MXPA00006124A - Disc assay devices and methods of use - Google Patents

Abstract

A culture device having hydrophilic liquid retaining discs for use in detecting and enumerating microorganisms. Methods of use therefor are also disclosed.

Description

DISCO VALUATION DEVICES AND UTILIZATION METHODS DESCRIPTION OF THE INVENTION This invention relates to disk titration devices and methods of use for dividing biological samples into a micron-volume aliquot, and detecting and enumerating microorganisms present within the samples. Detection and enumeration of microorganisms are practiced in numerous environments, including the food processing industry (tested for food contamination by microorganisms such as E. Coli and S. aureus), the health care industry (test the patient samples and other clinical samples for infection or contamination), the environmental testing industry, the pharmaceutical industry, and the cosmetic industry. Detection and enumeration based on the growth of microorganisms are practiced normally using any liquid nutrient medium (most likely number analysis (MPN)) or semi-solid nutritive medium (agar petri dish). The enumeration using the liquid MPN method is normally done by placing 10-fold dilutions in series of KEF.120956 a sample of interest in tube replication groups containing a selective medium and chemical indicators. The tubes are incubated at elevated temperature (24-48 hours) followed by examination for the growth of organisms. A statistical formula, based on the volume of the test sample and the number of positive and negative tubes for each group, is used to estimate the number of organisms present in the initial sample. This method of performing the MPN analysis has several disadvantages. It is an intensive task due to the multiple dilution and the pipetting stages necessary to perform the analysis. In addition, in practice it is only practical to use replicing groups of approximately three to five tubes for each dilution. As a result, 95% of the confidence limits for an MPN estimate for the microbial concentration are extremely broad. For example, an MPN estimate of three tubes of 20 has 95% confidence limits ranging from 7 to 89. In addition, it is usually not obtainable in less than twenty-four hours. In contrast to the method described above, a direct count of viable microorganisms in a sample can be achieved by expanding the sample over a defined area using a nutrient medium containing a gel-like agent. The agent in the form of gel (agar) prevents the diffusion of the organisms during the incubation (24-48 hours), producing a colony in the area where the original organism was deposited. There is, however, a limit to the number of colonies that can be placed in a given area of the nutrient medium before merging with neighboring colonies that make counting difficult. This makes it necessary to make several dilutions each sample. In addition, the classes of chemical indicator molecules that can be used to identify individual types of microorganisms present within a mixed population are limited to those that produce a product that is insoluble in the medium in gel form. In addition, rapid detection, that is, in less than approximately twenty-four hours, and enumeration are not feasibly used in this method. The present invention is directed to the disadvantages of the prior art. The invention provides devices and methods for rapid detection and enumeration of microorganisms. In one aspect, the invention provides a device that is capable of retaining sample microvolumes. The device has a substrate with a relatively hydrophobic surface. Within or on the surface is the hydrophilic sample that holds the discs. The discs may be comprised of hydrophilic fiber material projecting from the surface of the titration. Discs can be constructed from a variety of materials, including cellulosics, polyesters, polyolefins, and polyamides. A sample that is suspected of having microorganisms present is placed on the device's assessment surface. The hydrophobic / hydrophilic interaction between the discs and the substrate is allowed for the rapid inoculation of the discs such that the sample is substantially contained in the discs and substantially excluded from the substrate. The sample can be poured or otherwise released to the device, such as by immersing the device in the sample. The interaction acts to contain the sample in the disks and substantially excludes the sample from the substrate. This interaction also helps prevent indirect contamination in the discs that can occur if microorganisms are allowed to migrate from disk to disk. The device is then incubated to allow the growth of the suspected microorganisms.

The disks have a means provided thereon to facilitate the growth of the suspected microorganisms. The medium can be selective for one or more types of microorganisms. The discs are bicompatible with the microorganisms such that the materials do not interfere substantially with the growth or detection of the microorganisms. They can be covered with appropriate indicator substances or they can be deposited on the discs, or they can be mixed with the sample to be inoculated on the discs. Suitable indicators include, without limitation, chromogenic indicators, fluorescent indicators, luminescent indicators and electrochemical indicators. In a preferred embodiment, the indicators are fluorescent. The disks may be of uniform size, with each disk having a liquid holding capacity of about -0.01 to about 25 microliters, more preferably about 1 to about 2 microliters. The culture device may have, for example, about 10 to about 10,000 discs, more preferably about 400 to about 600 hydrophilic discs that retain liquid.

