MXPA00000953A - Method and devices for detecting and enumerating microorganisms - Google Patents

Abstract

A method for detecting a microorganism in a test sample is described. The method involves distributing microvolumes 0.01-25 microlitres of a sample to a plurality of microcompartments of a culture device, incubating for a time sufficient to permit at least one cell division cycle of the microorganism, then detecting the presence or absence of the microorganism in the microcompartments. Also disclosed are devices for carrying out these methods.

Description

METHOD AND DEVICES FOR DETECTING AND LISTING MICROORGANISMS DESCRIPTION OF THE INVENTION This invention relates to methods and devices that use microvolume compartments to effect rapid and accurate detection and enumeration of microorganisms. In the detection and enumeration of microorganisms is practiced in numerous scenarios, including the food processing industry (or test the contamination of food by microorganisms such as E. coli and S. aureus), the health care industry (to test samples of patients and other clinical samples to detect infections or contamination), the environmental control industry, the pharmaceutical industry and the cosmetics industry. Detection based on the growth and enumeration of microorganisms is commonly practiced using liquid nutrient media (most likely number analysis (MPN)) or semi-solid nutrient media (direct counting using, for example, Petri dishes with agar). Enumeration using the liquid MPN method is typically accomplished by placing 10-fold serial dilutions of a sample of interest in duplicate sets of tubes containing selective media and chemical indicators. The tubes are concealed at temperature REF .: 32679 elevated (24-48 hours) followed by the growth examination of the microorganism. A statistical formula is used, based on the number of positive and negative tubes per set, to estimate the number of organisms present in the initial sample. This method for performing the MPN analysis has several disadvantages. It is hard work due to the multiple steps of dilution and pipetting to perform the analysis. In addition, it is only practical to use duplicate sets of approximately 3 to 5 tubes per dilution. As a result, the 95% confidence limits for an MPN estimated for microbial concentration are extremely broad. For example, an estimated three-pipe MPN of 20 has 95% confidence limits ranging from 7 to 89. In contrast to the method described above, a direct count of viable microorganisms in a sample can be achieved by spreading the sample over an area defined using nutrient media containing a gelling agent. The gelling agent (agar) prevents the diffusion of the organisms during the incubation (24-48 hours), producing a colony in the area where the organism was originally deposited. There is, however, a limit on the number of colonies that can be placed on a given area of nutrient media before fusion with neighboring colonies, affecting the accuracy of counting. In this way, usually it is necessary to make several dilutions for each sample. In addition, the classes of chemical indicator molecules that can be used to identify individual types of microorganisms present with a mixed population are limited to those that produce a product that is insoluble in the gelled medium. In addition to these disadvantages, both of the MPN analysis and gel-based systems currently used require a relatively long incubation time before a positive result can be detected. The method of the present invention solves the problems associated with the systems currently used. In general, this invention provides a method for effecting rapid and accurate detection and enumeration of microorganisms based on the surprising result that the use of microvolumes substantially increases the detection rate. As used herein, the term "microorganism" includes all microscopic organisms and living cells, including without limitation bacteria, mycoplasmas, rikettsias, spirochetes, yeasts, molds, protozoa, as well as microscopic forms of eukaryotic cells, e.g. unique (cultured or derived directly from a tissue or organ) or small clusters of cells. Microorganisms are detected and / or enumerated not only when they are directly detected whole cells, but also when such cells are detected indirectly, such as through the detection or quantification of cell fragments, biological molecules derived from cells or cellular by-products. In one aspect, the invention features a method for detecting microorganisms in a liquid test sample. The method involves the step of: distributing microvolumes of the sample to a plurality of microcompartments of a culture device; incubating the culture device for a sufficient time to allow at least one cycle of cell division of the microorganism; and detect the presence or absence of the microorganism in the microcoparties. As used herein, the term "microvolume" refers to a volume of between about 0.01 and about 25 microliters, and the term "microcompartments" refers to a compartment that has a capacity, or volume, to hold a microvolume of a liquid test sample. . In preferred embodiments, the method further includes the step of quantifying microorganisms in the sample of the liquid test. The quantification may include the steps of determining the MPN in the sample, or it may involve enumerating the microorganisms in each micro-compartment of the culture device.

In other embodiments, the micro-compartments may contain a coating of nutrient medium, and the nutrient medium may further include at least one indicator substance. Alternatively, the liquid test sample may include at least one indicator substance. In any case, the indicator substance can be any indicator substance capable of providing a detectable signal in the liquid test sample. Such indicators include, but are not limited to, chromogenic indicators, fluorescent indicators, luminescent indicators and electrochemical indicators. For the purposes of this application, the term "electrochemical" means a chemical indicator that changes the resistance or conductance of the sample after the reaction with the microorganism. In another aspect, the invention features a method for detecting a microorganism in a liquid test sample. This method involves the steps of: distributing sample aliquots to a plurality of microcompartments of a culture device, where the culture device contains a plurality of sets of microcompartments, each set has microcompartments of uniform size and the sets vary in size of the microcompartment; incubate the culture device for a sufficient time to allow at least one cell division cycle of the microorganism; and detect the presence or absence of the microorganism in the microcompartments. In preferred embodiments, the microcompartments of those methods are of uniform size and each microcompartment has a volume of from about 0.01 to about 25 microliters. More preferably, each microparticle has a volume of about 0.1 to about 10 microliters, and even more preferably, about 1 to about 2 microliters. The culture device preferably contains from 1 to about 100,000 micro-compartments, more preferably from about 100 to about 10,000 micro-compartments, still more preferably from about 200 to about 5,000 micro-compartments, and more preferably from about 400 to about 600 micro-compartments. . In another aspect, the present invention presents an assay device. The device includes a substrate having the plurality of microcompartments therein, each microcompartment having an upper part and a lower part. The substrate may include a hydrophobic "land area" between the microcompartments. Preferably, the microcompartments include reagents from test, for example nutrients, gelling agents or indicator substances such as chromogenic indicators, fluorescent indicators, luminescent indicators or electrochemical indicators. To prevent the formation of air bubbles when the liquid sample is loaded into the wells, some of the microcompartments may have openings in both of their upper and lower parts. The openings in the lower surface are occluded by a material that is permeable to air but substantially impermeable to aqueous liquids. In still another aspect, the device contains microcompartments in the form of microchannels. The microchannels may be contained on the single layer or multiple layer substrate, such as the film. The device may or may not have a hydrophobic land area between the microchannels. The microchannels may comprise elongated holes that are formed in the substrate. In a preferred embodiment, the microchannels are covered with a film. The microchannels of the microcompartment may comprise capillary tubes. Discrete capillary tubes can be formed / joined together to form a device. The microchannels preferably have at least one test reagent coated thereon.

