MXPA98007574A

MXPA98007574A - Method for detection of contaminants

Info

Publication number: MXPA98007574A
Application number: MXPA/A/1998/007574A
Authority: MX
Inventors: C Miller David; Loomis Lawrence
Original assignee: New Horizons Diagnostics Corporation
Priority date: 1997-01-15
Filing date: 1998-09-15
Publication date: 1999-09-01

Abstract

A method and device for determining the presence and concentration of total microbial contamination or the presence and concentration of a specific microbial species is described. The method consists of a means of collection of the microbes from an air, liquid, surface or other sample and suspending them in fluid phase (3). An aliquot of the fluid phase (3) is introduced into a disposable test device which allows filtration of the sample to concentrate the microbes and to remove extraneous substances including somatic cells, and concentration of the microbes. The total concentration of microbes is determined by adding a somatic and bacterial releasing reagent to a disposable test device (25) which comprises a membrane (26) containing the luminescent reagents luciferin and luciferase, and introducing the disposable test device (25) into a luminometer that can read the luminescence from the underside.

Description

METHOD FOR THE DETECTION OF CONTAMINANTS Field of the Invention The present invention relates to a method for detecting the presence and determining the amount of contaminants. More particularly, the invention relates to a method for rapidly determining total microbial contamination or for determining the presence and amount of the specific microbial or chemical contaminant, present on a wide variety of surfaces including meat surfaces of carcasses or other foods. , surfaces of equipment, surfaces where food is being processed or prepared, and surfaces of equipment, gloves and materials in medical situations. Liquid pollutants and those that are carried by air are also detectable. In addition, the invention relates to a method for determining total microbial contamination or specific microbial contamination or chemical contamination, by bioluminescence or chemius inception.

Ref.28443 # Background of the Invention Microbial contamination is a significant cause of morbidity and mortality. The 5 fast and routine procedures for the quantitative determination of bacteria, particularly those present on surfaces, are frequently * vital importance, particularly in food processing and hospitals. The poisoning of food is often a result of microbial contamination of meat or food, which occurs during processing. Contamination can be spread through the contact of food with surfaces. In addition, the dispersion of Disease in hospitals and other facilities frequently occurs as a result of the passage of infectious microbes on the surface of clothes or equipment, or through air, water or other liquids. A key feature of these applications is the requirement for a rapid test over the course of minutes, a method that will overcome the potential contaminants of a variety of surfaces, liquids, and air, a requirement of no crossover in results from one test to a second, and a need for a general and specific test to verify the presence of microbes. There must be the ability to test contamination by both microbial counts and by the ability to test the presence of specific microbes. Several methods have been used to measure microbial contamination on surfaces. Traditional methods for evaluating bacteria on surfaces are based on swabbing the surface followed by a culture of the swab for 24 to 48 hours in or on a medium that supports the growth of the microbial species. The cultures are observed manually or with an automated instrumentation to determine the number of colonies that have formed as an indicator of the number of microbes present initially on the surface. The disadvantages of this methodology are prolonged evaluation times, specially trained personnel requirements, and the possible inadequate identification of certain potentially pathogenic microbes whose growth is not supported by the environment or the specific environment. In particular, it can be difficult to detect fungal contamination by this method. In addition, in many potential applications, the method does not provide results in the time frame required for a specific response.