In an alternative embodiment, the culture device can comprise a plurality of groups of hydrophilic discs that retain liquid, each of the groups have discs of uniform size, the groups that vary in liquid retention capacity, and the device that has two disk groups at least. In a preferred embodiment, the device has a total of 100 disks, with one group having 50 disks with a volume retention of approximately 2 micrclitres and another group having 50 disks with a volume retention of approximately 20 microliters. This device allows a device that has a relatively small number of disks but with a wide count interval. In another aspect, the invention provides sample release methods in the devices. The sample is divided into discrete disks based in part on the hydrophobic / hydrophilic interactions between the substrate and the disks and partly on the absorbency of the disks. As used herein, the term "microvolume" refers to a volume of less than about 25 microliters, and includes volumes in the sub-microliter range. The term "microorganism" includes all organisms and microscopic living cells, including without limitation bacteria, mycoplasmas, ricotrios spirochetes, yeasts, molds, protozoa, as well as the microscopic forms of eukaryotic cells, for example simple cells (cultured or derived directly from a tissue or organ) or small groups of cells. The microorganisms were detected and / or enumerated not only when whole cells were detected directly, but also when cells are detected indirectly, such as through the detection or quantification of cell fragments, biological molecules derived from the cell, or derivatives of the cell. The terms "hydrophobic" and "hydrophilic" herein provide the meanings commonly understood in the art. Thus, a "hydrophobic" material has relatively little or no affinity for water or aqueous medium, while a "hydrophilic" material has a relatively strong affinity for water or an aqueous medium. The relative hydrophobicites and hydrophilicites of the devices described herein are such as to ensure the division of liquid samples substantially into the hydrophilic discs retaining liquid described in the sample application. The required levels of hydrophobicity and hydrophilicity may vary depending on the nature of the sample, but can be adjusted rapidly based on the simple empirical observations of the liquid sample Ia7 when applied to the devices. The term "electrochemical" means a chemical indicator that changes the resistance or conductance of the sample in the reaction with the microorganism. The devices and methods provide a system for the detection and enumeration of microorganisms and other biological materials that solve the problems associated with the systems currently used. The system is a liquid-based system, allowing effectively and effectively dividing the sample into discrete microvolumes for testing, and allowing for rapid detection and enumeration. - In the case of MPN analysis for the detection and enumeration of microorganisms, the approaches described herein allow the use of water soluble indicator species, and reduce or eliminate the need for the various dilutions normally required in current MPN analyzes . Figure 1 is a perspective view of one embodiment of a microcompartment growing device. Figure 2 is a perspective view of a micro-compartment culture device having two groups of discs of different volume and a cover sheet. Figure 3 is a perspective view of a micro-compartment culture device having disks inside the wells. This invention relates to disk devices and methods of use thereof for dividing biological samples into liquid sample aliquots of microvolume and directing detection and enumeration based on signals from microorganisms et? the test. Among the problems encountered in the related art in testing liquid samples for microorganisms are relatively long incubation times, the need to undertake multiple pipetting operations for aliquots being tested, and the need for a relatively larger volume of sample for tests. . This invention addresses the difficulties in the prior art by providing a device that is easily inoculated with sample microvolumes. The invention provides absorbent disc materials which are absorbent and which are still biocompatible. In addition, these materials are compatible with fluorescent indicator systems. The materials lend themselves easily to the manufacturing process.