The microcompartments can be arranged in substantially parallel rows. Typically the volumes of the microcompartments in each row are uniform. Alternatively, micro-compartments can be arranged in several groupings or patterns to recognize and count positive signals more easily. The volumes of the microcompartments may range from about 0.01 to about 25 microliters, more preferably from about 0.1 to about 10 microliters, more preferably from about 1 to about 12 microliters. As described herein, the present invention has several advantages. First, the use of microvolumes in microcompartments allows the surprisingly rapid detection of a microorganism in a liquid test sample. Second, this rapid detection allows rapid enumeration or quantification of microorganisms from the liquid test sample. The invention is particularly useful in the MPN analysis of a liquid test sample for a particular microorganism, such as E. coli or S. to ureus. The invention allows the MPN analysis to be conducted conveniently in a single device, in the position to the separate tubes, and advantageously requires a substantially shorter incubation time to achieve a growth of the detectable microorganism. Third, the use of microvolumes in microcompartments allows the separation of a liquid test sample in a relatively large number of test volumes. In general, the use of microvolumes in microcompartments provides a much greater number of tests, or repetitions, of a test on the liquid sample. In the case of MPN analysis, the use of microvolumes in microcompartments provides a greater number of data points from which the MPN can be calculated, thus significantly narrowing the 95% confidence limits for a result of the MPN given. Fourth, the separation of the sample in a large number of test volumes allows a higher concentration of microorganisms to be enumerated, thereby reducing or eliminating the dilutions of the sample. Fifth, this invention allows the MPN analysis to be conducted in a single device that has the indicators and / or nutrients directly coated therein. Sixth, this invention allows a wide range of counting when performing the MPN analysis. Figure 1 is a perspective view of a modality of a device for cultivating itdcrocoi artimientos. Figure 2 depicts a plan view of a microcompartment growing device having sets of microcompartments that vary in the volume of the microcompartments.

Figure 3 is a side view of a microcompartment device having open microcompartment bottoms occluded by a nonwoven fabric material. Figure 4 is a graphic description of the increased enzymatic kinetics from the use of microcompartments. Figure 5 is a graphic description of the increased kinetics from the use of microcompartments. Figure 6 is an exploded perspective view of a single layer microcompartment device having microchannels. Figure 7 is a perspective view of a multilayer microcompartment device having microchannels. Figure 8 is a cross-sectional view of a single layer microcompartment device having microchannels. This invention relates to the use of aliquots of liquid samples at the microvolume level in microcompartments in the detection based on a signal of the microorganisms in liquid samples. Among the problems encountered in the technique related to the testing of liquid samples for the presence or quantity of microorganisms are the times of relatively long incubation, the need to use separate containers for the aliquots being tested and the need for a relatively large volume of sample for testing. The present invention provides a solution to those and other problems associated with such tests. The invention provides a method for detecting the presence, amount, or absence of a microorganism in a liquid sample by distributing microvoluner to microcompartments in a test device. A "microvolume" as the term used herein, refers to a volume less than about 25 microliters, and includes volumes in the submicroliter range. The inventors of the present have discovered that the use of microvolumes in the detection of microorganisms in liquid samples results in remarkably shorter incubation times required to produce a growth level based on a detectable signal. Because shorter incubation times are highly desirable in this field, this feature of the invention provides a distinct advantage. In addition, the use of a relatively large number of microvolume compartments significantly narrows the 95% confidence limits for the results and reduces the number of dilutions of the sample for concentrated samples.

In addition to the above advantages, the use of microvolumes in the testing of liquid samples may allow the use of substantially smaller test samples. Very small volume test samples are sometimes necessary due to very small volume or desirable sample sources for purposes such as ease of handling. The inventors of the present have developed a number of novel devices for microvolume-based tests of liquid samples. Non-limiting examples of such devices include a substrate, such as micro-patterned or pressed films having a plurality of micro-compartments and having various surface treatments to improve performance and convenience and micro-printed or pressed films having a plurality of mircro-compartments open at the bottom , wherein each opening at the bottom of the well is occluded by a material that is permeable to air but that is substantially impermeable to aqueous fluids. The open configuration at the bottom can help eliminate the potential problem of air bubbles that are trapped in the sample in the microcompartments. When viewed in a plan view, the microcompartment may have, for example, a generally circular, faceted, square, oval or elongated appearance. It should be noted that the microcompartments of these devices can have many possible ways, such as cylindrical, conical, pyramidal, hemispherical, tetrahedral, cubic, truncated and similar shapes, with open or closed bottoms. Another example includes a substrate, such as a plastic film containing microchannels, where the liquid sample can be moved to the microchannels by capillary action. The microchannels can be discrete capillary tubes that are formed or attached to a substrate. The cross section of each channel can take many forms, including circular, triangular, square and rectangular shapes and the like. In a preferred embodiment, the cross section of the ends of the microchannels is smaller than the cross section of the middle part of the microchannels. In this configuration, the sample is less likely to spill during handling of the devices. Advantageously, the devices summarized above allow testing liquid samples using aliquots of microvolumes in a single device, eliminating the need for separate containers. A test sample can be distributed among hundreds or thousands of discrete micro-compartments, thereby substantially increasing the number of data points in a liquid sample test. A particularly useful application of these methods and devices is in the detection and enumeration based on the growth of microorganisms in liquid test samples. Such growth-based detection and enumeration is very important for testing food, environmental, clinical, pharmaceutical, cosmetic and other samples to determine contamination by microorganisms. The methods and devices of this invention allow efficient, accurate, convenient, and inexpensive testing of such samples. The preferred use of the methods and devices of this invention is in the MPN. The amount of work is greatly reduced because it is not necessary to pipette into individual tubes. Instead, the liquid sample is distributed to the microcompartments by methods such as loading a single device and distributing the sample over the microcompartments. In addition, less dilution of the test is necessary due to the large number of microcompartments in the devices. The relatively large number of microcompartments also provides an estimate of the most accurate microbial concentration. This is due to the correspondingly greater number of data points provided and a corresponding narrower confidence limit interval. Accordingly, the present invention provides a method for detecting a microorganism in a liquid test sample. The method involves first distributing microvoids of the test sample to a plurality of microcompartments of a cultivation device. The culture device can be any device that contains a plurality of microcompartments and can be loaded with the liquid test sample. Non-limiting examples of such culture devices include those described herein. The micro-compartments in the culture device are preferably of uniform size and each microcompartment has the capacity to contain a volume of about 0.01 to about 25 microliters of the liquid sample. In a preferred embodiment, each microcompartment has a volume of from about 0.1 to about 10 microliters. In another preferred embodiment, each microcompartment has a volume of about 1 to about 2 microliters. The culture device preferably containing from about 1 to about 100,000 micro-compartments, more preferably from about 100 to about 10,000 micro-compartments, still more preferably from about 200 to about 5,000 micro-compartments and more preferably from about 400 to about approximately 600 microcompartments. The use of a device having from about 400 to about 600 Microcompartments is particularly useful in the context of testing a liquid sample for the concentration of a microorganism using MPN. Certain regulatory requirements may dictate that a test method must be capable of detecting a microorganism in a sample of about five milliliters. Such a sample size is standard in the food processing industry for microbiological testing. Thus, for example, a culture device having 500 microcompartments, where each microcompartment has a volume of approximately 2 microliters, would be very useful for testing a 1 ml sample. The size of a 2 microliter microcompartment allows rapid development of a detectable signal according to the invention, and the use of about 400 to about 600 microcompact provides a sufficiently large number of data points to substantially improve the confidence interval for a MPN calculation. In addition, it is feasible to carry out a manual count of positive tests of the microcompartments for the microorganism of interest. The liquid test sample can be any sample containing microorganism from any source. The sample can be distributed to the plurality of the microcompartment directly, or the sample can be diluted before distribution to the microcompartments.

