Patent ID: 8518664

Claim:
A method of determining the amount of microorganisms present in a test sample, the method comprising the steps of: a) incubating the test sample with enzyme substrate to form an incubated sample, wherein the enzyme substrate comprises an enzymatically hydrolysable group and a fluorescent group, wherein microorganisms present in the test sample include an enzyme that hydrolyzes the hydrolysable group from the fluorescent group to form a fluorescently detectable product, and wherein the fluorescently detectable product has both an acidic and basic species; b) exciting the fluorescently detectable product with light having a wavelength of Exλiso for a time sufficient for the fluorescently detectable product to emit light, wherein Exλiso is the absorbance isosbestic point of the fluorescently detectable product; c) detecting light emitted at a wavelength of Emλ1; and d) quantifying the light emitted at the wavelength of Emλ1, wherein the quantity of the light emitted at the wavelength Emλ1 is indicative of the amount of microorganisms present in the incubated test sample, wherein the quantity of the light emitted at the wavelength Emλ1 is substantially constant across a pH range of 2.5 to 8.0.