The methods and devices of the present invention provided for effectively dividing a liquid sample into microvolume compartments of a test device, with only minimal manipulation of the required liquid sample from the laboratory technician or other operator. The present inventors have discovered that the use of microwaves in the detection based on microorganism signals in the results of liquid samples in remarkably shorter incubation times required to produce a perceptible signal. Because shorter incubation times are very desirable in this field, this feature of the invention provides a distinct advantage. -1 In addition to achieving a shorter incubation time, 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 sample sources. Small-volume liquid test samples are also sometimes desirable, for example for easy handling or transport of the sample for ease of testing. ll The present inventors have developed new devices - and methods for dividing the biological liquid samples into discrete microvolumes within the liquid retaining discs. By sale, the devices allow the testing of liquid samples using aliquots of microvolume in a single device, eliminating the need to separate the vessels in the test. A test sample can be distributed among hundreds or even thousands of discs that retain discrete liquid, substantially increasing the number of data points in a test of the liquid sample. 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 detection and enumeration based on growth is very important in the verification of food, environment, clinical, pharmaceutical, cosmetic, and other samples for contamination by microorganisms. The methods and devices of this invention allow for efficient, accurate, convenient, and cost-effective testing. A preferred use of the methods and devices of this invention in microbiological testing is in MPN. In the traditional MPN, a sample of interest is diluted consecutively (10 times) and pipetted in equal amounts into the replication groups of tubes containing a selective growth medium and chemical indicators. The tubes are incubated at elevated temperature for approximately 24-48 hours followed by examination for the growth of organisms. A statistical formula, based on the volume of the sample and the number of positive and negative tubes for each group, is used to estimate the number of organisms present (by volume) in the initial sample. As it is generally used, this traditional method has several disadvantages. Being an intensive work due to diluting multiple and pipetting stages required to perform the analysis. As a practical matter; - Only replication groups of approximately three to five tubes are normally used for each dilution. As a result, the 95% confidence limits for an MPN estimate of the microbial concentration used by this method is extremely broad. For example, the MPN estimate of a nine tube (3 ten-fold dilutions) of 20 has 95% of the confidence limit ranging from 7 to 89_ The use of the methods and devices of the present invention in the MPN analysis exceeds some of the aforementioned disadvantages. The amount of labor is greatly reduced because no pipetting in the individual tubes is necessary, and very little or no agitation or other manipulations are required. Instead, the liquid sample is distributed in discs that retain microvolume liquid by simply contacting the liquid sample with the device. In addition, less dilutions of the sample are necessary when larger numbers of discs that retain fluid are present in the device. The relatively larger number of discs that retain fluid also provide a more accurate estimate of microbial concentration. This is because the corresponding greater number of data points provides a correspondingly narrow confidence limit interval. Therefore, the present invention provides a method for detecting (including enumeration) a microorganism in a liquid test sample. The method involves the distribution of microvolumes of the test sample for a plurality of hydrophilic discs that retain fluid from a titration device. The titration device can be any device that includes a titration surface having a plurality of hydrophilic discs that retain liquid, where each disc has a liquid retention microvolume capability. The device also includes a given area between the discs, which is hydrophobic and remains substantially free of liquid after the biological sample has been distributed in the liquid retaining discs. The examples do not limit the assessment devices including those described herein. The discs that retain liquid in the titration device are preferably of a uniform size and each disc has a liquid retention capacity of about 0.01 to about 25 microliters of the liquid sample. Preferably, each disk has a liquid retention capacity of from about 0.1 to about 10 microliters, and more preferably about 1 to about 2 microliters. The titration device preferably contains between 1 and about 100,000 liquid retaining discs, more preferably about 10 to about 10.0O0 discs, even more preferably about 200 to about 5,000 discs and more preferably about 400 to about 600 discs. The device may also preferably contain disk groups with different volumes. In this preferred embodiment, the device preferably has 100 disks. Preferably, 50 discs have a volume of approximately 20 microliters and 50 discs with a volume of approximately 2 microliters. The device is particularly useful in the context of testing a liquid sample for the concentration of microorganism using MPN. Certain regulatory requirements may dictate that a method of testing must be able to detect a microorganism in a sample of one to five milliliters. The size of the sample is standard in the food processing industry for microbiological testing. In this way, for example, a titration device having 500 hydrophilic discs that retain liquid, where each disc has a liquid capacity of approximately 2 microliters, being very useful for testing a 1 ml sample. A liquid retention disk having a capacity of 2 microliters allows a rapid development of a detectable signal according to the invention, and the use of about 400 to about 600 disks provide a sufficiently large number of data points to substantially improve the confidence interval for an MPN calculation. In addition, it is feasible to perform a manual count of the discs that retain positive test fluid for the microorganism. The use "of devices that have substantially more than 400 discs that retain liquid may require; As a practical matter, the help of an automated instrument or counter. The disks may be attached to the substrate by various means known in the art, including without limitation, the use of adhesives. Preferred adhesives include water-insoluble isooctyl acrylate adhesives as described in U.S. Patent No. 5,409,838, the disclosure of which is incorporated herein by reference. ^ ~ The liquid test sample can be any sample of interest, from any source. The sample may be distributed directly in the plurality of discs that retain liquid, or the sample may be diluted prior to distribution in the discs. The determination as to whether dilution of the sample is necessary will depend on a variety of factors such as sample source and age, and determination is a matter of routine for those skilled in the art. The liquid test sample may include a selective nutrient growth medium for the microorganism of interest, and / or an indicator substance that produces a signal in the presence of the growing microorganism. Optionally, the nutrient medium can include a gel-like agent that helps "encapsulate" the growing microorganisms. Gel-shaped agents are known to those skilled in the art, and include any water-absorbing material that returns a gel in addition to an aqueous liquid. In any case, the nutrient growth medium is present as a coating or other deposition within or on the disk that retains liquid, in sufficient quantities to achieve the desired concentrations when a microvolume of the liquid test sample is distributed on the disk. A coating can be achieved, for example, by placing or distributing a solution of the nutrient medium (with or without agent in gel form) on the disc and drying the solution to produce a coating or deposition of the nutritive medium on the disc. The components of the medium may be present in the adhesive or other substance that mixes the discs in the substrate (if applicable). The medium is finally diffused in the disc material. A wide variety of selective growth medium is known for a wide variety of microorganisms of interest, such as a broad variety of indicator substances for a wide variety of microorganisms, and any of these means or indicator substances are suitable for use in the method of the invention. An advantage of the present invention is that the soluble indicators can be used, since the diffusion is prevented by confinement of the liquid of the aqueous biological sample in the hydrophilic discs that retain liquid. Various methods can be used to distribute a liquid test sample on the discs that hold liquid. More than one method can be applied to a particular device, although the preferred method may depend on some extension in the configuration of a particular evaluation device. The sample can be poured or pipetted onto the device and the sample extended into the discs that retain fluid by tilting or rocking the device. The hydrophilic / hydrophobic interaction acts to retain Aa sample in the disks and substantially excludes the sample from the substrate. Alternatively, the evaluation surface of the device can be immersed in the sample. In the removal of the surface of valuation of the liquid sample, liquid is retained in the hydrophilic discs that retain liquid and is likewise substantially excluded from the given hydrophobic area.

