RAPID PROCESS FOR DETECTING PATHOGENIC MICROORGANISMS

Abstract: A rapid process for detecting pathogenic microorganisms in products for human consumption comprises contacting the microorganisms with a methylumbelliferone substrate. The substrate is hydrolyzed into methylumbelliferone by an enzyme given off by the microorganisms. Hydrolysis is accelerated by sodium lauryl sulfate, which renders the microorganisms more permeable to the substrate, the enzyme, or both. The methylumbelliferone is detected by its fluorescence, either in solution or on an agar medium supporting microcolonies formed from individual microorganisms.

Description: RAPID PROCESS FOR DETECTING PATHOGENIC MICROORGANISMS This invention relates to processes for detecting pathogenic microorganisms, such as colifor bacteria and heterotrophic microorganisms, and more specifically has reference to processes for such detection in products for hu consumption which operate in a sufficiently rapid time as to allow treatment of the product before such consumption if needed. It has long been recognized that the quality of product f° r human consumption can be significantly affected by the presence of pathogenic microorganisms. Such products includ drinking water, bathing water, food, food preparation equipment, and, in general, any vector which the microorgani may use to invade the human body. If present in sufficient numbers, such microorganisms can cause product deterioration spoilage, disease, and significant economic loss. However, conventional processes for detecting such microorganisms in such products generally require twenty-four to seventy-two hours to complete. This elapsed time is generally too great provide information about a decrease in product quality in t to take remedial measures. Three groups of microorganisms are commonly sought to detected. Total coliform (TC) bacteria are those normally present in the colon or intestine of humans or animals. Fe coliform (FC) bacteria are those TC bacteria which are generally present in the feces of humans or animals. Heterotrophic plate count (UPC) microorganisms are microorganisms which have an external source of nourishment In this they are like animals which eat food, but are unlik plants which use photosyn hesis to produce an internal sour of nourishment from mere sunlight. The two former groups (TC and FC) are indicators of sanitary quality. A process for their detection is conventionally considered to be "rapid" if it takes less t twenty-four hours to perform. The latter group (HPC) is a indicator of general microbiological quality. A process f its detection is conventionally considered to be "rapid" i takes less than forty-eight hours to perform. Recent development in rapid processes for use in wate analyses are presented in Part 919, pp. 1031-1033, Standar Methods for the Examination of Water and Wastewater (APIIΛ, 1985). They are generally not rapid enough to dete hygienically significant concentration of microorganisms w the ordinary work shift of eight hours. The prior art includes processes for detecting microorganisms and quantifying the concentration thereof by detecting and quantifying the enzymes which the microorganis give off as they metabolize. The enzyme in turn is detected and quantified by its ability to cleave certain chemicals, known as fluorogenic substrates, into products. At least o of these products, the fluorescent portion, fluoresces; tha is, it emits light at one wavelength when irradiated with l of a different wavelength. The fluorogenic substrate, howe does not fluoresce. The presence of fluorescence therefore indicates the presence of bacteria. See, e.g. , Findl et al, U.S. Patent No. 4,242,447. An example of the aforementioned fluorogenic substrate are 4-methylumbelliferone (4-MU) compounds. The 4-methylumbelliferone product fluoresces upon excitation wi light i h a waveleng h of about 365 nm to emit a light it wavelength of about 465 nm. MU derivatives have been used the prior art. Snyder et al. , Appl. Environ. Microbiol. , 5 (5): 969-977 (1986); Feng et al., Appl. Environ. Microbiol. , (6): 1320-1329 (1982); and oumura et al., U.S. Patent No. 4,591,554. "Fluorescence" can be measured by measuring the amount fluorescence after a given incubation time, or by measuring incubation time needed to obtain a given amount of fluorescence. Processes which do this generally take enough time for the microorganisms to multiply exponentially throug several generations. There is, therefore, an exponentially larger amount of fluorescence available to be measured at t end of the incubation period, whether that period is fixed variable. "Fluorescence" can also be measured by frequently samp the amount of fluorescence during the first part of the incubation period, and calculating therefrom the fluorescen velocity; that is, the time rate of change in the amount of fluorescence. Processes which do this generally do not tak enough time for the microorganisms to multiply exponentiall through several