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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

Uncorrected growth kinetics of columns 11 (panelA) and 12 (panelB) of the 96-well plate in Experiment 1. In these two columns (n=16), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0989 ± 0.0043, which corresponds to a %RSD of 4.4. The line marked “0.1” is approximately at the position of the threshold ΔOD650 of 0.02.
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f2: Uncorrected growth kinetics of columns 11 (panelA) and 12 (panelB) of the 96-well plate in Experiment 1. In these two columns (n=16), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0989 ± 0.0043, which corresponds to a %RSD of 4.4. The line marked “0.1” is approximately at the position of the threshold ΔOD650 of 0.02.

Mentions: In Experiment 1, configured as shown inFigure 1B, all eight wells in column 12 turned turbid and produced growth curves with the same growth rate and doubling times as the other growth curves on the same microplate (Figure 2). Colony morphologies of samples from these wells also matchedE. coli ATCC 25922. A comparison of threshold times indicated almost the same difference between input and output controls in columns 11 and 12 (Table 1). There was a roughly 70-minute difference in input and output threshold times in the input and output control wells in Experiment 1, which agreed closely with another roughly 70-minute difference in the threshold times of the adjacent wells. Contamination caused by viable environmental strains would have been expected to produce widely varying threshold times, if not visible differences in the appearance of the turbid wells. Therefore, the 70-minute difference indicated that the cross-contamination occurred at the same time that cells were transferred.


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 11 (panelA) and 12 (panelB) of the 96-well plate in Experiment 1. In these two columns (n=16), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0989 ± 0.0043, which corresponds to a %RSD of 4.4. The line marked “0.1” is approximately at the position of the threshold ΔOD650 of 0.02.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Uncorrected growth kinetics of columns 11 (panelA) and 12 (panelB) of the 96-well plate in Experiment 1. In these two columns (n=16), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0989 ± 0.0043, which corresponds to a %RSD of 4.4. The line marked “0.1” is approximately at the position of the threshold ΔOD650 of 0.02.
Mentions: In Experiment 1, configured as shown inFigure 1B, all eight wells in column 12 turned turbid and produced growth curves with the same growth rate and doubling times as the other growth curves on the same microplate (Figure 2). Colony morphologies of samples from these wells also matchedE. coli ATCC 25922. A comparison of threshold times indicated almost the same difference between input and output controls in columns 11 and 12 (Table 1). There was a roughly 70-minute difference in input and output threshold times in the input and output control wells in Experiment 1, which agreed closely with another roughly 70-minute difference in the threshold times of the adjacent wells. Contamination caused by viable environmental strains would have been expected to produce widely varying threshold times, if not visible differences in the appearance of the turbid wells. Therefore, the 70-minute difference indicated that the cross-contamination occurred at the same time that cells were transferred.

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