Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i
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DISSOLVED AND PARTICULATE LIPOPOLYSACCHARIDE
SUMMARY: LPS, an obligate constituent of the cell walls of bacteria and cyanobacteria, varies in proportion to total bacterial biomass. Particulate (bound) and dissolved LPS samples are collected from standard hydrocasts and are measured using the Limulus amoebocyte lysate (LAL) reaction. Bound-LPS can be extrapolated to bacterial biomass using laboratory-derived LPS/C relationships.
The outer membrane of all gram-negative bacteria, including photosynthetic prokaryotes (e.g., sulfur and nonsulfur purple bacteria, green sulfur bacteria, cyanobacteria) contains lipopolysaccharide (LPS). Total LPS (T-LPS) exists in seawater in both a particulate or bound form (P-LPS) and a dissolved form (D-LPS). The measurement of P-LPS has been suggested as an indicator of bacterial biomass in the marine environment (Watson et al., 1977; Watson and Hobbie, 1979). Watson has adapted the Limulus amoebocyte lysate (LAL) test for the in vitro detection of bacterial LPS as endotoxin (or pyrogen) and a commercially- available automatic system now exists for the quantitative determination of LPS. During the assay, LPS reacts with LAL which causes an increase in the optical density of the seawater sample. The rate of optical density increase is proportional to the pyrogen concentration. In this analytical procedure, the rate of optical density increase is converted to LPS concentration using lipopolysaccharide standards made up in pyrogen-free seawater. Further extrapolation of LPS to total prokaryote biomass relies upon empirically-determined LPS-to-carbon relationships.
Contamination is the primary concern with the LPS assay, and great care should be used to ensure that the samples are not contaminated. All glass and plastic sampling gear must be pyrogen free. Niskin bottles should be acid-washed before use. The primary source of contamination is from fingertips, therefore plastic gloves should be worn when collecting samples at sea and when analyzing the samples in the laboratory.
3. Sampling and Storage
4. Lab Procedures
5. Data Reduction and Calculations
The quantity of LPS in each sample is determined by the computer software program. LPS concentrations are corrected for dilution in these calculations. P-LPS is calculated by difference between "total" and "dissolved" determinations.