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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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.

1. Principle

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.

2. Precautions

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

3.1. Total LPS (T-LPS)
3.1.1. A 20 to 40 ml sample is collected directly from each Niskin bottle into a previously unused, 50 ml plastic centrifuge tube. Do not use a drawing tube as this too is a potential site for LPS contamination. It is important that only pyrogen-free, disposable tubes are used. It is not necessary to rinse the centrifuge tubes.
3.1.2. As soon as the samples have been collected, pipette a 1 ml aliquot from each 50 ml centrifuge tube into a 1.5 ml microcentrifuge tube. Use a new, clean pipette tip for each sample and wear gloves during this operation. After the 1 ml subsample has been removed, tighten the cap securely on the 50 ml sample and store at -20 °C for subsequent laboratory analysis. The 1 ml subsample contained in the centrifuge tube is then immediately processed for D-LPS (see Chapter 17, section 3.2).
3.2. Dissolved LPS (D-LPS)
3.2.1. The 1.0 ml subsample (Chapter 17, section 3.1.2) is immediately centrifuged for 10 minutes in a Beckman microfuge. The supernatant is then decanted into a second pyrogen-free microcentrifuge tube and stored frozen (-20 °C) until assayed.

4. Lab Procedures

4.1. Turn on computer-assisted LAL-5000 and follow menu to set up the instrument. The LAL-5000 must reach the appropriate operating temperature before the assay can begin, this can take up to 2 hours. The instrument should, therefore, be turned on and set up to accept samples before samples are prepared for analysis.
4.2. Dilute the commercially-available pyrotell-GT reagent with 5 ml of pyrogen-free seawater, as described on the label.
4.3. Set up an endotoxin standard curve in combusted 10x75 mm glass culture tubes. The standard curve should contain two pyrogen- free blanks and one tube each of the following concentrations: 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1 ng endotoxin ml-1. Each tube should contain a total of 0.4 ml of the appropriate solution.
4.4. Dilute three replicate aliquots of each sample into a combusted 10x75 mm glass culture tube. Dilute each sample 1:20 with pyrogen-free seawater to make a total of 0.4 ml of diluted sample per tube (i.e., 380 µl of pyrogen free seawater and 20 µl of sample). Extreme caution must be taken to avoid contamination. Use new pipette tips for each sample and wear gloves throughout the sample preparation procedure.
4.5. Immediately before positioning the samples and standards in the LAL-5000 for analysis, add 100 µl of diluted pyrotell- GT to each tube and vortex for approximately 10 seconds. Process each tube sequentially and immediately place the tube in the instrument after vortexing. Try to do this part of the assay as reproducibly as possible. Variability between samples in this procedure will directly contribute to variability in measured LPS concentrations between replicates.

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.

6. Equipment/Supplies

  • new, pyrogen-free 50 ml and 1.5 ml centrifuge tubes

  • LAL-5000 (Associates of Cape Cod) and MS-DOS computer

  • automatic pipettes and tips

  • combusted 10x75 mm glass culture tubes and appropriate rack

  • parafilm

7. Reagents

  • pyrogen-free seawater (prepared by UV irradiation of surface seawater)

  • pyrotell-GT (Associates of Cape Cod)

  • endotoxin standard (Associates of Cape Cod)

8. References

  • Watson, S. W. and J. E. Hobbie. 1979. Measurement of bacterial biomass as lipopolysaccharide. In: Native Aquatic Bacteria: Enumeration, Activity and Ecology, J. W. Costerton and R. R. Colwell, editors, American Society for Testing and Materials, Baltimore, pp. 82-88.

  • Watson, S. W., T. J. Novitsky, H. L. Quinby and F. W. Valois. 1977. Determination of bacterial number and biomass in the marine environment. Applied and Environmental Microbiology, 33, 940-946.