After the sample is distributed to the liquid retention hydrophilic disks of the titration device, various titrations can be carried out depending on the desired uses. For microbial detection or enumeration, the titration device can be incubated for a sufficient time to allow at least one cell division cycle of the microorganism. For these purposes, the device is generally incubated at about 25 ° C to about 45 ° C, more preferably at about 30 ° C to about 37 ° C. The incubation time for bacterial detection will vary. The detection time will also vary depending on the growth rate and the present number of microorganisms in the sample. Considering these considerations, detection time for enumeration purposes can be as small as approximately 10 hours. This relatively short incubation time represents a distinct advantage over the currently used detection methods, which normally require incubation times of approximately 24 hours or more. In the next incubation of the titration device, the presence or absence of the microorganism is detected in the discs (and also in the sample of the liquid test). The detection mode depends on the type of indicator substance used in the method. Any indicator substance that is capable of providing a perceptible signal can be used. Indicators include but are not limited to fluorescent, chromogenic, luminescent, and electrochemical indicators. The presence or absence of a microorganism in a disc can be detected visually, with the eye naked or microscopically, if a luminescent or chromogenic indicator is used. The indicator can be coated or otherwise incorporated into the discs. The indicators can also be included in the adhesive or other substance that mixes the discs (if applicable) to the substrate. In this case, the indicator is finally diffused in the disc material. If a fluorescent indicator substance is used, the equipment and methods for detecting a fluorescent signal for detection can be employed. - There are numerous indicator substances and signal detection systems, including systems for detecting electrochemical changes, known in the art for detecting microorganisms, and any substance or system may be used in accordance with the present invention.

