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( A ) Role of SdpA in β-lactam resistance. β-Lactam antibiotic susceptibility assay was performed via spotting dilutions. Diluted cultures (6 µL) of strains CB15N, RP48 (CB15N/pJS14-Pxyl- sdpA ), RP47 (∆ sdpA ), RP52 (∆ sdpA /pJS14/Pxyl/ sdpA ), RP53 (∆ sdpA /pJS14/Pxyl/ sdpB ), RP54 (∆ sdpA /pJS14/Pxyl/ sdpC ), and RP55 (∆ sdpA /pJS14/Pxyl/ sdpAQ544E ) were spotted on PYE + 0.3% xylose containing antibiotics at varying concentrations (ampicillin 10 and 30 µg/mL, cephalexin 5 and 8 µg/mL, mecillinam 10 and 15 µg/mL, vancomycin 20 µg/mL). PYE plates with no antibiotic were used as a control. All plates were incubated at 30°C for 36 h before imaging. ( B ) Overexpression of SdpA leads to morphological and cell division defects. Strain RP48 (CB15N/pJS14-Pxyl- sdpA ) was grown to the exponential phase with 0.3% xylose added to the media. The culture was immobilized on a PYE agarose pad and imaged by DIC microscopy after 6 h post-induction at 30°C (scale bar = 2 µm). ( C ) Distribution of cell length in a population of RP48 after the overexpression of sdpA . Micrographs of strain RP48 were subjected <t>to</t> <t>automated</t> image analysis by <t>Oufti</t> . The data obtained ( n = 874 cells per condition) are shown as a scatter plot where the bar represents the median. ( D ) Growth rate of the sdpA overexpression (induced with xylose) strain compared to CB15N and sdpA deletion strain was analyzed. OD 600 was measured at 2 h interval for a total period of 8 h. Data represent the mean of three biological replicates.
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( A ) Role of SdpA in β-lactam resistance. β-Lactam antibiotic susceptibility assay was performed via spotting dilutions. Diluted cultures (6 µL) of strains CB15N, RP48 (CB15N/pJS14-Pxyl- sdpA ), RP47 (∆ sdpA ), RP52 (∆ sdpA /pJS14/Pxyl/ sdpA ), RP53 (∆ sdpA /pJS14/Pxyl/ sdpB ), RP54 (∆ sdpA /pJS14/Pxyl/ sdpC ), and RP55 (∆ sdpA /pJS14/Pxyl/ sdpAQ544E ) were spotted on PYE + 0.3% xylose containing antibiotics at varying concentrations (ampicillin 10 and 30 µg/mL, cephalexin 5 and 8 µg/mL, mecillinam 10 and 15 µg/mL, vancomycin 20 µg/mL). PYE plates with no antibiotic were used as a control. All plates were incubated at 30°C for 36 h before imaging. ( B ) Overexpression of SdpA leads to morphological and cell division defects. Strain RP48 (CB15N/pJS14-Pxyl- sdpA ) was grown to the exponential phase with 0.3% xylose added to the media. The culture was immobilized on a PYE agarose pad and imaged by DIC microscopy after 6 h post-induction at 30°C (scale bar = 2 µm). ( C ) Distribution of cell length in a population of RP48 after the overexpression of sdpA . Micrographs of strain RP48 were subjected to automated image analysis by Oufti . The data obtained ( n = 874 cells per condition) are shown as a scatter plot where the bar represents the median. ( D ) Growth rate of the sdpA overexpression (induced with xylose) strain compared to CB15N and sdpA deletion strain was analyzed. OD 600 was measured at 2 h interval for a total period of 8 h. Data represent the mean of three biological replicates.