The determination of whether dilution of the sample is necessary will depend on a variety of factors such as the source and age of the sample, and such determination is a routine matter for those skilled in the art. The liquid test sample may include selective nutrient growth media, optionally including a gelling agent, for the microorganism of interest and / or indicator substance that produces a signal in the presence of the growing microorganism. A gelling agent is a material that absorbs water that becomes a gel after the addition of water. If a gelling agent is used, the gel will preferably be encapsulated, or will contain the growing microorganism. One or both of the selective nutrient growth media and the indicator substance may be present in the micro-compartments, in sufficient quantities to reach the desired concentrations when a microvolume of liquid test samples is distributed in the micro-compartments. The coating can be achieved by placing or distributing a solution of the nutrient medium and / or indicator substance in the micro-compartments and dehydrating the solution to produce a coating of the nutrient medium and / or indicator substance in the micro-compartments. A wide variety of selective growth media is known for a wide variety of microorganisms of interest, as well as a wide variety of indicator substances for a wide variety of microorganisms, and any of those indicating means or substances may be suitable for use in the method of the invention. Soluble indicators can be used in the present invention, because diffusion is prevented by confinement within the microcompartments. The method by which the liquid test sample distributed to the plurality of microcompartments depends on the particular culture device employed in the method. If a film device containing micro-compartments is used, the sample can simply be poured or pipetted onto the device and the sample distributed over the micro-compartments with, for example, stirring, a blade or other tool. If microchannels are used, the sample can be distributed in the microchannels via capillary action. After the sample is distributed to the micro-compartments of the culture device, the culture device is optionally covered or sealed to close the micro-compartments and then incubated for a sufficient time to allow at least one cycle of cell division of the microorganism. In general, the incubation of the culture device is conducted at about 25-45 ° C, preferably at about 30-37 ° C. In the practice of this invention, in which micro-compartments are used, the incubation time will vary. For example, when bacteria are detected and enumerated mostly, the incubation time will typically range from about 20 minutes to about 24 hours to produce detectable growth as demonstrated by the indicator substance in the incubated liquid test sample. The relatively short incubation time represents a distinct advantage over currently used detection methods, which typically require incubation times of about 24 hours or more. After incubation of the culture device, the presence or absence of the microorganisms in the micro-compartments (and thus in the liquid test sample) is detected. The mode and sensitivity of the detection depends on the type of indicator substance used in the method. In some cases, the presence or absence of microorganisms can be detected visually without the help of the indicator substance that generates the signal, visualizing the turbidity or clarity of the sample in each microcompartment. Any indicator substance that provides a detectable signal in the liquid test sample can be used, including but not limited to chromogenic indicators, fluorescent indicators, luminescent indicators, electrochemical indicators, and Similar. The presence or absence of a microorganism in a microcompartment can be detected visually, with the naked eye, microscopically, or with the help of other equipment or methods. There are numerous indicator substances and signal detection systems known in the art to detect microorganisms, and any such substance or systems according to the present invention can be used. The detection of the icroorgans in the liquid medium can also involve the enumeration of a microorganism count in the liquid test sample. In a preferred embodiment, the enumeration is performed using MPN. Once the number of microcompartments containing a microorganism of interest has been determined, the MPN can be calculated using the known MPN techniques. If desired, the number of microorganisms in an individual microcompartment can then be determined using known techniques, for example, signal strength compared to a known standard, or by plating the contents of the microcompartment. Advantageously, the largest number of compartments used in the method of the invention allows narrower ranges for the 95% confidence limits in an MPN analysis of a liquid test sample. Due to the greater number of microcompartments in a single device that provides the methods and devices of the present invention, it is possible to use a only device in the detection and enumeration of multiple microorganisms of interest, still retaining the advantages of the invention. For example, a single liquid sample could be tested for the presence or concentration of E. coli and S. aureus. A portion of a culture device could contain micro-compartments for the detection and enumeration of one of those microorganisms by, for example, coating a set of micro-compartments with a selective growth medium and a first indicator substance selected to detect that microorganism. A second set of macro-compartments could be coated with a selective growth medium and a second indicator substance selected to detect another microorganism of interest. Alternatively, any microcompartment of a culture device can be coated with assay reagents designed for the simultaneous detection of multiple microorganisms. For example, E. coli could be detected with a fluorescent indicator substance while, at the same time, coliforms could be detected with a chromogenic indicator substance. In another embodiment, the invention relates to a method for detecting a microorganism in a liquid test sample. The method is similar to the method described above, except that the distribution step involves distributing microvolumes from the liquid test sample to a plurality of microcompartments of the culture device, wherein the culture device includes a plurality of microcompartment assemblies. Each set of microcompartments has compartments of uniform size, and the device has at least two sets of microcompartments. For example, the culture device could include a plurality of lanes or other groupings, each containing microcompartments of a particular volume. This feature allows the distribution of the liquid test sample in different test volume sizes, including volume sizes greater than the microvolume size within a single device. In the MPN, this feature provides a significant advantage since, for a highly concentrated sample, the appropriate volume size can be selected and the MPN analysis can be performed using a single distribution step in a single device without the need for serial dilutions. As stated above, the method of this invention can be practiced using any culture or test device that contains microcompartments, depending on the particular modality that is being practiced. The inventors of the present invention have developed novel devices suitable for use in the methods of this invention. The following are non-limiting examples of such devices Referring to Figure 1, a device 10 may comprise a substrate 12 having a plurality of compartments in the form of micro-compartments .14. The substrate 12 can be manufactured from any material in which micro-compartments can be made. The substrate 12 can be manufactured, for example, from polymeric films or other suitable materials. Suitable polymers include without limitation polyethylene, polypropylene, polyamides, fluoropolymers, polycarbonates, polyesters, polyurethanes and polystyrenes. The micro-compartments 14 can be formed by any process appropriate for the material of the substrate 12. Such processes include without limitation thermal embossments, molding, laser punching and etching with reactive materials. Alternatively, the device can be prepared by laminating a sheet of designed material containing a plurality of small openings on a support film, where a microcompartment is formed by combining the opening and the supporting film. The polyethylene or polypropylene films may be, for example, pressed or extruded stamped, and may include various pigments and surfactants. The device 10 can include any desired number of microcompartments. Additionally, the device 10 may include relatively reservoirs Large or other compartments adapted to contain large volumes of liquid for maintaining a moisture level. appropriate inside the device. Although the number of microcompartments may be relatively small (eg, 2-50), the small sizes of the microcompartments allow relatively large numbers of microcompartments to be manufactured on a single device 10. Preferably, the device has approximately 1 to 100,000 microcompartments. , more preferably from about 100 to about 10,000 micro-compartments, still more preferably from about 200 to about 5,000 micro-compartments, most preferably from about 400 to about 600 micro-compartments. The device 10 can have a population of micro-compartments of uniform size 14, although the micro-compartments do not need to be of uniform size. For example, a device 16 such as that described in Figure 2 may have assemblies (e.g., rows) of microcompartments in which volumes are constant within a set, but may vary between sets. As described in Figure 2, the volumes may vary increasingly in an array of microcompartment assemblies, with the smallest microcompartment 18 containing volumes of submicroliters and the larger micro-compartments 20 containing multiple volumes of the order of microliters. It is still possible for the larger microcompartments in a device as described in Figure 2 to include microcompartments 22 that would not be classified as "microcompartments". Such microcompartments may contain, for example, substantially more than 25 microliters up to volumes of the order of milliliters. The test reagents can be covered within the microcompartments of the device. Such test reagents may include without limitation nutrients for the growth of microorganisms, gelling agents, indicator substances such as chromogenic indicators, fluorescent indicators, luminescent indicators and electrochemical indicators. The assay reagents can be immobilized in the microcompartments by any of numerous methods for immobilizing assay reagents on solid substrates known to those skilled in the art. Such methods include for example drying the liquids containing the assay reagent in the microcompartments, as well as other methods for non-covalently binding biomolecules and other assay reagents to a solid substrate. In a preferred embodiment, the microcompartments are manufactured to prevent entrapment Air bubbles when the micro-compartments are loaded with liquids from aqueous samples. This can be achieved, for example, by making openings in the bottom of the well which are permeable to air and which at the same time are substantially impervious to aqueous liquids. One such embodiment is described in Figure 3. In this embodiment, a test device 24 contains microcompartments 26 having upper portions 27 and lower portions 29 which are prepared with holes 28 through the bottoms. These orifices are occluded (clogged or covered) with material 30 which is permeable to air but substantially impervious to liquids of aqueous samples. The occluder material can be any composition having the desired permeability characteristics. In the preferred embodiment described in Figure 3, the occluder material is a non-woven fabric 30 attached to the bottom 29 of the microcompartment 26. Preferably the non-woven fabric 30 is an easily bonded blown fiber pressure sensitive adhesive material. at the bottom 29 by means of pressure. During the loading of the sample in the microcompartments 26, the bubbles are not formed or rapidly dissipated after their formation, leaving each well with approximately the same volume of the liquid sample as described in Figure 3. As discussed above, The presence of microcompartments in a test device allows the separation of a liquid test sample in a relatively large number of test microvolumes. The ability to separate a liquid sample in microcompartments and to perform MPN or other tests without cross-contamination between the compartments is a major advantage of the devices present. Various additional manufacturing methods can be used, however, to further improve the separation function of the microcompartments, as described below. Referring again to Figure 1, the area 13 between the microcompartments 14 ("land area") can be manufactured to be hydrophobic. This serves to prevent aqueous fluids from forming bridges between the micro-compartments 14, thus preventing cross-contamination. The land area 13 can be made hydrophobic in several ways. For example, the land area on an extrusion-stamped polyethylene film, which has been rendered hydrophilic by the incorporation of a surfactant, may become hydrophobic by transferring a thin layer of acrylated silicone or other hydrophobic material to the land area. Referring to Figure 6, the microcompartments can be fabricated as microchannels 32 into a substrate 34. The shape of the microchannel 32 may vary. The microchannel can be of square bottom, in the form of U and V or comprise elongated holes. Preferably, the microchannel 32 is converted to prevent evaporation of the channel and contamination of the channel. The coating 36 can be prepared from any suitable material that is at least partially impervious to water vapor. For example, the coating may comprise a silicone pressure sensitive adhesive film or a heat sealable film. The test reagents can be coated in the microchannel. Preferably, at least one of such reagents is coated in each microchannel. In a preferred alternative embodiment, as described in Figure 7, the individual layers of the microchannel film 32 therein can be laminated together to form a multilayer structure 38. This structure has many advantages, including having a large number. of microcompartments in a small area and ease of inoculation of a greater number of microcompartments. As described, shaded channels 33 represent channels that have a positive indication for the target microorganism. Alternatively, as described in Figure 8, the device 40 may comprise a plurality of capillary tubes 42 that are attached or formed together on a substrate, as described in Figure 7. The capillary tubes may be open ended or may be partially closed at one end. The following examples are offered to help understand the present invention and should not be construed as limiting the scope thereof. Unless otherwise indicated, all parts and percentages are by weight.