In the present invention, fluorescent indicators are preferred because they can be detected at relatively low concentrations. Suitable indicators include 4-methylumbelliferyl phosphate, and 4-methylumbelleferyl-B-D-glucopyranoside, L-phenylalanine-T-amido-4-methylcoumarin. Others may include 4-methylumbelliferyl acetate and 4-methylumbelliferyl sulfate. The detection of microorganisms in the liquid sample may additionally involve the enumeration of the microorganism count in the liquid test sample. In a preferred embodiment, enumeration is performed using MPN. Once the number of liquid retention discs containing the microorganism of interest is determined, an MPN calculation can be made using the known MPN techniques. If desired, the number of microorganisms in a single disk can then be determined using known techniques, for example the signal strength compared to a known standard, or the plating of the contents of the disk. Advantageously, the larger number of liquid retention discs used in the method of the invention is allowed for narrower ranges for 95% confidence limits in an MPN analysis of a sample of the liquid test.

Due to the greater number of liquid retention discs that can be manufactured in a single device, it is possible to use a single device in the detection and enumeration of multiple microorganisms of interest, while retaining the advantages of the invention. For example, a single sample of the liquid test can be tested for the presence or concentration of E. coli and S. aureus. A portion of a titration device may contain the hydrophilic liquid retention discs for the detection and enumeration of one of these microorganisms, while a second group of discs may be directed for the detection and enumeration of another microorganism of interest. For example, this is accomplished by including nutrients from a specific microorganism and / or indicator substances in the respective groups of the liquid retention disks. Alternatively, all liquid retention disks may contain titration reagents designed for the simultaneous detection of multiple microorganisms. For example, E. coli can be detected with a fluorescent indicator substance while, at the same time, other coliforms with a cramogenic indicator substance are detected. Subsequent tests may be conducted. For example, the discs can be removed from the device and transferred into a test tube to differentiate the growths of specific microorganisms therein. In another embodiment, the distribution step may involve the aliquot distribution of the liquid test sample to a plurality of liquid retention hydrophilic disks of a titration device, wherein the titration device includes a plurality of disk arrays. Each group has disks of uniform size, and the device has at least two disk groups. For example, the titration device may include a plurality of paths, with the hydrophilic liquid retaining discs in a particular path having the same liquid retention capabilities. This feature allows the distribution of the liquid test sample in the different test volume sizes within a single titration device. In MPN, this feature provides a significant advantage in that, for a highly concentrated sample, an appropriate volume size can be selected and the MPN analysis is performed using a single distribution step in a single device without the need for serial dilutions. .

As stated above, the methods of this invention can be practiced using any titration device containing the hydrophilic liquid retention discs and a given hydrophobic area, depending on the particular embodiment to be practiced. The present inventors have developed various novelty devices suitable for use in the methods of this invention. The following examples are not limiting of the devices. Referring to Fig. 1, a device 10 comprises a substrate 12 having a plurality of hydrophilic liquid retaining discs 14. In a preferred embodiment, the discs 14 are hydrophilic and absorbent Discs 14 can be made from a variety of materials, preferred are those including cellulosics, polyolefins, polyesters, and polyamides, with cellulosics Suitable cellulosics include paper, wood pulp, and rayon, and chemically modified cellulosics, such as cellulose esters, may be included Appropriate polyolefins include hydrophilic polyethylene or the hydrophilic polypropylene fibers Convenient polyamides include nylon Suitable polyesters include polylactic acid.

The materials of the present invention 10 are biocompatible and can be used with fluorescent indicators. The materials do not exhibit significant inherent fluorescence that interferes with the use of the indicators. In addition, the disks 14 do not exhibit a significant absorption at the wavelength of the emission of the indicators. The substrate 12 can be made of any material that is relatively hydrophobic and provides a convenient or support surface for the disks 14. The substrate 12 can be made, for example, of polymeric films or other suitable materials. Suitable polymers include, without limitation, polyethylene, polypropylene, polyimides, fluoropolymers, polycarbonates, polyurethanes, and polystyrenes. A particular polymer must not be sufficiently hydrophobic, to be able to be treated to impart hydrophobicity. For example, a thin layer of acrylated silicone or other hydrophobic material can be added to the substrate. The substrate film 12 should not exhibit fluorescent or light absorbing properties that would interfere with any fluorescent indicator system used. Those skilled in the art will recognize other means "for imparting surface hydrophobicity.

