Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains

Fontana, Célia; Crussard, Steve; Simon-Dufay, Nathalie; Pialot, Daniel; Bomchil, Natalia; Reyes, Jean

doi:10.1016/j.mimet.2016.10.013

Cited by 9 publications

(4 citation statements)

References 28 publications

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“…In the application perspective of this technique, we used the food industry analysis strategy to quantify bacteria. There is a LIVE/DEAD TM kit containing SYTO 9™‐PI [23] used to enumerate total and viable bacteria in beverages [24], probiotic Lactobacillus rhamnosus in chocolate [25] or Oenococcus oeni in wines [26]. For this purpose, we chose to monitor mortality on long‐time cultures [27], that is, cultures incubated for an extended period at 37°C without changing the medium.…”

Section: Resultsmentioning

confidence: 99%

A flow cytometry method for safe detection of bacterial viability

Servain‐Viel,

Aknin,

Domenichini

et al. 2023

Cytometry Pt A

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Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris‐Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non‐CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability‐DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram+, Gram−) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization.

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Section: Resultsmentioning

confidence: 99%

A flow cytometry method for safe detection of bacterial viability

Servain‐Viel,

Aknin,

Domenichini

et al. 2023

Cytometry Pt A

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show abstract

“…Although different methods were reported to indicate for cell lysis, for instance by determination of cytoplasmic proteins; βgalactosidase (Bao et al, 2016) and glucose-6phosphate dehydrogenase (Li et al, 2010) that are expected to be present in very low concentration in the culture medium, they differ in respect to sensitivity and accuracy. In this study, a flow cytometry method was adopted to quantify the concentrations of live and dead cells where it considered as a rapid, sensitive and highly reproducible approach (Fontana et al, 2017). This approach could also replace the cells quantification method under microscopy that is time-consuming and lacks reproducibility.…”

Section: Effect Of Glycine Supplementation On Cell Viability To Confmentioning

confidence: 99%

Improved extracellular secretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis

Nik-Pa

Abd‐Aziz

Ibrahim

et al. 2019

APJMBB

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The use of an effective inducer feeding strategy without causing cell lysis presents significant advantage to enhance the secretion of an enzyme to the culture medium of Escherichia coli. The cgt gene encoding β-cyclodextrin glycosyltransferase (β-CGTase) was cloned into pQE30xa as an N-terminal His-tagged protein and transformed into E. coli. The induction strategy was applied towards enhancing the extracellular secretion of the recombinant β-CGTase by increasing permeability of the outer membrane of E. coli. The supplementation of 1.2 mM glycine following 2 h of fermentation at 37°C enhanced the activity of β-CGTase to 38.295 U/mL, which was approximately 1.3-fold higher than the control (without induction). Further flow cytometry analysis was adopted as a rapid and highly reproducible approach to determine the effect of glycine supplementation on the viability of E. coli cells. The supplementation of glycine did not contribute to apparent cell lysis, with no adverse effects on cell viability, hence indicating the effectiveness of glycine in enhancing the extracellular secretion of β-CGTase. The recombinant β-CGTase was then purified through a combination of diafiltration and Ni-NTA affinity chromatography with 18.4-fold increase in purity. An effective glycine feeding strategy could enhance the extracellular secretion of β-CGTase without adverse effects on cell viability. This strategy could be applied potentially to enhance the secretion of a recombinant protein to the culture medium from E. coli cells without having cell lysis.

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“…In biorefineries, quantification is crucial for monitoring fermentation processes and controlling contaminating bacterial cells (Ceccato-Antonini, 2012., Ceccato-Antonini 2018). The pharmaceutical industry relies on precise quantification to optimize yields and guarantee the safety and efficacy of drugs and vaccines produced through bioprocessing (Deshmukh et al, 2016; Fontana et al, 2017). In environmental applications, quantifying microbial populations is essential for assessing wastewater treatment efficiency and environmental bioremediation efforts (Douterelo et al, 2014; Lee et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses

Martins,

Jacobus,

Conceição

et al. 2024

Preprint

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In scenarios where yeast and bacterial cells coexist, e.g. the food and bioethanol industries, it would be of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanolSaccharomyces cerevisiaePE-2 yeast strain and cells from the probioticLactiplantibacillus plantarumstrain in microbial suspensions. Individual suspensions were prepared (∼107, yeast cells/mL or ∼109, bacterial cells/mL) and mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries. The following methods were used: bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter counter (at 1:1 and 100:1 ratios). By subjecting the same mixed cell suspension to each technique, we observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (P>0.05). In contrast, the Coulter counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (P<0.05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn’t possible due to an observed overlap between yeast cell debris and bacterial cells. The results from this work indicate that each method has limitations, advantages, and disadvantages, meaning that the best option will always depend on the application. We present a comparison of the techniques, in terms of time-to-results, cost of analysis and equipment, range of detectable cell/particle diameters, adequacy for simultaneous enumeration, and general pros and cons.Graphical AbstractThis study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.

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Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains

Cited by 9 publications

References 28 publications

A flow cytometry method for safe detection of bacterial viability

A flow cytometry method for safe detection of bacterial viability

Improved extracellular secretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis

Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses

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