EXAMPLE 1 Stamped Film Culture Devices Stamped film culture devices containing a plurality of microcompartments and can be used for the detection of microorganisms in a liquid test sample were constructed as described in this example. Microcompartments can be formed into a substrate by a process number, examples of which are thermal embossing, molding, laser punching and etching the surface with a reactive material. Detailed descriptions of how to make cavities (i.e., "microcompartments") in polymeric films is provided in U.S. Patent 5,192,548; 5,219,462; 5,344,681; and 5,437,754. The following descriptions are representative of farming devices of specific stamped film used in the later examples.

A. Press Stamped Films Containing a Plurality of Microcompartments Be molded by polyethylene extrusion (Eastman Chemical Company Resin # 18B0A) with a content of 10% by weight of Ti02 (50% TiO2 / 50% Polyethylene Pigment Concentrate) and 0.5% by weight of Triton X-35 surfactant (Sigma Chemical Company) in a film (4 mils thick (101.6 μm)). The film was cut into sheets and stacked (-20 sheets) onto a photolithographically recorded magnesium alloy tool prepared as described in U.S. Patent 5,192,548, designed to form a plurality of microcompartments. The engraved magnesium tool contained projections or protuberances arranged in the patterns described in the later examples. The stacked polyethylene sheets were stamped on a hot hydraulic press (132 ° C, 120 second drying time) as described in US Patent 5,219,462. The samples were allowed to cool, at which time the tool was removed to provide a single layer film containing the "negative" image of the tool.

B. Extruded Stamped Films Containing a Plurality of Microcompartments A magnesium-engraved master tool was photolithographically attached to a steel roller using a pressure sensitive transfer adhesive. The polyethylene / pigment and surfactant composition described in Example IA were mixed and molded by extrusion onto the roll as described in US Pat. No. 5,192,548, which is incorporated herein by reference. Samples without Triton X-35 were also prepared in this manner.

