Applications of Flow Cytometry to Characterize Bacterial Physiological Responses

Ambriz-Aviña, Verónica; Contreras‐Garduño, Jorge; Pedraza-Reyes, Mario

doi:10.1155/2014/461941

Cited by 119 publications

(98 citation statements)

References 128 publications

(163 reference statements)

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“…However the role of spontaneous lesions, including deaminated and oxidized bases in modulating this process in B. subtilis is currently unknown. Results have demonstrated that in a bacterial culture there is cell heterogeneity [6,16], and that the batch methods employed for physiologic characterization are limited, since the contribution of small populations could be overlooked [2,9]. Since mutation rates are low, even in the stressful starving conditions of stationary phase, a method of single-cell analysis such as Flow Cytometry (FCM) may be better suited for measuring mutations with the appropriate reporter system.…”

Section: Introductionmentioning

confidence: 99%

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations

et al. 2016

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Compelling evidence point to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry based methodology that employs a fluorescent reporter system to measure at single cell level the occurrence of transcription-associated mutations (TAM) in nutritionally stressed B. subtilis cultures. Using this approach we demonstrate that i) high levels of transcription correlates with augmented mutation frequency, and ii) mutation frequency is enhanced in non-growing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

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Section: Introductionmentioning

confidence: 99%

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations

et al. 2016

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“…Hence, it can be applied only in cases where the spores are still able to germinate. On the other hand, flow cytometry is a useful, rapid and high (20). Flow cytometry has been employed for many years as a method for single cell analysis and separation of eukaryotic cells.…”

Section: Introductionmentioning

confidence: 99%

“…Flow cytometry has been employed for many years as a method for single cell analysis and separation of eukaryotic cells. However, so far it has remained challenging to distinguish among bacterial species or small size particles (20,21). Hence, studies reporting on flow cytometry as a tool for discriminating vegetative cells from spores of Bacillus subtilis remain scarce.…”

Section: Introductionmentioning

confidence: 99%

Quantification and Isolation ofBacillus subtilisSpores using Cell Sorting and automated Gating

Karava

Bracharz

Kabisch

2019

Preprint

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The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of Bacillus subtilis form sub-populations which include vegetative cells, spore forming cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Moreover we tested different fluorescent dyes as well as different conditions in order to develop a method for optimal separation of distinct populations during sporulation. Automated gating procedures using k-means clustering and thresholding by gaussian mixture modeling were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in strains harboring different genome modifications. We identified the different subpopulations formed during sporulation by employing sorting and microscopy. Finally, we employed the technique to show that a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA results in faster spore formation.

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“…Many different modalities have been established to quantify bacteria in planktonic and biofilm cultures, including direct cell enumeration (plate counting, microscopy, Coulter counting, and flow cytometry) [15][16][17][18][19][20][21][22][23][24][25], optical density [26][27][28], dry mass analysis [29][30][31], protein assays [32][33][34][35][36][37][38], and several staining methods [16,22,23]. The current gold standard method to quantify bacteria is cell enumeration by plate counting, offering multiple advantages.…”

Section: Introductionmentioning

confidence: 99%

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

et al. 2018

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A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP). The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD 600 ) and plate counting (colony-forming units (CFUs)). While both OD 600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

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Applications of Flow Cytometry to Characterize Bacterial Physiological Responses

Cited by 119 publications

References 128 publications

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations

Quantification and Isolation ofBacillus subtilisSpores using Cell Sorting and automated Gating

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

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