertheless, in this work all L. monocytogenes strains revealed to be weak biofilm producers. Conclusion: Overall, this studys results contribute with important initial information on L. monocytogenes growth and biofilm formation to further assist predictive growth modeling in food matrices and environments, also enabling subsequent quantitative microbial risk assessment, to improve pathogen’s control.

The role of the macular pigment optical density measurement in the diagnosis of eye diseases

2016 ◽  
Vol 16 (4) ◽  
pp. 197-200
Author(s):  
E.N. Eskina ◽  
◽  
E.A. Egorov ◽  
A.V. Belogurova ◽  
А.А. Gvetadze ◽  
...  
Keyword(s):  
Optical Density ◽  
Density Measurement ◽  
Macular Pigment ◽  
Eye Diseases ◽  
Macular Pigment Optical Density ◽  
Optical Density Measurement

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

scholarly journals Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

2006 ◽  
Vol 72 (5) ◽  
pp. 3482-3488 ◽  
Author(s):  
M�nica Ordax ◽  
Ester Marco-Noales ◽  
Mar�a M. L�pez ◽  
Elena G. Biosca
Keyword(s):  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Control Plant ◽  
Viable Cell ◽  
Epifluorescence Microscopy ◽  
Survival Strategy ◽  
Vbnc State ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Cell Counts

ABSTRACT Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

scholarly journals The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress

2015 ◽  
Vol 12 (106) ◽  
pp. 20150069 ◽  
Author(s):  
Hiroki Takahashi ◽  
Taku Oshima ◽  
Jon L. Hobman ◽  
Neil Doherty ◽  
Selina R. Clayton ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Zinc Concentration ◽  
Systems Approach ◽  
Model Simulation ◽  
Dynamic Balance ◽  
Model Fitting ◽  
Viable Cell ◽  
Population Heterogeneity ◽  
Cell Counts ◽  
Import And Export

Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli . We measured the response to zinc of the main specific zinc import and export systems in the wild-type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: first, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments, we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesize that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments.

In Vitro Anti-Biofilm Activity of Hydrogen-Peroxide Generating Electrochemical Bandage Against Yeast Biofilms

2021 ◽  
Author(s):  
Yash S. Raval ◽  
Abdelrhman Mohamed ◽  
Jayawant N. Mandrekar ◽  
Cody Fisher ◽  
Kerryl E. Greenwood-Quaintance ◽  
...  
Keyword(s):  
Membrane Damage ◽  
Microscopic Analysis ◽  
Viable Cell ◽  
Wound Infections ◽  
Unique Challenge ◽  
Cell Membrane Damage ◽  
Cell Counts ◽  
Extensive Cell ◽  
Fluorescence Microscopic Analysis

Wound infections are caused by bacteria and/or fungi. The presence of fungal biofilms in wound beds presents a unique challenge, as fungal biofilms may be difficult to eradicate. The goal of this work was to assess the in vitro anti-biofilm activity of a H 2 O 2 -producing electrochemical bandage (e-bandage) against 15 yeast isolates representing commonly-encountered species. Time-dependent decreases in viable biofilm CFU counts of all isolates tested were observed, resulting in no visible colonies with 48 hours of exposure by plate culture. Fluorescence microscopic analysis showed extensive cell membrane damage of biofilm cells after e-bandage treatment. Reductions in intracellular ATP levels of yeast biofilm cells were recorded post e-bandage treatment. Our results suggest that exposure to H 2 O 2 -producing e-bandages reduce in vitro viable cell counts of yeast biofilms, making this a potential new topical treatment approach for fungal wound infections.

scholarly journals Analysis of Sensitometry in Radiographic Films using Optical Density Measurement

2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Oladotun A. Ojo ◽  
Peter A. Oluwafisoye ◽  
Charles O. Chime
Keyword(s):  
Quality Control ◽  
Optical Density ◽  
Density Measurement ◽  
Absorbed Dose ◽  
Good Prediction ◽  
Radiographic Film ◽  
X Ray ◽  
Dose Curve ◽  
Optical Density Measurement ◽  
Before And After

The sensitivity of radiographic films is an important factor to the clarity and accuracy of X-ray exposure to patients during treatment or diagnostic periods. It is therefore important to do a thorough analysis of the sensitivity of the radiographic film before and after exposure to enhance the Quality Assurance (QA) and the Quality Control (QC), of the exposure procedures. The optical densities (OD) of each film was measured, with a densitometer model MA 5336, made by GAMMEX. These values were then converted to the absorbed dose (X mGy), which is the amount of dose absorbed by each patient. The optical density versus the dose curve, followed the expected pattern, showing a good prediction from the General model, that the films employed in the exposures were of good quality and standard. Hence the optical density versus dose sensitometric curves depicts the outcome of the various films sensitivity after an exposure to the X-ray radiation through the patients.

scholarly journals Statistical Study of the Spot-Plate Technique for Viable-Cell Counts

1963 ◽  
Vol 11 (4) ◽  
pp. 305-309
Author(s):  
A. F. Gaudy ◽  
F. Abu-Niaaj ◽  
E. T. Gaudy
Keyword(s):  
Statistical Study ◽  
Viable Cell ◽  
Plate Technique ◽  
Cell Counts
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