The device 10 may include any desired number of disks 14. Additionally, the device 10 may include relatively large reservoirs or other compartments adapted to retain larger volumes of liquid for maintaining an appropriate humidity level within the device 10. Although the number of discs 14 can be relatively small (eg, 2-50) for certain applications such as a preliminary coating, the small sizes of the microvolume discs 14 allow relatively large numbers of discs 14 to be in a single device 10. The device 10 it can have a population of disks with uniform size 14, although the disks are not necessarily of uniform size. For example, referring to Figure 27, the device may have groups (e.g., rows) of microvolume disks 14, 16 where the volumes are constant within a group, but would be between the groups. The volumes may vary incrementally with respect to a series of disk groups 14, 16, with the smaller disks 14 retaining sub-microliter volumes and the larger disks retaining the sub-microliter 16 volumes. It is even possible for the Larger discs in a device such as the one described in Figure 2, include disks 16 that would not be classified as microdisks. The discs 16 could have a liquid holding capacity, for example, of substantially more than 25 microliters. Optionally, the device 10 can include a liner sheet 18 to protect the discs 14 from contamination or desiccation once the sample has been added to the device 10. The liner sheet 18 can be further sealed to the device along its edges with a pressure sensitive adhesive. ~ In an alternative mode, as described in Figure 3, the device 10 may include the disks 14 contained in microwells 22 that have been made in the substrate 12 of the device 10. As with other embodiments, the numbers and size of the disc may be varied. The discs can be of any form. For example, the discs may be circular, oval, square, or polygonal or other appropriate shapes. The titration reagents are covered or on the other hand they are deposited inside the liquid retention discs of the titration devices. The titration reagents include without limitation the nutrients for the growth of microorganisms.

Other reagents may include, but are not limited to, gel-like agents and indicator substances such as chromogenic indicators, fluorescent indicators, luminescent indicators, and electrochemical indicators. Reagents can be immobilized on liquid retention disks "by any of the numerous methods for immobilizing titration reagents on solid substrates known to those skilled in the art.The methods include for example low drying of liquids containing reagents in discs, as well as other methods for non-covalently binding biomolecules and other titration reagents to a solid substrate Alternatively, various methods can be employed to covalently bind titration reagents to the discs by methods well known to those skilled in the art. As discussed above, the presence of hydrophilic discs that retain liquids with the ability to retain microvolume liquids in a titration device allows the separation of a sample from the liquid test in a relatively large number of test volumes. to separate a liquid sample in a microvolume and to perform the MPN or other valuations without indirect contamination between aliquots, is an advantage of the method and devices present. All references and publications cited herein are expressly incorporated by reference in this disclosure. The particular embodiments of this invention will be discussed in detail and reference is made to possible variations within the scope of this invention. There are a variety of alternative techniques and procedures available to those skilled in the art that will allow one to successfully practice the desired invention. The following examples are offered to assist in understanding the present invention and will not be understood as limiting the scope thereof. Unless otherwise indicated, all parts and percentages are by weight. EXAMPLE 1 Absorbent Disc Culture Devices [0113] Constructed as described in this example, the absorbent disc culture devices containing a plurality of hydrophilic absorbent discs are formed on a hydrophobic surface and capable of being used for the detection and enumeration of microorganisms in a sample of the liquid test A. Cultivation Devices Made with Absorbent Paper Discs A sheet of absorbent material. { Schleicher & Schuell Grade 903 Paper; absorbs approximately 4.5 g of water / 100 cm2) was laminated to a film coated with Rexam silicone (Grade # 15819 D 2MIL CL PET MM34P / ooo having a clear polyester film thick of 2- il as a substrate, Rexam Résele, Oak Brook, IL) with an acrylate pressure sensitive adhesive (PSA) containing the chromogenic indicator of 2,3,5-triphenyl-2iT-tetrazolium chloride (ITC) (Amresco, Solon, OH). The material was saturated with a growth medium of Tryptic Soy Broth (TSB) containing 0.5% of the fluorescent indicators of 4-methylumbelliferyl phosphate (100 μg / ml, Sigma, St. Louis, MO) and 4-methylumbelliferi aD-glucoside (50 μg / ml, ~~ Sigma), cleaned with a wire wing rod, and dried at 110 ° C for 10 minutes. Circular discs approximately 0.635 cm in diameter were punched from the laminate and the silicone-coated film is removed again. The discs with PSA are then adhered to another sheet of Rexam silicone coated film so that the discs are molded in equally spaced parallel rows. The film and the assembled discs were irradiated with gamma rays at a level of 8.9 kGy, cut according to size, and then recorded in a petri dish such that each box contains a piece of film with 20 discs. Based on the gravimetric measurements, each disk in the resulting culture devices had a holding capacity of approximately 40 μl of liquid.

