Assay for chemicals
AssigneeNew Mexico State University Technology Transfer Corporation
InventorJames L. Botsford
Life of this patent
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Abstract
A microbiological assay for chemicals, which uses a cell and a reducing dye to quantitatively measure inhibition of electron transport in the cell membrane as a function of chemicals in the substance being tested, is disclosed. This assay and method is reliable, simple, fast, and inexpensive, requires a minimum amount of durable equipment, and avoids the need for the use of live animals as the indicator organisms. The assay is particularly useful for testing for toxicity in food products, environmental, medical and industrial processes, sewage treatment, effluent, agricultural wastes, and chemical dumps.
Claims37
A method of testing for toxicants in a substance comprising the steps of:
- (a) adding a cell to the substance to form a mixture, wherein the cell is selected from the group consisting of plants;
- fungi;
- animals;
- and bacterial cells selected from the group consisting of Bradyrhyzobium japonicum, Arthrobacter crystallopoites, Rhizobium meliloti, Rhizobium leguminosarum, Streptococcus lactis, and Rhodorseudonomas sphaeroides;
- (b) adding to the mixture a dye which undergoes chemical reduction in the presence of the cell;
- and
- (c) quantitatively measuring inhibition of electron transport in the cell as a function of toxicants in the mixture.
A method of testing for toxicants in a substance comprising the steps of:
- (a) adding a cell to the substance to form a mixture, wherein the cell is selected from the group consisting of plants;
- fungi;
- animals;
- and bacterial cells selected from the group consisting of Bradyrhyzobium japonicum, Arthrobacter crystallopoites, Rhizobium meliloti, Rhizobium leguminosarum, Streptococcus lactis, and Rhodopseudonomas sphaeroides;
- (b) adding to the mixture a dye which undergoes chemical reduction in the presence of the cell and changes color;
- (c) incubating the mixture during reduction of the dye;
- (d) quantitatively measuring a change in color intensity of the dye during the incubating step, the change being indicative of the inhibition of electron transport and the decrease in reduction of the dye over time;
- and
- (e) calculating the concentration of toxicants in the mixture from data obtained in the measuring step.
A method of testing for the presence of toxicants in a substance comprising:
- (a) adding a cell having a cytoplasmic membrane to the substance being tested to form a mixture, wherein the cell is selected from the group consisting of plants;
- fungi;
- animals;
- and bacterial cells selected from the group consisting of Bradyrhyzobium japonicum, Arthrobacter crystallopoites, Rhizobium meliloti, Rhizobium leguminosarum, Streptococcus lactis, and Rhodopseudonomas sphaeroides;
- (b) suspending the mixture;
- (c) measuring absorbance in the mixture at an appropriate wavelength at time=0;
- (d) adding to the mixture a dye capable of chemically reducing and changing color in the presence of the cell;
- (e) incubating the mixture, thereby causing reduction of, and a resulting color change in, the dye;
- (f) measuring absorbance of the dye in the cell membrane during the incubating step, the absorbance decreasing over time from the time=0 absorbance to an endpoint absorbance equivalent to an endpoint of substantial reduction of the dye, as demonstrated by a change in color intensity of the dye over time;
- (g) subtracting the time=0 absorbance from the endpoint absorbance to arrive at an absorbance difference;
- (h) plotting the absorbance difference on a Y axis versus the toxicants on an X axis;
- (i) calculating a regression line, and thereby determining a slope of the line, a Y intercept, and a regression coefficient;
- (j) solving an equation Y/2=mX+B for an X value, where Y is the absorbance of a control sample without a toxic chemical, m is the slope, B is the Y intercept, and X is a concentration of toxicant causing a percentage decrease in reduction of the dye;
- and
- (k) calculating the x value to quantitate the toxicity of the toxicants in the substance being tested.
Dependencies are inferred from the claim text.