The effect of extraction,storage, and analysis techniques on the measurement of airborne endotoxin from a large dairy |
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Authors: | Robert S Dungan April B Leytem |
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Institution: | (1) USDA-Agricultural Research Service (ARS), Northwest Irrigation and Soils Research Laboratory, 3793 North 3600 East, Kimberly, ID 83341, USA |
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Abstract: | The objective of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis
of airborne endotoxin, with a specific focus on samples from a dairy production facility. We utilized polycarbonate filters
to collect total airborne endotoxins, sonication as the extraction technique, and 0.05% Tween 20 in pyrogen-free water (PFW)
as the extraction solution. Endotoxin concentrations were determined via the Limulus amebocyte lysate (LAL) assay. The endotoxin concentrations in extracts after 15 and 30 min of filter sonication were similar,
while the concentration in 60 min extracts was about twofold lower. Rapidly vortexing samples for up to 15 min after sonication
did not increase the endotoxin concentration. However, concentrations were 13 and 26% lower in extracts that were centrifuged
at 1,000 and 10,000g for up to 15 min, respectively. Field samples and endotoxin standard were also sonicated in glass or polypropylene tubes
for up to 120 min. Regardless of the extraction vessel, a decrease in endotoxin concentration occurred when sonicated for
>30 min. Samples and endotoxin standard subjected to 12 freeze–thaw cycles at −20°C only showed a slight but not significant
decrease in endotoxin concentration. Our results also demonstrate the importance of simultaneously adding LAL reagent to 96-well
plates before initiating the LAL assay. |
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