Luminescent reactions have been used in various ways to detect bacteria in fluids and processed materials. In particular, bioluminescent reactions based on the reaction of adenosine triphosphate (ATP) with luciferin in the presence of the enzyme luciferase to produce light (the reaction of the "firefly") have been used. Since ATP is present in all living cells including all microbial cells, this The method can be used in a trial or rapid assessment to obtain a quantitative estimate of the number of living cells in a sample. The initial discourses on the nature of the reaction, the history of its discovery, and its general area of applicability are provided by E. N. Harvey (1957), A History of - * © - Luminiscence: From the Earliest Times Until 1900, Amer. Phil. Soc., Philadelphia PA and W.D. McElroy and B. L. Strehler (1949) Arch. Biochem. Biophys. 22: 420-433. Alternatively, detection has been used chemoluminescent by isoluminol or similar compounds. This method is based on the detection of substances that contain iron in microbes. The test procedures that exemplify the 'use of bioluminescent reactions for bacterial determinations and, the specialized instrumentation for the measurement of the associated light emission, are already known and have already been described. Plakas (US Patent Nos. 4,013,418, 4,144,134, and 4,283,490) teaches a bioluminescent assay for the detection of bacteria in a sample, which includes the steps of using non-bacterial cells, effecting filtration by means of positive pressure, washing, lysis of bacterial cells and detection of ATP released with a luciferin / luciferase / Mg2 + reagent. The art in this patent does not address the specific problems associated with the collection of the material from a surface or with the detection of specific bacteria. The issue of timing is not mentioned anywhere and the invention as described could require significant time. Chapelle in U.S. Pat. No. 4,385,112 describes a method for the detection of water-based bacteria, based on bioluminescence. This test requires several hours to be done and is specifically aimed at detecting the total bacterial content in the water. Clendenning in its U.S. Patent No. 3,933,592 describes a method for the bioluminescent detection of microbial contamination and in the examples refers to carrying out the process in less than 2 minutes. The procedure does not involve pretreatment phases and the removal of ATP from somatic cells. Aegidius (US Patent No. 5,258,285) describes a method for the detection of bacterial concentration in a sample that uses a filtration step, a washing step to remove foreign material including ATP from somatic cells, the establishment of a extraction chamber in which luciferin / luciferase / Mg2 + is added and the reaction measure. This method does not mention time. In addition, it uses separate chambers for washing, the extraction of bacterial ATP, and the measurement of the reaction. This can potentially lead to reduced sensitivity due to loss of material in the process of transferring the solution from one camera to the other. In addition, the method does not describe a means of collecting a sample from a surface. The detection of bacteria on the surfaces raises additional issues not mentioned in these previous methods. First of all it is the method to collect a sample that will be compatible with these devices and test materials. The method must effectively recover the bacteria from the surface and lead to a liquid suspension of the microbes.

