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Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols.

Ericksen B - F1000Res (2014)

Bottom Line: The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 and BSL-2 bacteria including Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Enterobacter aerogenes.  In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen Bacillus anthracis.  Both studies were published in the journal Antimicrobial Agents and Chemotherapy.  Here I report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK).  The QGK threshold time, or T t, equivalent to the cycle time C t reported in 1996 by Heid et al., precisely identifies when wells were inoculated.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland School of Medicine, Institute of Human Virology, 725 W. Lombard St., Baltimore, MD, USA.

ABSTRACT
The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 and BSL-2 bacteria including Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Enterobacter aerogenes.  In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen Bacillus anthracis.  Both studies were published in the journal Antimicrobial Agents and Chemotherapy.  Here I report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK).  The QGK threshold time, or T t, equivalent to the cycle time C t reported in 1996 by Heid et al., precisely identifies when wells were inoculated.

No MeSH data available.


Related in: MedlinePlus

Safer, more effective, and more useful adaptation of the method of transferring cells depicted inFigure 5.A five-fold more concentrated inoculum is added in one-fifth the volume beneath buffer rather than as droplets added from above.
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f6: Safer, more effective, and more useful adaptation of the method of transferring cells depicted inFigure 5.A five-fold more concentrated inoculum is added in one-fifth the volume beneath buffer rather than as droplets added from above.

Mentions: Next, in Experiment 2, the controls lacking antimicrobial agents were moved from column 11 to column 10, and columns 11 and 12 contained 16 uninoculated contamination control wells. Wells E10-H10 contained output controls and wells A10-D10 contained input controls (Figure 5). These controls are designed such that comparing the difference in threshold times between the input and output controls, relating that difference to the calibration curve elsewhere on the same 96-well plate, and assuming that adhesion or cohesion and lag phases in exponential growth were the same for all cells, the growth of the cells during the two hour incubation on the plate could be quantified. Enumerating the change in cell concentration during that step would allow the calculation of the difference in virtual survival values that would correspond to bacteriostatic activity.Figure 6 depicts the improved methodology, requiring a fivefold more concentrated cell inoculum in buffer added in one-tenth, rather than one-half, of the 100 µL total volume of the 2-hour incubation step. This method has not been tested, and it is unknown whether the additional step of pipetting up and down 15 times to mix, as depicted inFigure 4, would be necessary to ensure proper mixing. The volumes shown inFigure 6, rather thanFigure 5, used for the addition of cells in the red portions of the 96-well antimicrobial assays as designed inFigure 1, would lessen the probability of cross contamination.


Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols.

Ericksen B - F1000Res (2014)

Safer, more effective, and more useful adaptation of the method of transferring cells depicted inFigure 5.A five-fold more concentrated inoculum is added in one-fifth the volume beneath buffer rather than as droplets added from above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4309163&req=5

f6: Safer, more effective, and more useful adaptation of the method of transferring cells depicted inFigure 5.A five-fold more concentrated inoculum is added in one-fifth the volume beneath buffer rather than as droplets added from above.
Mentions: Next, in Experiment 2, the controls lacking antimicrobial agents were moved from column 11 to column 10, and columns 11 and 12 contained 16 uninoculated contamination control wells. Wells E10-H10 contained output controls and wells A10-D10 contained input controls (Figure 5). These controls are designed such that comparing the difference in threshold times between the input and output controls, relating that difference to the calibration curve elsewhere on the same 96-well plate, and assuming that adhesion or cohesion and lag phases in exponential growth were the same for all cells, the growth of the cells during the two hour incubation on the plate could be quantified. Enumerating the change in cell concentration during that step would allow the calculation of the difference in virtual survival values that would correspond to bacteriostatic activity.Figure 6 depicts the improved methodology, requiring a fivefold more concentrated cell inoculum in buffer added in one-tenth, rather than one-half, of the 100 µL total volume of the 2-hour incubation step. This method has not been tested, and it is unknown whether the additional step of pipetting up and down 15 times to mix, as depicted inFigure 4, would be necessary to ensure proper mixing. The volumes shown inFigure 6, rather thanFigure 5, used for the addition of cells in the red portions of the 96-well antimicrobial assays as designed inFigure 1, would lessen the probability of cross contamination.

Bottom Line: The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 and BSL-2 bacteria including Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Enterobacter aerogenes.  In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen Bacillus anthracis.  Both studies were published in the journal Antimicrobial Agents and Chemotherapy.  Here I report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK).  The QGK threshold time, or T t, equivalent to the cycle time C t reported in 1996 by Heid et al., precisely identifies when wells were inoculated.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland School of Medicine, Institute of Human Virology, 725 W. Lombard St., Baltimore, MD, USA.

ABSTRACT
The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 and BSL-2 bacteria including Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Enterobacter aerogenes.  In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen Bacillus anthracis.  Both studies were published in the journal Antimicrobial Agents and Chemotherapy.  Here I report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK).  The QGK threshold time, or T t, equivalent to the cycle time C t reported in 1996 by Heid et al., precisely identifies when wells were inoculated.

No MeSH data available.


Related in: MedlinePlus