Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: ASSESSING NUTRIENT LOSSES, EMISSIONS, AND PATHOGEN TRANSPORT FROM MANURE APPLICATION AND ANIMAL PRODUCTION SITES IN THE WESTERN U.S. Title: Optimizing the extraction, storage, and analysis of airborne endotoxins

Authors
item Dungan, Robert
item Leytem, April
item Verwey, Sheryl

Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2009
Publication Date: N/A

Technical Abstract: While the Limulus amebocyte lysate (LAL) assay is part of most procedures to assess airborne endotoxin exposure, there is no universally agreed upon standard procedure. The purpose of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis of endotoxins. Airborne endotoxins were collected from a large open lot dairy in southern Idaho. We utilized polycarbonate filters to collect endotoxins, sonication extraction, and 0.05% (v/v) Tween 20 in pyrogen-free water (PFW) as the extraction solution. Endotoxin concentrations were determined via the 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 2-fold 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 10,000 x g for up to 15 min, respectively. Field samples and endotoxin standard were 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 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. The endotoxin concentration of replicate samples that received LAL reagent 4 min earlier was 2.5-fold higher. We recommend that endotoxins extracted from polycarbonate filters with PFW-Tween be sonicated for 30 min in glass or polypropylene tubes. Although we routinely vortex our samples before analysis, vortexing for up to 15 min after sonication had no effect on the concentration. The extracts can also be subjected to multiple freeze-thaw cycles, with little or no effect on the final endotoxin concentration. To avoid analytical errors, we also recommend the use of a 96-channel pipette when dispensing LAL reagent into 96-well plates.

Last Modified: 11/27/2014
Footer Content Back to Top of Page