Limits...
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

Uncorrected growth kinetics of columns 10 (panelA), 11 (panelB) and 12 (panelC) of the 96-well plate in Experiment 2. In these three columns excluding well A12 (n=23), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0988 ± 0.0053, which corresponds to a %RSD of 5.4. The biphasic curve in well A12 was unique among the 96 wells analyzed in this assay, and is caused by an initial phase of optical density increase caused by condensation on the lid followed by a second phase caused by increased turbidity due to cell growth within the well.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Uncorrected growth kinetics of columns 10 (panelA), 11 (panelB) and 12 (panelC) of the 96-well plate in Experiment 2. In these three columns excluding well A12 (n=23), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0988 ± 0.0053, which corresponds to a %RSD of 5.4. The biphasic curve in well A12 was unique among the 96 wells analyzed in this assay, and is caused by an initial phase of optical density increase caused by condensation on the lid followed by a second phase caused by increased turbidity due to cell growth within the well.

Mentions: In Experiment 2, configured as shown inFigure 1C, the threshold times again reflected a roughly 70-minute difference between input and output controls. (Figure 3 andTable 2) However, this difference was not reflected in threshold times of the cells growing in column 12, suggesting that the contamination of those wells was the result of either a second contamination event unrelated to the timing of the transfer of cells into the wells in column 10 or a lower inoculum in each well. The only reasonable explanation of this agreement in threshold time differences between columns 10 and 11 and the far larger Tt values resulting from column 12 is that cross-contamination occurred while cells were expelled, and the aerosols thus formed travelled to the adjacent wells but not the intervening 96 contamination control wells in the contamination control plate, none of which turned turbid after overnight incubation at 37 degrees. These results indicate that 96-well plates and threshold times are useful for detecting contamination, and that cross-contamination occurs in experiments where cells are added as droplets from above.


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

Ericksen B - F1000Res (2014)

Uncorrected growth kinetics of columns 10 (panelA), 11 (panelB) and 12 (panelC) of the 96-well plate in Experiment 2. In these three columns excluding well A12 (n=23), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0988 ± 0.0053, which corresponds to a %RSD of 5.4. The biphasic curve in well A12 was unique among the 96 wells analyzed in this assay, and is caused by an initial phase of optical density increase caused by condensation on the lid followed by a second phase caused by increased turbidity due to cell growth within the well.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Uncorrected growth kinetics of columns 10 (panelA), 11 (panelB) and 12 (panelC) of the 96-well plate in Experiment 2. In these three columns excluding well A12 (n=23), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0988 ± 0.0053, which corresponds to a %RSD of 5.4. The biphasic curve in well A12 was unique among the 96 wells analyzed in this assay, and is caused by an initial phase of optical density increase caused by condensation on the lid followed by a second phase caused by increased turbidity due to cell growth within the well.
Mentions: In Experiment 2, configured as shown inFigure 1C, the threshold times again reflected a roughly 70-minute difference between input and output controls. (Figure 3 andTable 2) However, this difference was not reflected in threshold times of the cells growing in column 12, suggesting that the contamination of those wells was the result of either a second contamination event unrelated to the timing of the transfer of cells into the wells in column 10 or a lower inoculum in each well. The only reasonable explanation of this agreement in threshold time differences between columns 10 and 11 and the far larger Tt values resulting from column 12 is that cross-contamination occurred while cells were expelled, and the aerosols thus formed travelled to the adjacent wells but not the intervening 96 contamination control wells in the contamination control plate, none of which turned turbid after overnight incubation at 37 degrees. These results indicate that 96-well plates and threshold times are useful for detecting contamination, and that cross-contamination occurs in experiments where cells are added as droplets from above.

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