C. Extrusion-Stamped Films with Hydrophobic "Earth" Area Extrusion-stamped polyethylene films containing Triton Surfactant X-35 were prepared according to Example IB. The area between the microcompartments ("land" area) became hydrophobic by transferring a thin layer of acrylated silicone (Goldschmidt FC 711) containing 4.8% crosslinking agent (Daracur 1173) using a roll-to-roll coating device (Straub Design Con.). The hydrophobic coating was cured by exposing the film to ultraviolet radiation under a nitrogen atmosphere using a Fusion Systems UV lamp with a H bulb that provides a dose of 85 milijoules / cm2. An aqueous solution containing phenol red indicator (to provide contrast) was distributed over treated and untreated samples. The samples treated with the hydrophobic coating were shown to a partition liquid in individual microcompartments without formation of fluid bridges between the microcompartments.

D. Press Stamped Films Containing a Plurality of Microchannels The polyethylene film (Example IA) was cut into sheets and stacked (~ 10 four mil sheets (101.6 μm)) onto a magnesium tool designed to form a plurality of microchannels parallel, followed by stamping on a hot hydraulic press according to the following protocol; heat to 143 ° C, keep at 0.7 N / m2 for 1 minute, increase the pressure to 2.8 N / m2 and keep for 1 minute, decrease the pressure to 2.1 N / m2 and keep for 15 seconds, cool to 29 ° C , and release. The tool was removed to provide a single layer film containing the "negative" image of the tool. After stamping, a polyester (PE) support material containing a silicone pressure sensitive adhesive (PSA) was laminated (CW14HT, Specialty Tapes, Racine, Wl) to the top of the stamped film, thus creating a series of parallel covered microchannels.

EXAMPLE 2 Embossed Film Culture Devices with Bottom-Perforated Micro-compartments Embossed film culture devices containing a plurality of micro-compartments perforated in the bottom, and having the openings in the bottom coated with a non-woven fabric backing, are They built as described in this example. Filling efficiencies of the test sample and leakage of those culture devices were evaluated and are also described in this example.

A. Preparation of Stamped Film Culture Devices Perforated in the Bottom Polypropylene (18 mils (457.2 μm) thick, Conplex Co) was stamped as described in Example IA, but with a stamping tool designed to produce micro-compartments having very thin bottom layers (<1 thousandth of an inch (25.4 μm) thick). Then the heat of a torch was applied of propane to the lower surface of the microcompartments to generate an orifice (perforation). The diameter of the hole formed in this way is smaller than the diameter of the bottom of the original microcompartment.

Filling and Leaving Test of Stamped Film Culture Devices Perforated in the Bottom To test the filling and leakage efficiency, a test sample (1.5 - 3.0 ml) of Butterfield diluent (Weber Scientific, Hamilton, NJ) was applied. contained phenol red to assist visual inspection by means of a pipette on a polypropylene stamped film having a plurality of microcompartments, some of which were perforated as described above. Each microcompartment was in the form of an inverted truncated hexagonal cone, with a diameter of approximately 1.9 mm on the surface and 1.0 mm on its bottom, which was approximately 1.1 mm. The solution was distributed over the microcompartments by shaking the stamped film by hand. It was observed that almost all the microcompartments with perforated bottoms were filled with test solution and without bubbles apparently. In comparison, it was occasionally observed that microcompartments without perforated bottoms contained trapped air bubbles. However, when The bottom surface of the stamped film was placed in contact with a second surface, the test solution leaked from the microcompartments containing open bottoms. Also, during the inoculation process, it was observed that the test solution applied from a pipette at high speed sometimes leaked through some microcompartments located directly under the tip of the pipette.

Preparation of Films Stamped With Perforated Micro-compartments in the Bottom Coated with Non Woven Fabrics A pressure sensitive adhesive nonwoven fabric (PSA) was applied to the bottom surface of stamped propylene film culture devices having a plurality of microcompartments perforated in the bottom to eliminate leakage of the test sample solution. The non-woven fabric was constructed of Kraton 1112 (fabric weight = 50 g / m2) and containing a blown fiber PSA as described in the Patent Application.

European No. 94119851.7. The non-woven fabric of PSA was easily bonded to the film by means of pressure and, therefore, formed a covered bottom, but with pores of air on each of the microcompartments.

Filtration and leakage test of films with perforated micro-compartments in the bottom coated with non-woven fabrics Butterfield diluent containing phenol red, as described above, was damped onto a polypropylene stamped film having a plurality of microcompartments, one of which was perforated and coated on the bottom with a non-woven fabric. All the perforated microcompartments were filled by simple minor agitation of the film, and only the non-perforated microcompartments trapped air. Different amounts of the test solution (1-3 ml) were applied by means of a pipette without leakage during the inoculation process. No flow of the test solution was observed through the pores, the micro-compartments coated bottom, under an optical microscope, and where the film was placed in contact with a second surface, the formation of a solution wick was evident test.

B. Laminated Sheets with Open-Bottom Micro-compartments Coated with Non-woven Fabrics Laminated sheets containing a plurality of open-bottom micro-compartments were constructed, and they had openings in the bottom of the well covered with the support of non-woven fabric, according to what is described in this example. The laminated sheets can be cut to size and used in culture devices for the detection and enumeration of microorganisms. Polyethylene films containing a plurality of uniformly spaced, small apertures (Vispore 6607 and Vispore 6582, Tregedar Film Products, Richmond, VA) were laminated onto a laminated non-woven PSA fabric to provide laminated sheets containing a plurality of micro-compartments with Open bottoms covered with a non-woven fabric. The physical characteristics of the initial film material are summarized in Table 2B.

The laminated sheets were easily inoculated by adding a small volume of nutrient solution containing phenol red to improve the visualization of the filled microcompartments. Magnified optical images on laminated sheets after inoculation showed uniform filling of the microcompartments. No air bubbles or leaks were observed in the filled compartments, thus suggesting that the air, but not the liquid, could easily flow through the bottom openings of the microcompartments coated with non-woven fabric.

Example 3 Detection and Enumeration of Microorganisms (Method for Using a Plurality of Microcompartments) The feasibility of using stamped film culture devices containing a plurality of microcompartments to detect and enumerate E. coli was demonstrated in this example. A change of night cultivation of E. col i ATCC 51813 (~109 CFU / ml in a Tryptone Soy Broth medium (TSB)) was serially diluted in Violet Red Bile (VRB) media (7.0 g / 1 Bacto peptone, 3.0 g / 1 yeast extract, and 1.5 g / 1 of bile salts) containing 4-methylubeliferyl-β-D-glucuronide (0.5 mg / ml) (MUG, Biosynth International, Naperville, IL). Dilutions were prepared at the approximate bacterial concentrations shown in Table 3a. A diluted sample (ml) was applied by means of a pipette on a polyethylene stamped film culture device (Example IB, lacking Triton X-35) containing 525 microcompartments (approximately 1.9 μl / microcompartment). The microcompartments were arranged in a hexagonal network (approximately 19 microcompartments / cm2) and each microcompartment had the shape of an inverted truncated hexagonal cone, which had a diameter of approximately 1. 9 mm one surface and 1.0 mm in its bottom, which was approximately 1.1 mm. The microcompartments were filled as described in US Pat. No. 5,219,462 using the diluted sample solution down to the film with the edge of a scraper blade. A diluted sample (1 ml) was also placed on a Rapid Coliform Test Plate of PETRIFILMMR Series 2000 (3M Company, St. Paul, MN), incubated, and read according to the manufacturer's instructions. Of the inoculated stamped film culture devices were placed inside petri dishes, and incubated for 12 hours at 37 ° C. The number of microcompartments they exhibit was counted fluorescence for each sample. The most probable number was calculated using the formula MPN = N ln (N / N-X) where N is the total number of filled microcompartments and X is the total number of microcompartments that show a positive reaction. The results were compared with the counts of the PETRIFILMMR Series 2000 plates in Table 3a.

