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In-Field Implementation of a Recombinant Factor C Assay for the Detection of Lipopolysaccharide as a Biomarker of Extant Life within Glacial Environments.

Barnett MJ, Wadham JL, Jackson M, Cullen DC - Biosensors (Basel) (2012)

Bottom Line: In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities.Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls.Sixteen of these samples returned positive LPS detection.

View Article: PubMed Central - PubMed

Affiliation: Cranfield Health, Vincent Building, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, UK. m.barnett.s06@gmail.com.

ABSTRACT
The discovery over the past two decades of viable microbial communities within glaciers has promoted interest in the role of glaciers and ice sheets (the cryosphere) as contributors to subglacial erosion, global biodiversity, and in regulating global biogeochemical cycles. In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities. Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls. In-field measurements were performed using the recently commercialised PyroGene® recombinant Factor C (rFC) endotoxin detection system and used in conjunction with a handheld fluorometer to measure the fluorescent endpoint of the assay. Twenty-seven glacial samples were collected from the surface, bed and terminus of a low-biomass Arctic valley glacier (Engabreen, Northern Norway), and were analysed in a field laboratory using the rFC assay. Sixteen of these samples returned positive LPS detection. This work demonstrates that LPS detection via rFC assay is a viable in-field method and is expected to be a useful proxy for microbial cell concentrations in low biomass environments.

No MeSH data available.


Related in: MedlinePlus

Effect of non-standard incubation temperature on the rFC assay using LPS standards to determine modified rFC assay protocol for field deployment. The 10, 1, 0.1 and 0.01 EU·mL−1 standards are normalised by subtraction of the 0 EU·mL−1 standard. (a) Time evolution of the endotoxin standards, y-axis is truncated, the 10 EU·mL−1 standard starts to plateau after 4 h and 1 EU·mL−1 standard continues to increase linearly (RFU = relative fluorescence units); (b) Calibration curves at 1, 3 and 6.5 h (i.e., assay response between t = 0 and time of interest). Error bars represent ±1 SD from triplicates.
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f2-biosensors-02-00083: Effect of non-standard incubation temperature on the rFC assay using LPS standards to determine modified rFC assay protocol for field deployment. The 10, 1, 0.1 and 0.01 EU·mL−1 standards are normalised by subtraction of the 0 EU·mL−1 standard. (a) Time evolution of the endotoxin standards, y-axis is truncated, the 10 EU·mL−1 standard starts to plateau after 4 h and 1 EU·mL−1 standard continues to increase linearly (RFU = relative fluorescence units); (b) Calibration curves at 1, 3 and 6.5 h (i.e., assay response between t = 0 and time of interest). Error bars represent ±1 SD from triplicates.

Mentions: Due to the expected lower and fluctuating incubation temperatures encountered in the field laboratory, a lower rate of fluorescence intensity development for the assay was anticipated, therefore requiring a longer incubation period to develop a suitable signal for a given LPS concentration. In order to test this hypothesis and to determine the required length of incubation time, the rFC assay was conducted with LPS standards (10, 1, 0.1, 0.01 and 0 EU·mL−1) in an institutional laboratory with an ambient temperature of +20 °C to +22 °C over an incubation period of 6.5 h. Figure 2 shows the fluorescent intensities recorded at half hourly intervals using the handheld fluorometer, and the corresponding calibration curves if the assay was stopped after 1, 3 and 6.5 h. The 10 EU·mL−1 LPS standard starts to plateau after 4 h (not shown in Figure 2(a)), but the 1 EU·mL−1 LPS standard continues to increase in intensity linearly (by visual inspection) during this time. Figure 2(b) shows the assay response for the 0 and 0.1 EU·mL−1 LPS standards is greatest when the incubation time is limited to one hour, which is an artefact of the low RFU values for each standard at time zero, as seen in Figure 2(a). The lower time zero data points have a greater effect on the standards when the incubation time is short. Figure 2(a) is normalised to the blank, the standard deviations (SD) of triplicates of the standards including the blank are included to demonstrate the ability to discriminate between the standards.