generations. Instead, such processes rely the tendency of the microorganisms which are initially pres to produce the same amount of enzyme during each sample per thereby causing a linearly increasing amount of fluorescenc See Snyder et al., above. Such rapid processes have many benefits. First, they by definition, more rapid than those which wait several generations. Second, their accuracy is good, for two reaso One, they measure current activity of the initially present microorganisms, rather than having to make deductions from measurements of the activity of microorganisms several generations later. Two, they generate multiple data points, situation which lends itself well to linear regression analysis, rather than generating the single datum produced the slower processes. Third, the linearity of the data poi (or lack of it) is an indicator of the accuracy of the measurement . Such rapid processes have a drawback as well. Tl.e microorganisms must be concentrated enough to produce a measurable amount of fluorescence velocity in less th n n generation. When further concentration is ot practical, m must be found which enhance the per-organism fluorescence velocit . It is therefore an object of the present invention to provide a process which enhances the per-organism fluoresce velocity of an enzymatic measurement of a dilute sample of pathogenic microorganisms in a product for human consumptio It is a further object of the present invention to enh such velocity while using -methylumbelliferone as the fluorescent product. In accordance with one embodiment of the present invention, a process for assaying, in a brief period of time dilute concentration of living pathogenic microorganisms in sample of product for human consumption, comprises six steps The first step is contacting the microorganisms of the sample with an actuating medium. The actuating medium comprises: (1) a nutrient which is capable of supporting metabolism of the microorganisms; (2) a production agent whi is capable of inducin the production of an enzyme in said microorganisms when the microorganisms are metabolizing; (3) fluorogenic substrate which is capable of reacting with the enzyme to release the fluorescent portion thereof; and (4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorogenic substrate, or both. The second step is incubating the resultant mixture in environment which allows, during the period of incubation, metabolism of the microorganisms, production of the enzyme, contacting of the enzyme (whether inside or outside the microorganisms) with the fluorogenic substrate, and release the fluorescent portion of the fluorogenic substrate. The third step is irradiating, at intervals during the incubation period, the fluorescent portion with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as to cause the fluorescent portion t fluoresce . The remaining steps are measuring, at such intervals, amount of emitted fluorescent light; calculating the velocit of emitted fluorescence; and determining the concentration o microorganisms from such velocity and a preestablished velocity-to-concentration correlation schedule. In a preferred embodiment, the sample is liquid or has been liquefied, and the actuating medium is liquid. The preferred embodiment further comprises, before the contacti step, concentrating the sample by filtering it through a fil fine enough to retain the microorganisms, the subsequent contacting step being accomplishe by placing the filter in actuating medium. In this embodiment, the production agent comprises lactose, the fluorescent portion comprises 4-me hylumbelliferone , the permeability agent comprises sod lauryl sulfate, a d the fluorescent portion is caused to fluoresce with emitted light of a wavelength of about 465 n being excited with light of a wavelength of about 365 nm. When it is desired to measure heterotrophic plate coun microorganisms in a concentration of at least 500 microorganisms per 100 milliliters in at least 15 minutes, enzyme comprises lipase or esterase, the fluorogenic substr comprises 4-methylumbelliferone-heptanoate , and the environ comprises a temperature of about 20°C. When it is desired to measure coliform bacteria in a concentration of at least 60 coliforms per 100 milliliters at least 15 minutes, the enzyme comprises B-D-galactosidase and the fluorescent substrate comprises 4-methylumbellifero B-D-galactoside. For total coliforms the environment compr a temperature of about 35°C. For fecal coliforms, the environment comprises a temperature of about 41.5°C. In accordance with a second embodiment of the present invention, a process for assaying living pathogenic microorganisms in a liquid or liquefied sample of product f human consumption in a concentration of at least 1 microorganism per 100 milliliters, and in a time of at leas six hours, comprises five steps. The first step is filtering the sample through a filte fine enough to retain the microorganisms. The second step placing the filter and microorganisms