A second issue of primary interest is that surfaces or other areas that are checked frequently are contaminated with materials that can interfere with the detection of microbes. Interfering materials that may be present on surfaces, air, or liquids are somatic cells, from the food itself and that include both animal and plant cells, or from the hands of an individual in contact with the surface. Since all living organisms, including somatic cells, contain ATP, the presence of these cells can mask or alter the reading obtained. An additional source of interfering substances are those that interfere with the light production reaction itself. These substances include a wide range of chemical substances such as chlorine, oxidizing agents, free ATP, heavy metals, and other chemical substances. As some of these chemicals are used to disinfect a surface, it is obvious that a reliable method for analyzing microbial contamination should include a means to remove these substances from a sample. It is an additional requirement in many cases in hospital applications and food processing that the method to ve the microbial contamination of the surfaces is fast. For example, in processing carcasses of carcasses, carcasses are processed on a line and any evidence of material for microbial contamination must be made within the time frame required to move the carcass for further processing. The luminescence-based methodologies previously described for microbial detection, have not included any means to directly process a sample from a surface, solid, or -gas, nor the manufacture of a liquid suspension for testing or directly from a sample of air or liquid. In addition, the processes have required multiple devices or chambers for the containment, filtration and measurement of the reaction. Finally, the processes have not incorporated a disposable device that minimizes cross contamination. Finally, in these assays or assessments to specifically detect microbial ATP and other specific contaminants, the previously described inventions have relatively long time frames that are not consistent with on-line processing, quality control, and immediate vecation of the results.Brief Description of the Invention The present invention is a method and device for determining the presence and / or concentration, of the total microbial concentration or the presence and / or concentration of a substance to be analyzed, specific target or target. In one embodiment of the invention, the method comprises collecting a surface sample by rubbing a circumscribed area of a surface in a prescribed manner using a collection apparatus means comprised of an absorbent or adsorbent material. The medium of the collection apparatus is placed in a container containing a fluid and agitated to release the surface contaminants from the medium of the collection apparatus in the fluid. The means of the collection apparatus may be in the form of a sponge or swab and the container may be a bag, tube, or small cup. The fluid can be collected directly, preparing a liquid suspension of a solid sample, passing a gas through the collection fluid, or collecting the air directly on the test device. An aliquot of the fluid phase is subsequently transferred to a disposable test device comprised of a translucent hollow cylinder, open on top and having a porous filter fixed on the lower portion. The fluid phase is filtered through the disposable test device comprised of a translucent hollow cylinder, open on the upper part and having a porous filter fixed on the lower part. The fluid phase is filtered through the disposable test device by applying a positive or negative pressure that leads to the retention of the microbes or the microbes. substances of target or target to be analyzed, on the surface of the filter. The filtration process leads to the concentration of the substance to be analyzed and the removal of any interfering substances from the material collected prior to the test, such as inhibitors or any materials not ^ frr specific, to maximize the specificity and sensitivity of the test. The material retained from the filter can be washed by adding an appropriate washing solution and reapplying an appropriate pressure to force to the fluid phase through the filter. Another feature of the present invention is that the retained material captured on the filter of the disposable test device can be evaluated or assayed by a chemiluminescent test method or bioluminescent. The final step of the test method comprises the addition of the luminescent substrate to the retentate which leads to the chemiluminescent reaction and the measurement of the light output from the chemiluminescent reaction using a photometer accommodating the disposable device. In another embodiment of the invention, a sample of liquid or air can be tested to determine the level of contamination. The use of a liquid sample eliminates the need to scrub a surface and / or wash the sample in a fluid. Similarly, a sample of air can be tested, using conventional collection media, without the need to swab a surface of any kind. The present invention allows a contaminant to be identified and / or the concentration determined in less than 1 hour from the time of collection to the final result, and generally in less than 5 minutes. More specifically, the present invention comprises a method for performing chemiluminescent assays such as bioluminescent assays for ATP, chemiluminescent immunoassay, or DNA probe assays. One embodiment of the present invention is a method for determining total microbial contamination, comprising the steps of: a) collecting a sample with a collection medium, b) shaking the collection apparatus medium with a fluid phase to discharge pollutants in a fluid phase, the fluid phase that comes to be the material collected; c) placing an aliquot of the material collected in a disposable test device; d) adding a washing / lysis reagent that lyses any somatic cells present in the aliquot; e) applying a positive pressure to the upper part 15 of the disposable test device or a negative pressure to the lower part of the disposable test device to remove the liquid phase containing the free ATP and any inhibitors as well as the concentration of the bacteria in the interface; f) adding a bacterial lysis reagent that perforates the bacterial cell walls allowing the release of the microbial ATP; g) adding the ATP-free luciferin and luciferase reagents; and • h) determining the amount of ATP present by measuring the light emitted through the translucent sides of the disposable test device. 5 The selection of fluids for the collection is well known to those skilled in the art. In In general, the fluid is comprised of a detergent, salt or buffer substance or any combination thereof that maintains the integrity of the microbial cell walls. A fluid consisting of 0.15M sodium chloride containing 0.5% Tween 20 detergent is one such selection. It is possible to use other formulations including a salted solution buffered with phosphate or with HEPES and other detergents including zwitterionic detergents and nonionic detergents. It will be obvious to a person skilled in the art that the mixing of the reagent (s) can be accomplished in any of the steps through the use of a micropipette. The detection method of this invention specifically allows both the concentration of the substance to be analyzed and any resulting chemiluminescent reaction caused by the presence of the substance to be analyzed, which occurs within the chamber of the test device. disposable. An added feature of the disposable test device is that the diameter of the filter is from 0.5 to 2.0 cm, preferably approximately 1.0 cm, so that the volume of the solution 5 of the bioluminescent or chemiluminescent substrate is minimized to maximize the exit of the filter. signal towards the detector means. The final volume of the substrate must # is from 20 μl to 1000 μl, more preferably approximately 60 μl to 100 μl. The device The disposable test can be inserted into a complementary device comprising a larger chamber (liquid-tight), comprising at least two components, which can house the disposable test device and through which a volume of the material collected greater than 500 μl can pass through the filter under positive or negative pressure and retain the microbes or substances of interest to be analyzed on the filter surface. For example, the disposable test device can be inserted into the lower chamber of the two-component device, with the lower chamber having an output flow for filtering, to which is fixed a removable top chamber of the two-component device. The upper chamber comprises a liquid-tight seal for lower compartment and has an adapter or intake fitting. The adapter or admission fitting can be configured for an accessory or adapter with Luer tip, complementary, for attaching a syringe with a Luer tip. The syringe may include at least one series of pre-filter (s) to remove any larger debris that is introduced into the filter of the disposable test device. When the passage of the material collected through the filter of the disposable test device is complemented, the two-component device can be opened, and the disposable test device physically removed. The disposable test device then contains the retained materials from a large volume of the collected material (ie, 50 ml). The filtration of the large volume of the collected material makes possible the increased sensitivity for the detection of the substances to be analyzed of the fluid for the collection. The disposable test device is then processed as previously described. - The chemiluminescent reactions of luciferin / luciferase for ATP are well known. Other chemiluminescent reactions employing bacterial luciferase reactions, or luminols for total microbial determinations, can easily be adapted to the methods and devices of the present invention.