* TNTC = Too many numbers to count The results of this example show that microorganisms can be easily detected and enumerated using a stamped film culture device having a plurality of microcompartments and that the values obtained are comparable with those obtained from commercial Series PETRIFILMMR Series Counting Plates. In addition, this method provides a wider range of control per sample than currently available methods.

Example 4 Detection and Enumeration of Microorganisms (Method for Using Perforated Microcompartments in the Bottom) The feasibility of using stamped film culture devices containing a plurality of microcompartments, which were perforated and coated on the bottom with a non-woven fabric, to The detection and enumeration of Serra tia arcescans was demonstrated in this example. A nocturnal breeding ground of Serra tia marcescans that grew at 35 ° C in TSB was serially diluted in increments of times the Butterfield diluent. Dilutions of 10"4, 10" 5, and 10"6 were used to inoculate the polypropylene stamped film culture devices having perforated micro-compartments in the bottom coated with a non-woven fabric (Example 2). Each film was cut in circles of 5.1 cm in diameter and placed in polystyrene petri dishes (5.1 cm in diameter x 1.9 cm in height) for the inoculation, growth and detection of bacteria. The film disk was lifted from the bottom of the petri dish by means of a foam separating ring. Curable silicone was applied along the edge of the film disc to provide a seal between the film disc and the box. The seal dictated that the test sample solution leaked through the edge of the film disc during inoculation. Each of the plates of the resulting culture devices had approximately 300 microcompartments (approximately 2.0 μl / well) and were treated by immersing in isopropanol for 2 to 3 minutes and dried overnight at ambient conditions and in an oven at 60 ° C. for 5 minutes before use. A sample (0.1 ml) of the individual dilutions was mixed with nutrient medium of balanced aerobic count (0.9 ml) having the composition shown in Table 4a and containing 4-methylumbelliferyl phosphate (0.1 mg / ml). The resulting test sample (~ lml) was applied by means of a pipette on the plate of the culture device and the plate was shaken gently to deposit a portion of the sample in each of the microcompartments. The plate was then tilted to pour the excess sample volume on an absorbent pad that was attached to the edge of the polystyrene box. Additional distilled water (approximately 0.3 ml) was added to the absorbent pad to completely wet it to provide a reservoir of moisture. The inoculated plates were inverted, incubated for 24 hours at 35 ° C, and the number of fluorescent (positive) microcompartments was counted under the excitation of the use of (360 nm). The most probable number (MPN) was calculated according to Example 3. The MPN / ml was calculated by multiplying the MPN by 1.66 based on a sampled volume of 600 microliters contained within 300 microcompartments of the device. The results are given in Table 4b.

The results of this example show that microorganisms can be easily detected and enumerated using a stamped film culture device having a plurality of microcompartments, which they were perforated and coated on the bottom on a non-woven fabric. The fabric-covered openings in the bottom of the micro-compartments allowed the air to escape, but not the liquid, when the liquid sample was applied. The number of bacteria detected (positive micro-components or MPN) decreased correspondingly for each serial dilution.

Example 5 Detection and Enumeration of Microorganisms (od of Use of Microcompartment Sets) The feasibility of using stamped film culture devices containing a plurality of micro-compartments of different sizes ("sets") to detect and enumerate E. coli was demonstrated in this example. Diluted samples of E. coli ATCC 51813 were prepared according to Example 3. Two stamped film culture devices were prepared according to Example IB. The first contained a square array of micro-compartments of 0.8 μl (~ 16 microcompartments / cm2), each microcompartment in the shape of an inverted truncated cone, with a diar of approximately 1.2 mm on the surface and 0.7 mm in depth, which was approximately 1.0 mm. The second movie contained a square arrangement of larger 5 μl microcompartments (~ 4 microcompartments / cm2), each microcompartment in the form of a truncated square pyramid, having an aperture of 3.7 x 3.7 mm on the surface and 2.0 x 2.0 nm depth, which was approximately 1.0 mm . A 250 μl sample of each dilution was distributed in the micro-compartments using the procedure described in Example 3. The devices were incubated overnight at 37 ° C and a number of micro-compartments were counted which exhibited fluorescence for each ] 0 set of movies. The MPN values were calculated according to that described in Example 3. The MPN was calculated per milliliter by multiplying the value obtained by inoculum of 250 μl by 4. The results are given in Table 5a and compared with the counts obtained at from 5 standard tests with PETRIFILMMR 2000 Series Counter Plates. 0 The results of this example show that the microorganisms can actually be detected and enumerated using a stamped film culture device having a plurality of microcompartments of different sets and that the values obtained are comparable with those obtained from commercial Series 200 PETRIFILMMR Counter Plates. . This example further demonstrates that a broader counting interval can be obtained by using a set of "small" microcompartments paired with a set of "large" microcompartments.

Example 6 Detection and Enumeration of Microorganisms (High Count Interval) This example demonstrates a detection and enumeration od for a highly concentrated sample (> 200,000 CFU / ml). The VRB nutrient media containing 1 mg / ml of 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid (BCIG, Biosynth International, Naperville, IL) and glucose (1 g / 1) were prepared from according to Example 3. A nocturnal culture of E. coli ATCC 51813 was diluted in this media formulation to an appropriate concentration of 1,000 CFU / ml. A film containing approximately 2,330 micro-compartments per square inch (6.45 cm 2) (0.03 microliters per microcompartment) was prepared according to Example IA. Each microcompartment was in the form of an inverted truncated hexagonal cone, having a side-by-side diar of approximately 0.3 mm on the surface and 0.15 mm deep, which was 0.7 mm. The microcompartments were filled according to that described in Example 3 guiding the inoculum over the microcompartments with the edge of a doctor blade. For this example, a silicone pressure-sensitive adhesive tape (Product # CW-14HT, Specialty Tapes, Racine, Wl) was used to seal the parts superiors of the microcompartments. This method provided means to prevent the evaporation of small sample volumes in the microcompartments during the night incubation. The sealed film was incubated at 37 ° C overnight (18 hours) and subsequently removed from the incubator and observed under a microscope. The number of positive micro-compartments (blue color) was counted in a representative microscope field containing 520 micro-compartments, corresponding to a sample taking volume of 15.6 microliters. Nineteen positive microcompartments were observed in the field, corresponding to a calculated MPN value of 1.290 per ml. The maximum count interval for this example (519 samples for a sample volume of 15.6 microliters) was 208,000 CFU / ml.