In-Field Implementation of a Recombinant Factor C Assay for the Detection of Lipopolysaccharide as a Biomarker of Extant Life within Glacial Environments.

Barnett MJ, Wadham JL, Jackson M, Cullen DC - Biosensors (Basel) (2012)

Effect of non-standard incubation temperature on the rFC assay using LPS standards to determine modified rFC assay protocol for field deployment. The 10, 1, 0.1 and 0.01 EU·mL−1 standards are normalised by subtraction of the 0 EU·mL−1 standard. (a) Time evolution of the endotoxin standards, y-axis is truncated, the 10 EU·mL−1 standard starts to plateau after 4 h and 1 EU·mL−1 standard continues to increase linearly (RFU = relative fluorescence units); (b) Calibration curves at 1, 3 and 6.5 h (i.e., assay response between t = 0 and time of interest). Error bars represent ±1 SD from triplicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263545&req=5

f2-biosensors-02-00083: Effect of non-standard incubation temperature on the rFC assay using LPS standards to determine modified rFC assay protocol for field deployment. The 10, 1, 0.1 and 0.01 EU·mL−1 standards are normalised by subtraction of the 0 EU·mL−1 standard. (a) Time evolution of the endotoxin standards, y-axis is truncated, the 10 EU·mL−1 standard starts to plateau after 4 h and 1 EU·mL−1 standard continues to increase linearly (RFU = relative fluorescence units); (b) Calibration curves at 1, 3 and 6.5 h (i.e., assay response between t = 0 and time of interest). Error bars represent ±1 SD from triplicates.
Mentions: Due to the expected lower and fluctuating incubation temperatures encountered in the field laboratory, a lower rate of fluorescence intensity development for the assay was anticipated, therefore requiring a longer incubation period to develop a suitable signal for a given LPS concentration. In order to test this hypothesis and to determine the required length of incubation time, the rFC assay was conducted with LPS standards (10, 1, 0.1, 0.01 and 0 EU·mL−1) in an institutional laboratory with an ambient temperature of +20 °C to +22 °C over an incubation period of 6.5 h. Figure 2 shows the fluorescent intensities recorded at half hourly intervals using the handheld fluorometer, and the corresponding calibration curves if the assay was stopped after 1, 3 and 6.5 h. The 10 EU·mL−1 LPS standard starts to plateau after 4 h (not shown in Figure 2(a)), but the 1 EU·mL−1 LPS standard continues to increase in intensity linearly (by visual inspection) during this time. Figure 2(b) shows the assay response for the 0 and 0.1 EU·mL−1 LPS standards is greatest when the incubation time is limited to one hour, which is an artefact of the low RFU values for each standard at time zero, as seen in Figure 2(a). The lower time zero data points have a greater effect on the standards when the incubation time is short. Figure 2(a) is normalised to the blank, the standard deviations (SD) of triplicates of the standards including the blank are included to demonstrate the ability to discriminate between the standards.

Bottom Line: In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities.Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls.Sixteen of these samples returned positive LPS detection.

View Article: PubMed Central - PubMed

Affiliation: Cranfield Health, Vincent Building, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, UK. m.barnett.s06@gmail.com.

ABSTRACT
The discovery over the past two decades of viable microbial communities within glaciers has promoted interest in the role of glaciers and ice sheets (the cryosphere) as contributors to subglacial erosion, global biodiversity, and in regulating global biogeochemical cycles. In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities. Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls. In-field measurements were performed using the recently commercialised PyroGene® recombinant Factor C (rFC) endotoxin detection system and used in conjunction with a handheld fluorometer to measure the fluorescent endpoint of the assay. Twenty-seven glacial samples were collected from the surface, bed and terminus of a low-biomass Arctic valley glacier (Engabreen, Northern Norway), and were analysed in a field laboratory using the rFC assay. Sixteen of these samples returned positive LPS detection. This work demonstrates that LPS detection via rFC assay is a viable in-field method and is expected to be a useful proxy for microbial cell concentrations in low biomass environments.

No MeSH data available.


Related in: MedlinePlus