The invention also relates to a detection method in which the presence and quantity of specific microbes on a surface can be detected in a time frame of less than one hour. He The method comprises the steps of: a) providing a disposable, clean test device, comprising a part # upper open, translucent sides and a porous filter attached to the lower side; 10 b) add an aliquot of the collected material, the collected material is like the one described above; c) add an appropriate washing solution comprised of detergent, or buffered salts or a combination thereof; f d) applying a positive pressure to the top of the disposable test device, or a negative pressure to the bottom of the disposable test device to remove the fluid from the disposable test device; device and deposit the microbes or substances to be analyzed, target or target, directly or indirectly on the surface of the porous filter; e) add a specific labeled antibody directed against specific microbes that are • to be detected and incubated for an appropriate period of time; f) applying a positive pressure to the upper part of the disposable test device, 5 or a negative pressure with respect to the lower part of the disposable test device, to remove the fluid containing the antibody labeled from the unreacted enzyme from the device , 10 g) add an appropriate washing solution comprised of a detergent and buffered salts; h) apply positive pressure to the top of the disposable test device, or a negative pressure to the bottom of the disposable test device to further remove the fluid containing the labeled antibody that did not react from the device; e) add a chemiluminescent substrate and determine the amount of light emitted by the chemiluminescent substrate using a photometer that accommodates the disposable test device in a manner which allows it to be precisely positioned with respect to the surface of the photosensor and which avoids any possible loss of the final reaction mixture during and after the measurement cycle. The method described above can also be modified by adding capture particles such as latex beads coated with a binder such as antibodies specific for the antigens or antigens for the target or target microbe antibodies in the disposable test device prior to performing the step (d). The method can also be modified so as to capture the particles and antibodies labeled with enzyme and the collected materials are all reacted simultaneously within the disposable test device, prior to performing step (f). The particles can also be coated with a specific antigen which binds to an antibody specific for the virus. The particles are added with the fluid for collection to the disposable test device in step (c). The detection reagent can be an enzyme conjugated to the binder, wherein the binder is an antibody enhanced with ATP or an ATP-encapsulated liposome bound to the binder and wherein the binder itself can be antibodies, antigens, lectins, DNA fragments, viruses, and combinations thereof.

• The enzyme may be a peroxidase, a phosphatase, an oxidase, a luciferase, or combinations thereof. In yet another embodiment of the invention, all chemical substances and solutions are in a disposable membrane device. Such a device is easier to use, particularly in the field, than the use of a disposable test device or a large volume concentrating device. The use of the device with membrane reduces significantly the need for additional reaction reagents, thus leading to a mobile and more accurate test system. The membrane device also allows the processing of liquid and air samples directly on the membrane.

Virtually all the elements of the invention are essentially self-contained in the disposable membrane device. The membrane device preferably comprises a plastic, cardboard or paper support, with two sides, articulated, having a section upper and one lower, and a disc or absorbent pad placed on top of the inner side of the upper section. An upper part of the absorbent disc is a glass filter membrane which can be held in place by a rigid layer of plastic or paper.