against a solid or at least semi-solid substance. The substance comprises the sa constituents as are comprised in the actuating medium of t first embodiment of the present invention. The third step is incubating the substance, filter, and microorganisms in an environment which allows, during the period of incubation, metabolism and reproduction of the microorganisms, production of the enzyme, contacting of the enzyme (whether inside or outside the microorganism,s) with t fluorogenic substrate, and release of sufficient of the fluorescent portion of the fluorogenic substrate from each single microorganism and its descendants to form a visible microcolony under fluorescent conditions. The fourth step is irradiating the microcolonies ith li ht of a wavelength sufficiently close to ha of an excitation wavelength characteristic of the fluorescent portion, ani sufficiently intense, as to cause he microcolonies to fluoresce. Finally, one simply counts the number of fluorescent microcolonies. In a preferred embodiment an alkaline solution is added the microcolonies at the end of the incubation period, there enhancing fluorescence and facilitating counting; and the substance against which the filter and microorganisms is pla comprises agar. The remainder of the preferred embodiment o this second embodiment is the same as the remainder of the preferred embodiment of the first embodiment, and detects th same microor anisms. The above and other important features of the present invention will be better understood from the followin detai description, made with reference to the following drawings, hich: FIG. 1 is a graph showing fluorescence production by coliform bacteria in drinking water contaminated with raw sewage (solid circles), with the addition of lactose (open circles) , and with the addition of sodium lauryl sulfate (triangles) ; FIG. 2 is a graph correlating fluorescence velocity wit total coliform concentration, with error bars indicating th range obtained when replicate samples (open circles) are incubated from one original sample; FIG. 3 is a graph similar to FIG. 2, except that it correlates fluorescence velocity with fecal coliform concentration rather than with total coliform concentration FIG. 4 is a photograph of the agar of the secon embodiment, showing individually fluorescing microcolonies; FIG. 5 is a graph correlating measurements obtained fr the agar process with comparable measurements obtained from conventional M 7h FC process; FIG. 6 is a graph similar to FIG. 1, except that it sh heterotrophic plate count (HPC) activity rather than colifo activity; FIG. 7 is a graph similar to FIGS. 2 and 3, except tha correlates fluorescence velocity with IIPC activity rather t total coliforms, and omits replicate samples; and FIG. 8 is a graph similar to FIG. 7, except that, inst of drinking water, it shows results from water sampled from fish farm (open circles during start up; solid circles duri operation) and a nearby river (dashed line). The prese invention makes use of the en yme activity living microorganisms, particularly bacteria but including t fungi, to detect their presence and to quanti y them. En unique to a group of microorganisms can be further used to identify the group to the exclusion of o hers present in the sample . Specifically, the present invention uses enzyme activit as determined by the detection of MU-derived enzyme products detect and quantify two groups of microorganisms, the colifo group of bacteria and heterotrophic microorganism . As not d above, the coliform grou inclu es he total coliforms (TC) an the fecal coliforms (FC). These are considere indicators of he sanitary quality of oo , wate or process equipment; that is, their presence is generally thought to indicate contamination by fecal material. The coliform group constituents possess he en y e B-D-galactosidase . The present invention detects this enzy by the use of 4-MU-B-D-galactoside in the presence of known selective ingredients to exclude the activity and presence any other B-D-galactosidase positive non-coliforms . TC and are differentiated on the basis of temperature: TC are detected at 35°C; and FC are detected at 41.5°C. Heterotrophic activity is commonly measured by detecti heterotrophic bacteria or fungi as colonies appearing on various solid media (e.g. Nutrient agar) or as positive gro in liquid media (e.g. MPN Tube test) after an incubation pe of typically twenty-four to seventy-two hours for mesotroph heterotrophs. For bacteria the result is a heterotrophic p count (HPC) value which, as noted above, gives a general indication of the microbiological quality of the food, wate or equipment in question. The lipase/es erase group of enz is common to the heterotrophs during aerobic or anaerobic conditions of growth. The present invention therefore empl lipase/esterase activity, detecting it with 4-MU-heptanoate a similar manner as described for the coliforms herein. The general procedure for the detection of TC, FC, or heterotrophic