Journal: mBio

Article Title: Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

doi: 10.1128/mbio.02975-24

Figure Lengend Snippet: ( A ) Role of SdpA in β-lactam resistance. β-Lactam antibiotic susceptibility assay was performed via spotting dilutions. Diluted cultures (6 µL) of strains CB15N, RP48 (CB15N/pJS14-Pxyl- sdpA ), RP47 (∆ sdpA ), RP52 (∆ sdpA /pJS14/Pxyl/ sdpA ), RP53 (∆ sdpA /pJS14/Pxyl/ sdpB ), RP54 (∆ sdpA /pJS14/Pxyl/ sdpC ), and RP55 (∆ sdpA /pJS14/Pxyl/ sdpAQ544E ) were spotted on PYE + 0.3% xylose containing antibiotics at varying concentrations (ampicillin 10 and 30 µg/mL, cephalexin 5 and 8 µg/mL, mecillinam 10 and 15 µg/mL, vancomycin 20 µg/mL). PYE plates with no antibiotic were used as a control. All plates were incubated at 30°C for 36 h before imaging. ( B ) Overexpression of SdpA leads to morphological and cell division defects. Strain RP48 (CB15N/pJS14-Pxyl- sdpA ) was grown to the exponential phase with 0.3% xylose added to the media. The culture was immobilized on a PYE agarose pad and imaged by DIC microscopy after 6 h post-induction at 30°C (scale bar = 2 µm). ( C ) Distribution of cell length in a population of RP48 after the overexpression of sdpA . Micrographs of strain RP48 were subjected to automated image analysis by Oufti . The data obtained ( n = 874 cells per condition) are shown as a scatter plot where the bar represents the median. ( D ) Growth rate of the sdpA overexpression (induced with xylose) strain compared to CB15N and sdpA deletion strain was analyzed. OD 600 was measured at 2 h interval for a total period of 8 h. Data represent the mean of three biological replicates.

Article Snippet: Automated image analysis was carried out with MATLAB-dependent Oufti Software ( ).

Techniques: Drug Susceptibility Assay, Control, Incubation, Imaging, Over Expression, Microscopy

( A ) Role of AmpG permease in β-lactam resistance. β-Lactam susceptibility assay was performed by spotting dilutions of cultures CB15N, RP57 (CB15N/pJS14-Pxyl- ampG ), RP56 (∆ ampG ), and deletion mutant along with its complemented derivative RP58 (∆ ampG /pJS14-Pxyl- ampG ) on PYE solid media + 0.3% xylose containing antibiotics at varying concentrations. PYE plates with no antibiotic were used as a control. ( B ) Phase contrast imaging of ampG overexpression strain RP57 (CB15N/pJS14-Pxyl- ampG ) showing cell filamentation. Cells were grown to mid-exponential phase for 6 h in a medium containing (+) or lacking (–) xylose and were imaged by phase contrast microscopy (scale bar = 2 µm). ( C ) Distribution of cell length in a population of RP57 after the overexpression of AmpG. Micrographs from ( B ) were then subjected to automated image analysis by Oufti software. The values obtained are shown as scatter plots, where the bar indicates the median ( n = 964 per condition). ( D ) Growth rate of strains CB15N, RP56 (∆ ampG ), and RP57 (CB15N/pJS14-Pxyl- ampG ) confirmed growth defects. Cells were induced with xylose at mid-exponential phase, and OD 600 was measured at a 2 h interval at 30°C. Data represent the mean of three biological replicates.

Journal: mBio

Article Title: Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

doi: 10.1128/mbio.02975-24

Figure Lengend Snippet: ( A ) Role of AmpG permease in β-lactam resistance. β-Lactam susceptibility assay was performed by spotting dilutions of cultures CB15N, RP57 (CB15N/pJS14-Pxyl- ampG ), RP56 (∆ ampG ), and deletion mutant along with its complemented derivative RP58 (∆ ampG /pJS14-Pxyl- ampG ) on PYE solid media + 0.3% xylose containing antibiotics at varying concentrations. PYE plates with no antibiotic were used as a control. ( B ) Phase contrast imaging of ampG overexpression strain RP57 (CB15N/pJS14-Pxyl- ampG ) showing cell filamentation. Cells were grown to mid-exponential phase for 6 h in a medium containing (+) or lacking (–) xylose and were imaged by phase contrast microscopy (scale bar = 2 µm). ( C ) Distribution of cell length in a population of RP57 after the overexpression of AmpG. Micrographs from ( B ) were then subjected to automated image analysis by Oufti software. The values obtained are shown as scatter plots, where the bar indicates the median ( n = 964 per condition). ( D ) Growth rate of strains CB15N, RP56 (∆ ampG ), and RP57 (CB15N/pJS14-Pxyl- ampG ) confirmed growth defects. Cells were induced with xylose at mid-exponential phase, and OD 600 was measured at a 2 h interval at 30°C. Data represent the mean of three biological replicates.