• In this embodiment of the invention, the membrane device can serve as a collection device as well as a reaction system. For example, to estimate the concentration of bacteria 5 in a given amount of an air sample, the portion of the glass filter membrane of the ticket is inserted into a chamber through which a measured volume of air is passed through and the air is allowed to hit the surface of the glass membrane. He The device is removed and, after the addition of a bacterial release agent to release the cellular ATP, the device is bent or folded to initiate the reaction. In yet another embodiment of the invention, the bacterial release agent is attached to the glass filter membrane or other membrane of the device. The lower section of the membrane device preferably comprises a transparent window to which a membrane is fixed Semi-transparent (when wet) with immobilized luciferin-luciferase. Various buffers for extracting antigens and washing immune complexes are well known to those skilled in the art. 25 Brief Description of the Drawings Figure 1 is a side view of the collection apparatus means comprising an axis, an absorbent tip, and a container with fluid; Figure 2 is an angular side view of the collection apparatus means comprising a sponge and a bag with fluid; Figure 3 is a front view of a large volumetric concentration apparatus; Figure 4 is an exploded perspective view of a large volumetric concentration apparatus; Figure 5 is a cross-sectional side view of a negative pressure apparatus; Figure 6 is an exploded perspective drawing of a positive pressure apparatus, a disposable test device and a fastener with absorbent disc; Figure 7 is a drawing of the disposable test device, its respective positioning in the complementary traction carriage and the relation with respect to the photosensing means; Figure 8 is a graph of the count of the total plate obtained after 48 hours of incubation and the relative illumination units obtained from the 5-minute bioluminescent method described in the preferred embodiment with each data point representing the flesh of a single corpse of a res; Figure 9 is a cross-sectional side view of the membrane device; Figure 10 is an angled top view of the membrane device, and Figure 11 is a cross-sectional view of the membrane device positioned on the photomultiplier.

Detailed Description of the Preferred Modality Figure 1 is a drawing of a collection apparatus means comprised of an axis 1 and an absorbent tip 2. The absorbent tip 2 is moistened with an excess of the fluid for collection 3 and used to clean by rubbing a circumscribed area of a surface that is going to be verified. After rubbing the area, the absorbent tip 2 is placed in a container 4 and agitated to release any of the bacteria absorbed in the fluid for collection 3.