Example 7 Detection and Enumeration of Microorganisms (Method for Using a Plurality of Coated Microcompartments) This example demonstrates the method where the nutrient and the indicator are incorporated into a microcompartment of the film prior to inoculation with test sample. The VRB media containing MUG fluorescent indicator was prepared as described in Example 3. It was applied an excess of this solution to the surface of a film with the pattern and geometry of the microcompartment described in Example 3. The solution was distributed in the microcompartments by means of a blade to coat the solution on the surface of the film. The coated film was then dried in an oven at 52 ° C. An aqueous dilution of Serra tia liquefaciens was prepared from a night culture at a concentration of approximately 50 CFU / ml (Butterfield buffer, Fisher Scientific). A sample of this solution (300 μl) was applied to the nutrient coated film using the method of Example 3 to fill 420 micro-compartments. The sample was incubated overnight at 37 ° C. Thirty-six fluorescent microcompartments were observed, corresponding to a calculated MPN of 39 (130 / ml).

Example 8 Detection and Enumeration of Microorganisms (Detection Using pH Indicator and Incorporated Nutrient in Microcompartments) This example demonstrates the detection based on absorbance using an indicator that verifies the pH of the media.

The VRB media containing the phenol red pH indicator (1 mg / ml, Sigma Chemical Company) was prepared as described in Example 3. This solution was incorporated into the microcompartments of a film as described in Example 7. An aqueous dilution was applied (Butterfield buffer, Fisher Scientific) of Serra tia liquefa hundreds (approximately 50 CFU / ml) to the film as described in Example 3 followed by overnight incubation at 37 ° C. Of the 420 microcompartments that were filled, 21 exhibited a yellow color, which corresponds to an MPN value of 21 (70 / ml).

Example 9 Augmented Enzyme Kinetics Using Microcompartments (Enzyme + Fluorescent Indicator) In this example, the same number of enzyme molecules (2.5 ng) were placed in a plurality of microcompartments that ranged in size from 0.1 to 50 μl. Each well contained the same fluorescent indicator concentration (0.25 mM). The fluorescence production resulting from the enzymatic hydrolysis of the indicator was measured simultaneously for each microcompartment using a CCD camera. An image was stored at each point in time during the experiment. The quantitative fluorescence values for each microcompartment are obtained by loading the images stored in programs and processing programming systems and the intensity values were averaged over 4 pixels in the center of each microcompartment.

Detailed Procedure A photolithographically recorded magnesium tool was designed to provide inverted conical protrusions of increasing volume so that a stamped film prepared according to the procedure of Example IA was produced with micro-compartments of 0.1, 0.5, 1.0, 3.0, 14, 20, and 50 μl. A solution of alkaline phosphatase enzyme (0.1 mg / ml) in glycine buffer (50 mM, pH 10.4) was prepared and serially diluted in additional glycine buffer using the following sequential dilution scheme (in milliliters); 1: 1; 1: 4; 1: 1; 1: 2; 1: 1.3; 1: 1; 1: 0: 43; 1:15 An aliquot (100 μl) of each dilution was placed in adjacent micro-compartments of a microtiter plate of 95 micro-compartments. A multi-channel pipettor was used to add simultaneously 100 μl of 4-methylumbelliferyl phosphate indicator (0.5 mM in glycine buffer) to each microcompartment. An aliquot (0.1 μl) of the first dilution was placed immediately with a syringe in the microcompartment corresponding to this volume (0.1 μl). This was repeated for the next six dilutions corresponding to volumes of 0.5, 1, 3, 14, 20, and 50 μl. Using this procedure, each microcompartment was filled with a diluent containing 2.5 ng of enzyme in 0.25 mM of indicator. A background sample was also prepared that had microcompartments containing only indicator. After filling the microcompartments, the sample was placed in a covered petri dish and sealed with a tape to prevent evaporation. The box was placed inside a device for illumination and ultraviolet imaging (UltraLum Corporation, 365 nm). The CCD images were stored at the time intervals shown in Figure 4. The fluorescence intensity values for each point in time were obtained by averaging 4 pixels at the center of each microcompartment. The final values were obtained by averaging two experiments in duplicate. Figure 4 shows (1) that given the same number of enzyme molecules in each well, the kinetics of the reactions increased significantly in the smaller microcompartments, and (2) that the fluorescent signal for the detection system (CCD in this case) increased in the smaller microcompartments. For To illustrate this effect, the background fluorescence of the microcompartment containing only indicator was plotted (without enzyme) on the graph. On a per pixel basis, the signal is considerably larger (saturated for the smaller volumes) in the smaller microcompartments than in the larger microcompartments. Note that at the time point at 2 hours the intensity of the 50 μl is 2.3x on the background while the value 1 μl is 9.8 x greater.

Both effects of the increased reaction kinetics and the increased fluorescence signal led to a faster increase in detection against the size of the microcompartment decreased.

Example 10 Enhanced Microorganism Detection Using Microcompartments (Bacteria + Fluorescent Indicator) In this example, the same number of bacteria (~ 500 CFU) was placed in a plurality of microcompartments that ranged in size from 1 to 50 μl. Each microcompartment contained the same concentration of fluorescent indicator (0.25 mM) in a nutrient growth medium. The fluorescence production resulting from the enzymatic hydrolysis of the indicator was measured simultaneously for each microcompartment using a CCD camera Detailed Procedure Polyethylene stamped films according to Example 9 were prepared with microcompartments designed to contain 1, 3, 7, 14, 20, and 50 μl of liquid. A overnight culture broth of E. coli ATCC 51813 (~109 CFU / ml in TSB) was serially diluted in VRB media containing 4-methylumbelliferyl-β-D-glucuronide (0.5 mg / ml) as described in Example 3. Dilutions were prepared so that ~ 5000 CFU were initially present in each well before incubation. The inoculated films were placed in petri dishes followed by incubation at 37 ° C. The fluorescence relative to the points in time shown in Figure 5 was measured using the CCD imaging system described in Example 9. The calculated times to reach a relative fluorescence value of 80 for each of the microcompartments are provided. in Table 10a.

The results of this example as illustrated by the values in Table 10a and the graphical data of Figure 5 show that fluorescence was observed significantly faster in the smaller microcompartments than in the larger microcompartments.

Example 11 Detection and Enumeration of Microorganisms (Method for Using a Plurality of Microchannels) The feasibility of using film culture devices containing a plurality of coated microchannels to detect and enumerate batteries was demonstrated in Section A (Film Culture Device). Single Layer) and Section B (Single Layer Film Culture Device Coated with Medium) of this Example. The construction and inoculation of the film culture devices containing "assemblies" of multiple volumes of coated microchannels and containing multilayer film structures are described in Section C and Section D, respectively, of this Example.