activity in the present invention is as follo (a) the sample is concentrated by -passing it through membrane filter (0.2 urn to 0.80 urn pore size); (b) the microorganisms which are retained with the fi are aseptically placed in contact with a sterile medium containing the appropriate 4-MU-substrate; (c) the resulting fluorescence is measured and utiliz as (1) the rate of production of fluorescent produc the liquid medium associated with the sample determined at regular intervals over about fifteen minutes using a fluorescence detecti meter, or (2) the direct observation by eye of fluorescent product associated with colonies resulting f growth (division) of the bacteria originally retained on the filter uring the concentrat step . TC are detected in the manner described above by incubation of the sample plus 4-MU-B-D-galactoside at 35°C, whereas FC are detected by like incubation at 41.5°. Heterotrophic activity is detected in the manner descr above by incubation of the sample plus 4-MU-heptanoate at 20°C. The invention is illustrated by the following examples EXAMP E 1 Total coliform (TC) bacteria were analyzed in the following manner. Natural populations of coliforms were obtained from a river (Nidelven, Trondheim, Norway) receivin sewage effluent and from fresh raw domestic sewage. Samples were collected daily for experiments and diluted in tap wate in varying concentrations to model drinking water with fecal coliform (FC) and nonspecific heterotro hic plate count (HPC bacterial contamination. Concentration of coliforms ranged from 1/100 ml to 1000/100 ml. 500 ml of a water sample were filtered through a 0.45 u pore size 47 mm diameter membrane filter (Millipore) and aseptically placed in a 250 ml flask containing 18 ml of sterile buffered actuating medium. (The 500 ml sample size chosen as the maximum volume of surface v/ater of turbidity < NTU that could be filtered in a short period of time). The medium consisted of phosphate buffered saline (PBS; pll 7.3), 0.02% sodium lauryl sulfate (Sigma Chemical Co., St. Louis, Missouri), 0.56% nutrient broth (Difco Laboratories, Detroi Michigan), and 0.35% lactose. One of the fluorogenic substrates was added to each flask and to a sterile control flask containing the sterile medium, then placed in a shaki water bath. 4-methylumbelliferone-B-D-galactoside (4-MU-B-D-galactoside) was added at a concentration equal t 0.05 mg/ml. The samples were incubated at 35°C for determining TC activity, and at 41.5°C for determining FC activity. Flasks were removed from the bath and placed in a fluorimeter (Turne Model 111) every five minutes for thirty minutes to measure t initial fluorescence velocity. The excitation and emission wavelengths for methylumbelliferone are about 365 and 465 nm using light filters 7-60 and 2A+47B respectively. Fluorescen velocity was determined by least squares linear regression. The rapid fluorescence processes were compared with the conventional enumeration techniques described below. Heterotrophic plate count (HPC) bacteria were recovered on SP (Difco) or R2A agar and counted after three days incubation a 20°C. Total coliform (TC) bacteria were assayed with the membrane filtration technique using m-ENDO agar (Difco) and incubated for enty-four hours at 35°C. The seven hour and standard twenty-four hour membrane filtration processes (APH 1985) were used to recover fecal coliform (FC) at 41.5°C and TC at 35°C respectively. Peptone (0.1%) was used for all serial dilutions. All assays ere done according to Standard Methods (APIIΛ, 1985). Production of methylumbelliferone from 4-MU-B-D- galactoside by coliform bacteria in drinking water contaminat with raw sewage (solid circles) is shown in FIG. 1. Addition of lactose enhanced induction of R-D-galactosidase (open circles). Addition of sodium lauryl sulfate, a common selective ingredient for coliforms, further enhanced activity « (triangles). Enzyme assay temperature was 35°C. The addition of lactose and sodium lauryl sulfate to the medium enhanced galactosidase activity. The specificity of MU-glactosile for members of the coliform group was tested by pretreating water samples with common inhibitors employed in coliform media (Standard Methods, 1985). Most of the activit was retained after pretreatment suggesting that the galactosidase activity was attributable to the coliform grou as tlefine by he conditions of the assay. Replacement of sodium lauryl sulfate by the selective agents used in mT7h medium, polyethylene ether WU-1 and tergitol 7 did not enhan activity (data not shown). Linearity of the rate of product formation was generally high, r = .99 for most assays. Therefore the duration of the assay in the remaining experiments was decreased from 120 minutes to 30 minutes, although 15 minutes was sufficient. FIG. 2 shows the correlation between initial rate of hydrolysis of 4-MU-B-galactoside , as measured by initial fluorescence velocity, and the concentration of total colifor bacteria derived from raw sewage and mixed in