Referring to Figure 2, the means of the collection apparatus may be comprised of a sponge 5. The sponge 5 is moistened with the fluid for collection 3 and used to wipe a circumscribed area to be checked. After rubbing the area, the sponge 5 is placed in a plastic bag 6 containing the excess fluid and pressed several times to release any of the bacteria absorbed in the fluid for collection 3. The volume of the collected fluid may vary, depending on the size of the absorbent and the area cleaned by rubbing. The fluid for collection 3 is selected to ensure the transfer of microbial contaminants from the test surface to the collection device and then to a disposable test device. In general, the pH of the fluid for collection 3 is between 5 and 8, but preferably between 6.0 and 7.0. The fluid for the collection preferably contains salts such as sodium chloride between 0.1M and 0.3M, preferably approximately 0.25M NaCl to ensure the sval of the bacteria. The fluid for collection 3 should contain a detergent such as 0.05% Tween 20 to ensure that the bacteria are easily removed from the test surface and the collection apparatus. Referring to Figures 3-6, a large volumetric concentration apparatus 7 concentrates a quantity of fluid for collection in a disposable test device. A suitably dimensioned Luer tip syringe is attached to the inlet 8 # of the large volumetric concentration apparatus 7 and then a positive pressure is applied to the plunger of the syringe 10 causing the collection fluid to flow out of the outlet 9. The fluid for the collection flows through the lower part 11 of the filter of the disposable test device 10. The "0" rings 14 and 15 provide a leak proof seal. After complementing the Wß. concentration of the collected material, the upper compartment 13 is separated from the lower compartment 16 to expose the edge 12 of the disposable test device 10. The disposable test device is then removed manually from the lower compartment 16. The lower portion of the disposable test device is inserted into the holes 18. The appropriate volume of the solution for washing or to cause The lysis of the somatic cells can be added and a # vacuum can be applied to the outlet 19 to remove the fluid from the disposable test device 10. The positive test apparatus 20 is comprised of a plunger (19) and a barrel or cylinder 21, 5 a disposable test device 10, and a fastener 25 of the device is comprised of a disk or absorbent pad 26 for absorbing debris from the * > fluid. The disposable test device is inserted into the holder 24. An aliquot of the fluid for the Collection (ie 50 to 100 μl) is added and an appropriate volume of solutions can be added for washing or for lysis of the somatic cells. The rubber seal 23 of the positive pressure device is placed on top of the device of Disposable test 10. The application of pressure to the plunger 19 forces the air through the barrel or cylinder 20 and out through the outlet 22, displacing the fluid which passes to the absorbent disc 26. A solution of additional wash and it repeat the process. The disposable test device 10, its respective positioning 28 in the pulling carriage 27, and the relation with respect to the photosensor medium 30 is shown in Figure 7. The body of the test device disposable 10 is comprised of an optically clear molded plastic material, such as polystyrene, which is capable of almost complete transmission of light within a wavelength range of 500-600 nm. Fused to the bottom surface of the device is a semi-permeable membrane 11 which is characterized by its strength and lack of deformation under pressure, and a pore size distribution which # - ensures a superficial retention of the bacterial cells, while facilitating the complete passage of any associated liquid phase during pressurization. This membrane must also have sufficient surface tension to retain the measurement solution even after wetting. The traction car is an integral part of a luminometer instrument. The pull carriage is removed and the disposable test device is placed in the hole 28 so that a window for the translucent wall of the disposable test device is exposed to the photosensing means when the carriage traction is returned to a dark chamber complementary to the luminometer. In a general bacterial sieve or filter based on bioluminescence, after a microbial sample has been concentrated in the device disposable test, a bacteriological reagent is added to use the bacteria and release them from ATP. An appropriate volume of the luminescent substrate (i.e. luciferin-luciferase) is added to the disposable test device and the traction carriage is returned to the dark chamber of the luminometer. The measurement of the light emission is made by digitizing or converting the electrical signal of the photosensing means to a number of relative light units. If the method is going to be used to detect specific bacteria, a specific antibody conjugated to a chemiluminescent or enzyme probe is added. In the preferred embodiment, the antibody is placed in the disposable test device and allowed to react for 10 minutes. Additional washing steps can be carried out by adding a washing solution and evacuating the washing solution. Then a solution of the luminescent substrate is added. In the preferred embodiment such a substrate consists of a mixture of hydrogen peroxide and luminol. The traction car is returned to the dark chamber of the luminometer. The measurement of the light emission is made by digitizing or converting the electrical signal from the medium of the photosensor to a number of relative lighting units. In another embodiment of the invention, all of the chemicals and solutions may be in a disposable membrane device 100. As with the systems described above, all the systems and methods described below involve the detection and quantification of the the samples which may also contain somatic cells, free of ATP, and constituents such as chloride ions which are known to inhibit the reaction of the luciferin-luciferase enzyme. The membrane device 100 preferably comprises a hinged, plastic, cardboard, or paper stand 101 that has an upper section 102 and a lower section 103. An absorbent pad 104 is placed on top of the side inner 105 of the upper section 104. The absorbent pad 104 is comprised of a material made of cellulose. The material can be cotton, corn beard, possibly fiberglass, or other absorbent material. On top of the absorbent disc 104 is a glass filter membrane 106, which can be held in place by a rigid layer of paper or plastic 107. The lower section 103 of the membrane device 100 preferably comprises a transparent window 108. on the outer side 109 of the lower section, and a luciferin-luciferase solution immobilized on the disk 111 with membrane. The membrane disc fits or fits into a hole 113 in the lower section 103 of the device 100. In one embodiment of the invention, the somatic or bacterial cell releasing agent can be incorporated into the glass membrane 106 of a almost identical manner in which the luciferin-luciferase solution is incorporated into the discs 111 with membrane. To use the device 100 with membrane, a volume of the sample of 25 μl, collected by normal means, is applied through a hole 110 in the rigid layer 107 on the surface of the glass filter membrane 106. The glass filter membrane 106 retains the bacterial cells and somatic about surface of the glass filter membrane 106 while the fluids pass to the absorbent disk 104. In one embodiment of the invention, the somatic cell release agent is then added. on the surface of the glass filter membrane 106. The release agent of the somatic cells is added by dripping onto the surface of the glass membrane 106 to prevent the flooding of the membrane device and the washing of the cells out of the membrane glass filter 106.