B. Cultivating Devices Constructed with Various Polymer Absorbent Disc Materials A silicone-coated polyester release liner (as described in Example 1A) and a biaxially oriented polypropylene film (BOPP) (1.6-mil thickness, 3M Co., St. Paul, MN) were cut into pieces rectangular of 7.6 cm x 10.2 cm. The pieces of each material were attached to one end with a doubly coated adhesive tape.

SCOTCH T (No. 665, 3M Co with the silicone coated side of the release liner facing the BOPP film.) The release liner functions as the base of the culture device and the BOPP film functions as the part film. The sheets of the following polymeric absorbent materials were laminated to the separate layers of an acrylate adhesive (No. Y966, 3M Co.): Cellulosic Product No.10201-9 (Dexter, Windsor Locks, CT), Grade 903 paper of cotton lint (Schleichter &Schuell, Keene, NH), Product No. P-110 Polyol Superabsorbent (3M Co.), Product No. 9208283 Polyester (Veratec, Walpole, MA), Nylon Spunbond (4 oz. per square yard) ) Polyamide (Cerex Advanced Fabrics, Cantonment, FL), and polylactic polyester acid [meltblown network in absorbent non-woven fabric prepared from polylactic acid pellets (EPLON ™, Chronopol, Inc., Golden, CO) as described in the Patent North American No. 5,230,701, which is incorporated herein by reference] The circular discs (approximately 0.64-cm in diameter) were punched from the resulting sheet and adhere to the silicone-coated side of the polyester release liner. Each culture device contains the disks 12 equally spaced in a series of parallel rows of 3 x 4. After the construction was completed, the culture devices were irradiated with gamma raryos at a level of 8 kGy. Each disc had the retention capacity of approximately 10 μls.

Example 2 Inoculation Method (Method Utilizing Absorbent Disc Culture Devices) The method of inoculating absorbent disc culture devices containing a plurality of microvolume fluid retention discs with the medium that is shown in this example is demonstrated. contains the bacteria Inoculated devices constructed with absorbent paper discs were used to detect and enumerate E. coli bacteria.

A. Microbial Titration Using the Culture Devices Constructed with Absorbent Paper Discs (Example IA) A culture of E. coli ATCC 51813 was diluted to produce suspensions containing about 10 CFU / L and 1 CFU / L. Samples (1 to 2 ml) of the suspensions were applied by pipette to the absorbent disc culture devices described in Example IA. The excess liquid sample was poured, leaving approximately 0.8 ml retained in the device (20 disks, approximately 40 μl of liquid per disc). The inoculated devices were incubated at 35 ° C for 23 hours and inspected under ultraviolet light. The number of discs exhibiting fluorescence was counted for each device and "the most probable number (MPN) of the values was calculated using the formula MPN = N ln (N / NX) where N is the total number of discs inoculated and X is The total number of discs that show a positive reaction The MPN per milliliter was calculated by dividing the value obtained by the total volume of the sample (0.8 ml) The results are given in Table 2A and compared with the count obtained from the standard check with Coliform Count PETRELFILM ™ Plates (3M Co.) Fluorescent discs frequently showed red TTC, usually as discrete spots within the discs No indirect contamination between the absorbent discs was observed.

