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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

Position of pipette tips beneath liquid and in contact with the cross-sectional corners of the wells.Arrows indicate the flow of a cell suspension as the liquid is expelled, generating mild shear to disperse clumps and maximize mixing efficacy.
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f4: Position of pipette tips beneath liquid and in contact with the cross-sectional corners of the wells.Arrows indicate the flow of a cell suspension as the liquid is expelled, generating mild shear to disperse clumps and maximize mixing efficacy.

Mentions: On the other hand, pipetting up and down beneath liquid is undoubtedly an improvement. The mild shear resulting from the proximity of the well surfaces to the tip opening (Figure 4) would not only tend to disperse cohesive clumps, it would also yield a more homogeneously mixed suspension of single cells. Adding cells as droplets from above utilizes only the shaking of the plate within the plate reader to mix the cells with the buffer underneath. This shaking occurred in a linear fashion for about 15 seconds initially, then every five minutes for three seconds duration throughout both the two-hour and twelve-hour incubation steps of the assay in experiments at UMB using a Molecular Devices Vmax plate reader in a 37°C warm room between 2003 and November, 2011. Thereafter, the assay was adapted for a temperature-controlled Tecan Infinite M1000 plate reader, which allows for additional shaking options including either orbital or linear shaking and near-continuous shaking between readings. Sampling the volume of wells at various locations after the addition of cells, followed by plating and colony counting to compare the efficacy of various methods of mixing, might clarify this question further. It should be noted that the UCLA experiments published in 2011 utilized a Molecular Devices Spectramax plate reader, which is temperature-controlled with shaking features similar to the Vmax. Bacterial growth kinetics might vary somewhat in these three plate readers due to changes in aeration and temperature control, and airflow might in turn affect the magnitude of the influence of EFs on experimental results.


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

Ericksen B - F1000Res (2014)

Position of pipette tips beneath liquid and in contact with the cross-sectional corners of the wells.Arrows indicate the flow of a cell suspension as the liquid is expelled, generating mild shear to disperse clumps and maximize mixing efficacy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Position of pipette tips beneath liquid and in contact with the cross-sectional corners of the wells.Arrows indicate the flow of a cell suspension as the liquid is expelled, generating mild shear to disperse clumps and maximize mixing efficacy.
Mentions: On the other hand, pipetting up and down beneath liquid is undoubtedly an improvement. The mild shear resulting from the proximity of the well surfaces to the tip opening (Figure 4) would not only tend to disperse cohesive clumps, it would also yield a more homogeneously mixed suspension of single cells. Adding cells as droplets from above utilizes only the shaking of the plate within the plate reader to mix the cells with the buffer underneath. This shaking occurred in a linear fashion for about 15 seconds initially, then every five minutes for three seconds duration throughout both the two-hour and twelve-hour incubation steps of the assay in experiments at UMB using a Molecular Devices Vmax plate reader in a 37°C warm room between 2003 and November, 2011. Thereafter, the assay was adapted for a temperature-controlled Tecan Infinite M1000 plate reader, which allows for additional shaking options including either orbital or linear shaking and near-continuous shaking between readings. Sampling the volume of wells at various locations after the addition of cells, followed by plating and colony counting to compare the efficacy of various methods of mixing, might clarify this question further. It should be noted that the UCLA experiments published in 2011 utilized a Molecular Devices Spectramax plate reader, which is temperature-controlled with shaking features similar to the Vmax. Bacterial growth kinetics might vary somewhat in these three plate readers due to changes in aeration and temperature control, and airflow might in turn affect the magnitude of the influence of EFs on experimental results.

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