After the addition of the somatic cell release agent, the somatic cells have been used and the ATP released from the somatic cells, in the company of the free ATP and the inhibitory materials which could have contaminated the results, are trapped in the pad absorbent. In this stage only the bacterial cells are left intact on the surface of the disc 106 with glass filter membrane. In another alternative approach, the somatic release reagent can be placed on the swab used to scrape the surface area that is tested. In still another embodiment of the invention, the need to add a somatic cell releasing agent to the test sample is eliminated when the somatic cell releasing agent has already been attached to the glass membrane 106 prior to use of the membrane device. Next, 10 μl of the bacterial release agent is applied to the glass filter membrane 106, or the surface of a membrane 111 placed on the inner side 112 of the lower section 103 of the membrane device 100. The membrane 111 contains luciferin / immobilized luciferase. The luciferin / luciferase can be either saturated throughout the membrane 111, or foon the surface of the membrane 111. In another embodiment, the bacterial release agent can be immobilized on the glass filter membrane 106 or on the membrane 111 The upper section 102 and the lower section 103 of the membrane device 100 are then compressed together, preferably during insertion of the disposable membrane device into the luminometer pulling carriage. When the upper section 102 and the lower section 103. of the membrane device 100 make contact, the reaction that produces the light: Mg ++ 15 Luciferase + Luciferin + ATP - ^ oxy-Luciferin + AMP + light 02 is initiated. This leads to RLU 's during a ten-second integration period, which corresponds to the bacterial content of the sample. As shown in Figure 11, the membrane device 100 is preferably placed on the pull carriage 200 of the luminometer with the membrane of luciferin-luciferase turned downward, directly on a reading hole 201. The photomultiplier tube 30 is placed directly r the orifice 201. In another embodiment of this invention, the particles coated with a specific antigen are added with the sample fluid to the disposable test device, with the antigen that binds to an antibody specific for the virus. The methods described above can be used not only to test surfaces, but also to test fluids of all kinds, including air and liquids. To test the bacterial level of an air sample, the disposable membrane device or test device can be placed on an impactor or vacuum device of any kind, allowing air to be extracted through the collection device or membrane device . The bacterial air pollutants will then be trapped on the surface of the membrane devices, ready for testing. The invention is illustrated further by means of the following examples.

Example 1 - General Bacterial Filter on Hard Surfaces.

This example involves a procedure for testing a stainless steel surface to verify the presence of microbial contamination. The Escherichi coli was grown on tryptic soy agar for 18 hours at 30 ° C. A sample of the bacteria was introduced in 10 ml of soy broth peptic and incubated for an additional 18 hours. The bacteria were collected by centrifugation and washed three times in 0.9% NaCl which has been filtered under sterile conditions. The optical density of the solution was measured at 650 nm and the concentration was adjusted that the optical density was 0.300. Three 10-fold dilutions in series were prepared to reach a concentration of 105 microbes / ml. 100 μl of this solution was drained over a 10 x 10 cm area demarcated on the surface of a steel sheet stainless steel that has been previously cleaned with bleach, alcohol and sterile distilled water. The solution containing the bacteria was allowed to dry for 5 hours at room temperature. Distinguished control areas were prepared without bacteria. The individual sponges of 10 X 10 cm were pre-wetted with approximately 750 μl of a fluid for the collection comprised of 0.15 M NaCl containing 0.05% Tween 20 in a bag. This solution was only enough to moisten the sponge completely. Each sponge was removed from a bag and rubbed over the distinguished areas of the surface with 10 strokes in each direction. The sponge was then returned to the bag and # was manually pressed ten times producing a collected material. An aliquot (25 μl) of the collected material is removed from the bag and placed in a disposable test device. 25 μl of the bacteria release agent was added and 50 μl of a luciferin / luciferase / magnesium mixture was added. The traction carriage closed and the relative lighting units were determined. In a second set of experiments, the swabs were pre-wetted with approximately 300 μl of the fluid for collection in a bag as described above. The swabs were used for similarly rubbing the distinct areas of a stainless steel surface as described above. In each case, the control areas which had no bacteria seeded on the surface were also tested. In addition, the bacterial solution that has been seeded on the surface was placed directly in the collection fluid as a positive control. Each point of the data represents the average of three samples. Referring to Table 1, approximately 80% of the seeded bacteria could be detected using either a sponge or a swab as collection means. # Table 1 10 Example 2: Chemiluminescent Salmonella Assays This example involves a procedure to 15 • prove the presence of salmonella.

