An enzyme substrate which includes a target portion and a marker portion, hydrolysis of the substrate leading to separation of the target portion from the marker portion, and the target portion being specific for the enzyme activity being assayed. A formulation containing at least one such substrate, and a method for detecting the bacterial species Pseudomonas aeruginosa is also disclosed. The target portion of the substrate is specific for .beta.-alanine aminopeptidase activity and the marker portion is a compound which reveals whether a hydrolysis reaction has taken place or not. The invention is particularly applicable in the field of diagnosis.

The presence or absence of a predetermined target first generation environmental sourced microbe in an environmentally derived sample is determined by adding a testing medium to the sample, or vice versa. The testing medium provides a selective growth medium for the target microbe and includes a specific nutrient which only the target microbe can metabolize. This specific nutrient is modified by attaching a sample-altering moiety thereto, thereby converting the nutrient to a nutrient-indicator. The sample-altering moiety is activated to alter the sample only if the specific nutrient is metabolized by the target microbe. The sample-altering moiety can be a material which changes the color of the sample (visible or non-visible) or changes an electrical characteristic of the sample, or alters some other detectable characteristic of the sample. The testing media does not have to be kept sterile, and the testing procedure does not have to be performed in a sterile environment. The medium also includes an accelerant which hastens the advancement of the target microbes to the log phase of growth during the testing procedure.

The invention is a method for detecting the presence of bacteria in a sample. The method of the invention utilizes the steps of (a) admixing a fluorescent conjugate with a sample to be tested for the presence of bacteria in the sample, wherein a moiety of the fluorescent conjugate is metabolized to release the fluorescent moiety of the fluorescent conjugate; (b) inducing fluorescence of the fluorescent conjugate and the fluorescent moiety of the metabolized fluorescent conjugate at 325-345 nm; (c) simultaneously detecting the fluorescence of the fluorescent conjugate at 370-380 nm and the fluorescent moiety of the metabolized fluorescent conjugate at 445-480 nm; and (d) correlating the detected fluorescence of the fluorescent conjugate and the fluorescent moiety of the metabolized fluorescent conjugate to the presence of bacteria in the sample.

A rapid method for detecting the presence or absence of coliform bacteria in a liquid or liquified dairy sample, for example skimmed milk. A growth medium containing a fluorogenic substrate is combined with the sample and is incubated for a brief period of about 7-9 hours after which a single fluorescence value is measured. Total or thermotolerant coliform bacteria are determined to be present in the sample if the single fluorescent measurement exceeds a predetermined threshold value.

A rapid method for detecting the presence or absence of coliform bacteria in a liquid or liquified dairy sample, for example, skimmed milk, lowfat milk, or whole milk. A growth medium containing a fluorogenic substrate is combined with the dairy sample and is incubated for a brief period of time, for example for about 4 hours, after which a first fluorescence value of 4-methylumbelliferone is measured. The dairy sample is incubated again for about 2-8 hours after which a second fluorescence value of 4-methylumbelliferone is measured. Total, fecal, or thermotolerant coliform bacteria are determined to be present in the sample if the second fluorescent value exceeds the first fluorescent value by a predetermined 4-methylumbelliferone concentration threshold.