drinking water. Enzyme assay temperature was 35°C. Error bars , associated with open circles represent the range of fluorescence velocit obtained when replicate samples (5 < n < 8) are incubated fro one original sample. EXAMPLE 2 Fecal coliform (FC) bacteria were determined in the sam manner as in Example 1 e cept that an incubation temperature equal to ' 41.5°C was used. The results are shown in FIG. 3. Initial rate of hydrolysis of 4-MU-B-D-galactoside by fecal coliform bacteria derived from raw sewage and mixed in drinking water, as measured by initial fluorescence velocity, is compared with fecal coliform bacterial concentration. Enzyme assay temperature was 41.5°C. Error bars associated with open circles have the same meaning as in FIG. 2. EXAMPLE 3 This example demonstrates direct counting of FC. Concentrations of FC < 100/100 ml were analyzed by direct counting. Water samples were filtered and placed on a solid at least semi-solid substance, namely M 7h FC agar (Standard Methods, 1985) lacking the pll indicators and d-mannitol but containing 4-MU-B-D-galactoside. Fluorescent galactosidase positive microcolonies were visible after six hours incubati at 41.5°C with a 366 nm long wave ultraviolet light source (UVL-56, Ultraviolet Products, San Gabriel, California). Th addition of 0.2 ml of an alkaline solution, namely 0.1 N NaO after the incubation period incre sed the fluorescence and facilitated counting. FIG. 4 shows an example of the appearance of the microcolonies after si hours o incubation. FIG. 5 com are concentrations as measured by the agar direct counting proce with concentrations measured by the conventional M 7h FC process. Pluses represent raw sewage contaminated drinking water stored at 15°C and sampled after twenty-four to seventy-two hours for coliforms, and solid circles represent river water samples. The dashed line represents the ideal correlation, and is not fit to the data. The average ratio MU 6h MF to M 7h FC was 1.21 to 1 for thirty-four samples. Sixty-t o flu rescing colonies randomly selected from three experiments showed 100% confirmation as FC by gas production in lauryl tryptose broth incubated at 35°C for twenty-four hours and in EC broth incubated at 44°C for twenty-four hours. In all experiments HPC concentrations ranged from 300/ml to 50,000/ml. EXAMPLE 4 Heterotrophic plate count (IIPC) activity was determined water samples from a river and a drinking water tap. 500 ml sample was filtered through a 0.45 um membrane filter and placed in a flask containing phosphate buffered saline (pll 7.3). The flasks were placed in a shaking water bath at 20°C and 4-MU-heptanoate was added at a concentration equal to 0.0 mg/ml. Fluorescence production was measured in the same mann as in Example 1. The rate of production of methylumbellifero is shown in FIG. 6, and is compared with HPC enumeration in FIG. 7. EXAMPLE 5 Another source of heterotrophic plate count activity was chosen, namely, the water in a recirculation fish farm. Fluorescence velocity is compared with heterotrophic activity in FIG. 8. The samples were taken during a start-up period i 1986 (open circles) and several months later in 1987 (solid circles) when the bacterial population composition had shifte as a function of the changing aquatic environment. The fish farm results are compared with the heterotrophic plate count activity from a nearby river, marked as dashes in FIG. 8 and labeled "Nidelva". The samples were handled in the same man as described in Example 4.

Claims: P A T E N T C L A I M S A process for assaying, in a brief period of time, a dilute concentration of living patl ogenic microorganisms in a sample of product for human consumption, the proces comprising : (a) contacting the microorganisms of the sample with an actuating medium comprising: (1) a nutrient which is capable of supporting metabolism of the microorganisms: (2) a production agent which is capable of inducin the production of an enzyme in said microorganisms when the microorg nisms are metabolizing ; (3) a fluorogenic substrate which is capab1e of reacting wi h the enzyme to release the fluorescent portion hereof; and (4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorescent substrate, or both; (b) incubating the resultant mixture in an environment which allows, during the period of incubation: (1) metabolism of the microorganisms; (2) production of the enzyme; (3) contacting of the enzyme (whether inside or outside the microorganisms) with the fluoresce substrate; and (4) release of the fluorescent portion of the fluorescent conjugate; (c) irradiating, at intervals during the incubation period, the fluorescent portion with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as cause the fluorescent portion to fluoresce; (d) measuring, at such intervals, the amount of emitte fluorescence; (e) calculating the velocity of emitted fluorescence; (f) determining the concentration of microorganisms fr such velocity and a preestablished velocity-to- concentration correlation schedule. 