The results of the example can be easily inoculated which show that the culture devices of the absorbent disc have a plurality of absorbent discs formed in a hydrophobic film easily with the liquid samples containing the bacterium and where the inoculated devices can be used for the detection and enumeration of E. col i, with the values obtained being comparable with those obtained from Coliform Count PETRIFILM ™ Plates commercial.

B. Microbial Titration Using the Devices of Cultivation Constructed with Various Absorber Disc Polymer Materials (from Example IB) The cultures of different bacterial strains (Table 2B) were grown overnight at 35 ° C in 5 ml of the TBS medium. A 0.01-ml volume of each culture was diluted in 99 ml of sterile Butterfield's diluent (Fisher Scientific, Pittsburgh, PA), to obtain initial dilutions of 10% of the original bacterial suspensions. Three subsequent 10-fold dilutions (10 ~ 3, 10", and 10" 7) of the bacterial suspensions were prepared in standard Methods Broth containing the following ingredients: Casein Pancreatic Compendium (10.0 g / 1, Difco Labs), Yeast Extract (5.0 g / 1, Difco Labs), Glucose (2.0 g / 1, Becton Dickinson and Co., Cockeysville, MD), and the fluorescent indicator of 4-methylumbelliferylphosphate (0.05 g / 1, Biosynth International). With the top covers raised from the growing devices (from Example IB), three aliquots of 0.01-ml dilutions of 10 ~ ", 10 ~ fi, and 10" were transferred by pipette into nine individual disks in each of the devices. An equivalent volume of sterile media was transferred for the permanence of three disks in each device to serve as sterility controls. The top covers of the inoculated culture devices were closed, and the devices located in the GLAD-LOCK® ZIPPER ™ storage bags (First Brands Corp., Danbury, CT), each contain a moistened paper towel. The bags were placed in an incubator at 35 ° C - for 24 hours, after which the culture devices were examined under a long wave ultraviolet light source. The growth and positive detection was evidenced by a bluish fluorescence. The results are given in Table 2B.

The results of this example showed that culture discs constructed with a series of discs made of different absorbent materials can be used for the detection of various bacterial stresses. Especially effective in this example were the absorbent discs made of cellulosic, polyamide and polyolefin materials. Various modifications and alterations of this invention will be clear to those skilled in the art without departing from the scope and spirit of this invention, and it should be understood that "this invention is not limited to the illustrative embodiments indicated herein.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or ucts to which it refers.

Claims (10)

1. Having described the invention as above, the content of the following claims is claimed as property: 1. A culture device for the detection or "enumeration of microorganisms, the device comprises a substrate having the liquid retention discs wherein the substrate "it is hydrophobic in relation to the liquid retention discs and where the discs have growth media of microorganisms and the disks have a liquid retention microvolume capacity.
2. 2. The culture device according to claim 1, characterized in that the discs are "constructed at least in part from a material selected from the group consisting of cellulosics, polyolefins, polyamides and polyesters.
3. 3. The culture device according to claim 2, characterized in that the discs are constructed at least in part from alpha cellulose.
4. 4. The culture device according to claim 2, characterized in that the discs are constructed at least in part of rayon
5. 5. The culture device according to claim 2, characterized in that the discs are constructed at least in part of nylon.
6. 6. The culture device according to claim 2, characterized in that the discs are constructed at least in part from polylactic acid.
7. 7. The culture device according to claim 1, characterized in that each of the discs has a liquid retention capacity of about 1 to about 2 microliters.
8. 8. The culture device according to claim 1, characterized in that it has an indicator substance in the discs.
9. 9. A method for dividing an aqueous liquid sample into discrete microvolumes, characterized in that it comprises: a) providing ain device as claimed in claim 1 for the culture of a microorganism, the device has a titration surface, the titration surface comprises hydrophilic discs of liquid retention and a given hydrophobic area between the disks, each of the disks has a liquid retention microvolume capacity and the medium for the growth of microorganisms; and k) Etnsr in the liquid sample with __ the evaluation surface such that the liquid sample is divided into the hydrophilic liquid retention discs.
10. 10. The method according to claim 9, characterized in that the discs are constructed of a material selected from the group consisting of cellulosics, polyolefins, polyamides and polyesters.