Article Snippet: Automated image analysis was carried out with MATLAB-dependent Oufti Software ( ).

Techniques: Drug Susceptibility Assay, Mutagenesis, Control, Imaging, Over Expression, Microscopy, Software

Synthetic effects of AmpG and SdpA deficiency. ( A ) Antibiotic susceptibility was tested by spot dilution assay. Strains CB15N, RP47 (∆ sdpA ), RP56 (∆ ampG ), and RP60 (∆ sdpA ∆ ampG ) strains were serially diluted and spotted on medium (PYE + xylose) or lacking inducer (PYE + glucose) containing antibiotics at different concentrations (ampicillin 10 µg/mL, cephalexin 5 µg/mL, and mecillinam 10 µg/mL). No-antibiotic PYE plates were used as a control. ( B ) Strain RP60 (∆ sdpA ∆ ampG ) was imaged in the presence of 0.2% glucose to deplete ampG and glucose + ampicillin (30 µg/mL) with a phase contrast microscope. Cells grown in 0.3% xylose were used as a control (scale bar = 3 µm). ( C ) Cell length analysis of SdpA and AmpG deficient cells. The micrographs described in ( B ) of strain RP60 were analyzed in control (+ xylose) and depletion conditions (+ glucose) at different time points by Oufti Software. The values obtained are shown as scatter plots, plotted as cell length vs cell width ( n = 215 cells per condition). ( D ) Growth rate analysis of double-deletion mutant RP60 (∆sdpA∆ampG) along with strains CB15N, RP47 (∆ sdpA ), and RP56 (∆ ampG ), indicating reduced viability. Strains were grown in PYE + xylose and PYE + glucose (lacking inducer), and OD 600 was measured every 2 h. Data represent the mean of three biological replicates.

Journal: mBio

Article Title: Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

doi: 10.1128/mbio.02975-24

Figure Lengend Snippet: Synthetic effects of AmpG and SdpA deficiency. ( A ) Antibiotic susceptibility was tested by spot dilution assay. Strains CB15N, RP47 (∆ sdpA ), RP56 (∆ ampG ), and RP60 (∆ sdpA ∆ ampG ) strains were serially diluted and spotted on medium (PYE + xylose) or lacking inducer (PYE + glucose) containing antibiotics at different concentrations (ampicillin 10 µg/mL, cephalexin 5 µg/mL, and mecillinam 10 µg/mL). No-antibiotic PYE plates were used as a control. ( B ) Strain RP60 (∆ sdpA ∆ ampG ) was imaged in the presence of 0.2% glucose to deplete ampG and glucose + ampicillin (30 µg/mL) with a phase contrast microscope. Cells grown in 0.3% xylose were used as a control (scale bar = 3 µm). ( C ) Cell length analysis of SdpA and AmpG deficient cells. The micrographs described in ( B ) of strain RP60 were analyzed in control (+ xylose) and depletion conditions (+ glucose) at different time points by Oufti Software. The values obtained are shown as scatter plots, plotted as cell length vs cell width ( n = 215 cells per condition). ( D ) Growth rate analysis of double-deletion mutant RP60 (∆sdpA∆ampG) along with strains CB15N, RP47 (∆ sdpA ), and RP56 (∆ ampG ), indicating reduced viability. Strains were grown in PYE + xylose and PYE + glucose (lacking inducer), and OD 600 was measured every 2 h. Data represent the mean of three biological replicates.

Article Snippet: Automated image analysis was carried out with MATLAB-dependent Oufti Software ( ).

Techniques: Dilution Assay, Control, Microscopy, Software, Mutagenesis