2. The process of Claim 1, wherein the sample is liquid or has been liquefied, and wherein the actuating medium is liquid; the method further comprising, before the contacting step, concentrating the sample by filtering i through a filter fine enough to retain the microorganism the subsequent contacting step being accomplished by placing the filter in the actuating medium. The process of Claim 2, herein: (a) the production ent comprises lactose; (b) the fluorescent portion comprises 4-methylumbelliferone; (c) the permeability agent comprises sodium lauryl sulfate; and (d) the fluorescent portion is caused to fluoresce wi h emitted light of a wavelength of about 465 nm by being excited with light of a wavelength of about 3 nm. 4. The process of Claim 3, wherein: (a) the enzyme comprises lipase or esterase; (b) the fluorogenic substrate comprises 4-methylumbelliferone-heptanoate; (c) the microorganisms comprise heterotrophic plate co microorganisms; (d) the environment comprises a temperature of about 20°C; and (e) the concentration is at least 500 microorganisms p 100 milliliters, and the period of time is at leas 15 minu es. 5. The process of Claim 3, wherein: (a) the enzyme comprises B-D-galactosidase; (b) the fluorogenic substrate comprises 4-methy1u belliferone-B-D-galactoside; (c) the microorganisms comprise coliform bacteria; and (d) the concentration is at least 60 microorganisms pe 100 milliliters, and the period of time is at leas 15 minutes. The process of Claim 5, wherein: (a) the microorganisms comprise total coliforms; and (b) the environment comprises a temperature of about 35°C. 7. The process of Claim 5, wherein: (a) the microor anisms comprise fecal coliforms; and (b) the environment com rises a temperature of about 41.5°C. 8. A process for assaying living pathogenic microorganisms a liquid or liquefied sample of product for human consumption in a concentration of at least 1 microorgan per 100 milliliters, and in a time of at least six hour the process comprising: (a) filtering the sample through a filter fine enough retain the microorganisms; (b) placing the filter and microorganisms against a so or at least semi-solid substance comprising: (1) a nutrient which is capable of supporting metabolism and reproduction of the microor anisms ; (2) a production agent which is capable of induci the production of an enzyme in said microorganisms when the microorganisms are metabolizing ; (3) a fluorogenic substrate which is capable of reacting with the enzyme to release the fluorescent portion thereof; and (4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorogenic substrate, or both; (c) incubating the substance, filter, and microorganism in an environment which allows, during the period o incubation : (1) metabolism and reproduction of the microorganisms ; (2) production of the enzyme; (3) contacting of the enzyme (whether inside or outside the microorganisms) with the fluorogen substrate; and (4) release of sufficient of the fluorescent porti of the fluorogenic substrate from each single microorganism and its escen ants to form a visible microcolony un er fluorescent conditions ; (d) irradiating the microcolonies wi h ligh of a avelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as t cause the microcolonies to fluoresce; and (e) counting the number of fluorescent microcolonies. The process of Claim 8, further co prising adding an alkaline solution to the microcolonies at the end of the incubation period, whereby fluorescence is enhanced and counting is facilitated. 10. The process of Claim 9, wherein: (a) the production agent comprises lactose; (b) the fluorescent portion comprises 4-methylumbelliferone; (c) the permeability agent comprises sodium lauryl sulfate; (d) the enzyme comprises lipase or esterase; (e) the substance against which the filter and microorganisms is placed comprises agar; and (f) the fluorescent portion is caused to fluoresce with emitted light of a wavelength of about 465 nm by being excited x^i h light of a wavelength of about nm. 11. The process of Claim 10, wherein: (a) the enzyme comprises lipase or esterase; (b) the fluorogenic substrate comprises 4-me hylumbelliferone-heptanoate; (c) the microorganisms comp.rise heterotrophic plate co microorganisms; and (d) the environment comprises a temperature of about 20°C. 12. The process of Claim 10, wherein: (a) the enzyme comprises B-D-galactosidase ; (b) the fluorogenic substrate comprises 4-me hylumbelliferone-B-D-galactoside ; an (c) the microorganisms comprise coliform bacteria. 13. The process of Claim 12, wherein: (a) the microorganisms comprise total coliforms; and (b) the environment comprises a temperature of about 35°C. 14. The process of Claim 12, wherein: (a) the microorganisms comprise fecal coliforms; and (b) the environment comprises a temperature of about 41.5°C.

Inventors: BERG JAMES D, BERG, JAMES, D.
Applicants: BERG JAMES D, BERG, JAMES, D.
Application year: 1988
Main classification: C12Q1
IPC codes: C12Q1