A. Single Layer Film Culture Device A stamped film containing parallel, V-shaped grooved microchannels was prepared according to what is described in Example ID and in US Pat. No. 5,514,120. The resulting film was coated with an upper PSA / PE silicone film (Example ID), thereby creating a series of parallel, coated microchannels having a triangular cross section with a base of approximately 0.6 mm and a height of approximately 0.75 mm. A flat "ground area" of approximately 0.3 mm separated each micro channel, and provided a bonding surface for the coated film. The coated films were cut into strips 2 cm high by 5 cm wide with each strip (film culture device) containing 50 parallel microchannels of 2 cm in length. Each channel had a volume of approximately 5 μl (total sample volume of approximately 250 μl). A night broth culture of E. coli ATCC 51813 was serially diluted in VRB media (Example 3), which contained phenol red (0.5 mg / ml). Dilutions were prepared at the following approximate bacterial concentrations (CFU / ml): 10,000; 1,000; 100; and 10. An edge of the stamped film culture device was immersed in the sample and the fluid was allowed to advance towards the microchannels by means of capillary action. The upper edge of the device was then sealed by immersing in molten paraffin to slow the evaporation during inoculation. The bottom edge was left open. The samples were incubated overnight at 37 ° C in a wet petri dish and then observed to determine color changes from red to yellow. A yellow color along a single channel indicated acid production of the growth of the bacteria (glucose fermentation) within the channel. At dilutions of 10,000 CFU / ml and 1,000 CFU / ml, the samples showed a yellow color in the 50 channels, suggesting that at least one organism was distributed in each channel during the capillary fusion process. At the dilution of 100 CFU / ml, 29 channels were yellow (bacteria present) and 21 channels were red (no bacteria), which corresponded to a calculated MPN of 179 (the formula for MPN is given in Example 3). At the dilution of 10 CFU / ml, only 3 yellow channels were observed, which corresponded to an MPN of 12. No color change was observed in the control samples that were prepared without the addition of E. coli.

B. Single-Layer Film Culture Device Coated with Media A stamped film strip (Example 11A) without silicone PSA / PE top film was immersed in VRB media containing phenol red. The V-shaped slotted microchannels were allowed to fill, after which the film was removed from the nutrient media and dried at room temperature for approximately 30 minutes. The top PSA / PE film was applied to the stamped film to provide a media coated film culture device, which was then immersed in an aqueous dilution of E. coli ATCC 51813 (-50 CFU / ml). The bacterial solution was diffused capillary into the microchannels coated with medium via capillary action.

After incubation overnight at 37 ° C, it was observed that the stamped film culture device had 10 yellow channels and 40 red channels, which corresponds to an MPN value of 44. This example serves to demonstrate that the nutrients Bacteria can be incorporated into the microchannels of a film culture device, and that the device can be used to take samples directly from an aqueous test solution.

C. Microchannel arrays containing the Single Layer Film Culture Device A single layer stamped film culture device was constructed that contained multiple volume sets of closed microchannels having volumes of 20 μl, 2 μl and 0.2 μl as follow. The films of each set were stamped with tools of different configuration, covered with an upper film according to that described in Example HA, and cut into strips of specific widths to give the desired microchannel volumes. The dimensions of the strips and volumes of microchannels for each set are given in Table Ia.

The final single-layer film culture device was assembled by adhering two strips from each volume set (30 micro-channels per strip) adjacent to one at the base of a square petri dish ("Integrid" 100 X 15 mm.) Becton Dickenson , Lincoln Park NJ). The strips were attached using transfer tape (Scotch 300LSE Hi Strength Adhesive, 3M Co.) and placed approximately 2 mm apart. The device was inoculated using a solution containing a food coloring dye to provide contrast. A transfer pipette was used to place the test solution in the "channel area" between each set of strips. Dripping the device, the fluid drained downward toward the "canal area" and filled the open microchannels at the end (placed perpendicular to the "canal area") by capillary action. The excess solution was contained by a strip of paper towel placed at the base of the device. Using the device of this example (60 microchannels per set) and the formula of the MPN set forth in Example 3, the counting intervals for each of the three sets were calculated and given in Table Ia. This example serves to demonstrate that a single single-layer film culture device contains sets of microchannels that can provide the basis for a bacterial enumeration test that is highly sensitive and covers a very wide counting interval.

D. Multilayer Film Culture Device Multilayer film structures were constructed to increase the total volume of the liquid sampled and the number of individual closed microchannels in the culture device. Two constructions were prepared by laminating together single layer patterned films and are described in Table llb. Single layer films were used in the construction of multiple layers DI contained in parallel microchannels that had a square cross section with sides of approximately 0.2 mm by 0.2 mm. Each microchannel was separated approximately 0.1 mm. The single layer films were cut into strips 1.5 cm wide x 1 cm high. A thin layer of adhesive (RD 1273, 3M Co.) was applied to the back of each strip, and the strips were stacked to form a multi-layered structure containing a plurality of microchannels. Construction D2 was assembled using the single layer films described in Example HA laminated together using an adhesive layer (Super Strength Adhesive, 3M Co.).

The detection of E. coli ATC 51813 was demonstrated with the multi-layer film device D2 (Table llb) using serial dilutions of bacteria in .VRB media containing phenol red (Example HA). One end of the device was immersed in the media, thereby filling each microchannel by means of capillary action. After incubation overnight at 37 ° C, color changes from red to yellow were observed in the microchannels containing bacterial growth by observing the device over the rim.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (10)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A test device for detecting microorganisms, in a liquid sample, characterized in that it comprises: a substrate having a plurality of microchannels of microcompartments therein, the microchannels have at least one test reagent coated thereon.

2. A test device for detecting microorganisms, characterized in that it comprises: a plurality of microcompartment capillary tubes formed together, the capillary tubes have at least one test reagent coated thereon.

3. The test device according to any of the preceding claims, characterized in that the different microcompartments include different test reagents coated thereon.

4. The assay device according to any of the preceding claims, characterized in that the assay reagent comprises nutrient media.

5. The test device according to any of the preceding claims, characterized in that it has multiple rows of microcompartments. The assay device according to any of the preceding claims, characterized in that it has a plurality of microcompartment assemblies, each set is of uniform size and the assemblies vary in the size of the microcompartment. 7. The test device according to any of the preceding claims, characterized in that it has multiple layers of microcompartments. The test device according to any of the preceding claims, characterized in that it is adapted to be inoculated with a liquid sample through the capillary action. 9. A method for conducting an analysis of the most probable number using any of the devices of the preceding claims, characterized in that it comprises: a) distributing a sample to a plurality of microcompartments of a culture device; b) incubating the culture device for a sufficient time to allow at least one cycle of cell division of the microorganisms; c) detect the presence or absence of microorganisms in the microcompartment; Y d) conduct a more likely analysis based on the number of wells where a microorganism is detected. The method according to claim 9, characterized in that the step of distributing the sample to the device comprises introducing the device into a liquid sample, where the sample inoculates the microcompartments via capillary action.