Dr. Wade McGillis, Department of Applied Ocean Physics and Engineering,Woods Hole Oceanographic Institution
Dr. Scott Gallager, Biology Department, Woods Hole Oceanographic Institution
Dr. Robert Jaffe, Environmental Toxcology Laboratory

Introduction:
    A novel approach has been developed for the detection of ultra low levels of biologically active compounds for the purpose of identifying hostile introduction of toxic agents into air and drinking water sources. The new technique builds on the Tetramitus Growth Inhibition Test developed by the Environmental Toxicology Laboratory (ETL) (www.envirolab.com), and adds enhanced sensitivity and rapid (real-time) analysis. Scientists at ETL and the Woods Hole Oceanographic Institution (WHOI) have teamed up to find that the swimming pattern produced by the 10 micron diameter flagellate, Tetramitus rostratus is extremely sensitive, in a dose-dependant way, to compounds which influence flagellar motor activity, including heavy metals, endotoxins, ion channel blockers, and a variety of other noxious compounds. The key was to develop a real-time method for tracking individual cells within a population, quantify motion characteristics such as displacement, velocity, speed, and bearing and then use those characteristics to separate normal cells from those that had been affected by a toxin.  The result is a sensitive, rapid assay for biologically active admixtures and individual toxins which could not be detected by conventional means.
    Although techniques such as mass spectrometry and high pressure liquid chromatography are used to characterize admixtures of toxic compounds, they do not provide information on the bio-toxicity of such mixtures.  In addition, new toxins with unknown mass spectra would not be detected by conventional techniques.  Bioassays are required to determine the relative toxicity of known and unknown compounds to biological systems.

Objectives:
The objective of this preliminary set of experiments was to determine the time dependent change in swimming behavior of the heterotrophic flagellate Tetramitus rostratus following exposure to a relatively low concentration of a standard toxicant.

Methods:
Toxicant: 4-Nitroquinoline-N-Oxide
Concentration and sample points:
control: t0, t49 min
10 ug/mL: t6 min, t24 min, t65 min
100 ug/mL: t0, t42 sec

Results:
Associated with each of the treatments shown below are
1) A video sequence of Tetramitus rostratus cells under dark field illumination
(this is a large avi file [2 Mb] so it may take a few seconds to minutes to download depending on your internet access)
2) Cell tracks or paths of each individual cell within the population automatically identified and labeled.
3) Some motion statistics used to characterize motility of the cell populations. These include:
a. Frequency distribution of the Integral Length Scale.  This is a measure of how auto correlated each path is with itself and what the characteristic length scale is for each path.
b. Frequency distribution of the mean speed along each path
c. Frequency distribution of the Net to Gross Displacement Ratio (NGDR) for each path. The NGDR is a measure of how circuitous a path may be: A NGDR ratio of 1 would indicate a straight line while a ratio near zero would indicate a circular path. This statistic is probably the most sensitive to changes in swimming pattern associated with loss of flagellar activity.
d. Frequency distribution of cell size in microns
e. Speed of each cell along its path.  In this case the cells were monitored for 250 frames or 25 s at 10 fps.
f. The bottom right panel shows some population statistics.  Percentage normal and abnormal are extracted from the NGDR ratio assuming cells exhibiting a ratio greater than 0.5 are normal.

Control
    video
    cell tracks
    6 - panel data plots

Tetramitis cells exposed to 10 ug/mL
6 min Exposure
    video
    cell tracks
    6 - panel data plots

24 min exposure to 10 ug/mL
    video
    cell tracks
    6 - panel data plots

65 min exposure to 10 ug/mL
    video
    cell tracks
    6 - panel data plots

Tetramitus cells exposed to 100 ug/mL
42 seconds
    video
    cell tracks
    6 - panel data plots

Conclusions:
* The Net to Gross Displacement ratio (NGDR) is very sensitive to changes in flagellate swimming pattern and appears to be a good measure of cytotoxicity
* Control populations typically exhibited NGDR above 80%
* Cell populations exposed to toxins showed a rapid shift in the frequency distribution towards zero
* Time scale for measurable changes in behavior were on order seconds for the 100 ug/mL dose and minutes for the 10 ug/mL dose.

Planed activities and those in progress:
With appropriate funding, we plan to complete the following objectives over the next few months:
* Optimize protocols for flagellate culture and evaluate several flagellate species for their sensitivity
* Optimize the flagellate-toxin reaction system by testing at least 10 standard toxicants.
* Design and construct a bench-top Swimming Behavior Spectrometer as a rapid, accurate, and simple assay for cytotoxicity.  To date we have developed a rather large and complex optics based machine vision system for characterizing swimming patterns.  The hardware needs to be reduced in size and the software optimized for user-friendly operation.
* Verification of the SBS and the chosen flagellates within a DoD laboratory using toxins relevant to US security.