Determination of toxicity

Details Determination of toxicity Determination of toxicity

1996-03-21

1998-04-07

Leary, Louise

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

435 34, 435 4, 435808, 436164, 356 401, 356213, C12Q 102, C12Q 104, G01N 2100

Patent

active

057363544

DESCRIPTION:

BRIEF SUMMARY
This invention relates to a method of monitoring the toxicity of a fluid sample by means of measuring the light output of light emitting microorganisms which have been exposed to the sample. The metering and mixing of the sample medium with a fluidic suspension of the said microorganisms is controlled automatically and the resulting light output interpreted using kinetic theory to relate these data to parameters of toxicolo- gical significance, such as the EC.sub.50 value, and thereby provide a quantitive estimate of the sample toxicity.
A number of procedures for the determination of toxicity using luminescent bacteria are well known. For example, one such procedure involves the addition of a suspension of the bacterium of the genus Photobacterium Phosphoreum to a number of serial dilutions of an aqueous sample. Each diluted aliquot is measured in sequence for the light flux due to P. Phosphoreum metabolism at two preset time intervals. A blank, containing no portion of the sample is included for comparison. The light emission, sample concentration, and time data are used to estimate parameters of toxicological significance, such as the well known EC.sub.50, using a predetermined mathematical algorithm. It is well known that such measurements must be carried out at constant temperature, such that the monitoring apparatus supporting the samples and their dilutions is thermostatically controlled. The bacterium being a naturally occurring species in sea water, requires careful adjustment of sample salinity to maintain constant ionic strength, and thereby constant osmotic conditions, throughout the sample dilution aliquots.
Such toxicity testing is usually applied to aqueous samples, where only 0.5 cm.sup.3 of sample is required for a typical test. The bacterium is used as a liquid suspension reagent that is generated daily by reconstitution from a freeze-dried standard product. The popular embodiment of such a measurement device relies upon intensive manual manipulation of sample, osmotic adjuster and bacterium suspension aliquots. It is typical for the sample cuvettes to be loaded manually into a detection device sensitive to the wavelength of light emitted from the bacteria in each test suspension. A common feature of these procedures is the problem associated with other properties of the sample solution which interfere with the detection of the emitted light. Examples of such properties include samples which are coloured or turbid. In this way the light emission from the admixture between the test sample and the bacterium suspension reagent may be attenuated and lead to false estimates of sample toxicity. Techniques to overcome the effects of the above mentioned attenuation usually involve additional equipment, such as multichambered measurement cuvette, and additional complexity in respect of the optical measurement apparatus and data interpretation procedures.
The present invention is directed towards toxicity testing, employing the well known principle of utilising light emitting organisms in conjunction with kinetic rate theory to provide a measurement approach that reduces the complexity of the toxicity assay and at the same time enhances measurement precision. A further feature is that the kinetic rate approach affords a superior means of fully automating toxicity testing, based upon light emitting organisms.
According to the present invention there is provided a method of determining the toxicity of a fluid sample comprising the steps of: period of time (t.sub.o -t.sub.n) using a photodetector device sensitive to the wavelength of the emitted light; and ##EQU1## to give a measure of toxicity concentration.
The method of determining the toxicity of a fluid sample may comprise the further steps of: a suspension of light emitting organisms; period of time of equal duration to the period (t.sub.o -t.sub.n) to determine the decay in light output due to natural and environmental factors, and output value (E') for use in step (iii).
The steps (iv) and (v) may be carried out simultaneously with steps (i) a

REFERENCES:
patent: 3959081 (1976-05-01), Witz et al.
patent: 4808517 (1989-02-01), Blondin et al.
patent: 5426035 (1995-06-01), Greene et al.
patent: 5441873 (1995-08-01), Knight et al.
Database WPI--Week 9019, Derwent Publications Ltd., London, GB; AN 90-146619 & SU, A, 1 497 218 (Moscow Lomonosov University) 30 Jul. 1989--see abstract.

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