Bacteria, either Salmonella typhimurium, ATCC 14028 or Aeromonas hydrophila, ATCC 7966, were scraped from frozen storage materials on tryptic soy agar plates and incubated for 18 hours at 26 ° C. Bacterial colonies were collected in 0.9% sterile NaCl. The optical density of the solution was measured at 650 nm and the concentration was adjusted so that the optical density was 0.300 by diluting the bacteria in 0.05M Tris, 0.05M EDTA, 0.15M NaCl, pH 8.2. An aliquot (10 μl) of a 0.5% solution of latex microspheres coated with antibodies to the salmonella was added to the disposable test device. An aliquot, 100 μl, of the diluted bacteria was placed in a disposable test device with a filter on the lower surface composed of Biodyne C of 1.2 microns. After the aliquot of the bacteria was added, the solution was allowed to settle for 10 minutes. A positive pressure was applied and the fluid was evacuated on an absorbent pad. Trapped antigens were washed by adding 200 μl of wash solution consisting of 0.01M PES, pH 7.2 containing 0.05% Tween 20. The positive pressure was applied again and the fluid was evacuated on an absorbent disc. An antibody labeled with horseradish peroxidase directed against Salmonella was added to the disposable test device and allowed to sit for 10 minutes at room temperature. A positive pressure was applied again and the fluid was evacuated from the disposable test device. A wash solution was added and evacuated with positive pressure twice more. The disposable test device was placed in a luminometer. 100 μl of the Lumiglo Chemiluminescent substrate (Kirkegaard and Perry Laboratory Gaithersburg, MD) was added, the traction car was closed immediately and the emission of the light was determined. The results shown in Table 2 indicate that concentrations as low as 105 organisms could be easily distinguished from a negative control using this system.

Table 2: Results of a Test for Salmonella A second procedure similar to that detailed above was used, except that none of the latex beads were added to the disposable test device prior to the introduction of the aliquot of the bacteria. In this case, the signal to noise ratio for a solution of S. Typhimurium (108 organisms): A. Hydrophila (108 organisms) was 5.91. 10 A third procedure was also tried. In this method, 40 μl of the sample, and 40 μl of anti-salmonella antibody labeled with horseradish peroxidase were added to a disposable test device. The mixture was incubated for 20 minutes at room temperature. Positive pressure was used to evacuate the fluid from the test device. The trapped material was washed three times by the introduction of 200 μl of a 0.01M phosphate buffered solution of pH 7.2 containing 0.05% Tween 20 followed by the evacuation of the disposable test device 5 fluid using a positive pressure. The disposable test device was placed on the luminometer and 100 μl of the Lumiglo substrate * Chemiluminiscent (Kirkegarrd and Perry Laboratories, Gaitherburg, MD) were added. The traction carriage 10 closed immediately, and the light emission was determined. The ratio of the signal with respect to noise for a solution of S. Typhimurium (106 organisms): A hydriphila (106 organisms) was 1.83.

Example 3: Detection of Bacterial ATP from a Powdered Food Sample A suspension of a powdered food sample was prepared by mixing 25 g of a sample of food in 225 ml of an SRA solution in a laboratory beaker. 50 μl of the suspension are placed in the filtravette filter device. Four (4) drops of SRA are added to the suspension. The FiltravetteR is placed on the plastic pedestal are the paper for the transfer by absorption placed below. The suspension is filtered using the positive pressure device, by which the entire ATP is extracted from the somatic cells and allowed to be absorbed by the paper for transfer by absorption while retaining all the bacterial cells on the filter paper. Six more drops of the SRA are placed in the filtervetteR and the step is repeated * of the filtration using the positive pressure device. 10 The filtravette is then placed in the microluminometer, and 50 μl of BRA is added to release the bacterial ATP. The tip of the pipette is used to mix the BRA and the suspension. 50 μl of the reconstituted LL is added and mix 2 to 3 times, using the tip of the pipette. The sample extractor is closed immediately to work for a period of 10 seconds. The reading of the relative light units (RLU's) are recorded from the digital reading of the microluminometer. 20 Example 4: Method of Determination of the ATP Amount of the Yeast Using Various Membranes Strains of Escherischia coli and Streptococcus pyrogenes were grown on Tryptic Soy Agar and the Saccharomyces cerevisiae strain was grown on Rose Bengal Agar. Colonies that have recently grown from each test organism were taken in 0.01M PBS. The suspension was adjusted to an OD650 value of 0.3 representing ca. 3 x 108 bacterial cells / ml and 3 x 106 yeast cells / ml. Two ten-fold dilutions were made in 0.01M PBS of each suspension. A Filtravette® was placed on a plastic pedestal with a paper for absorption transfer stacked under it. A 50 μl sample of each suspension (ca 106 bacterial cells / ml and 104 yeast cells / ml) were added in the Filtravette®. 4 drops of SRA were added. The solution was filtered using the positive pressure device. This was repeated by adding 6 drops of SRA again. The filtravette was then placed in the microluminometer extractor. Fifty μl of BRA was added to the filtravette. Fifty μl of reconstituted LL is then added followed by mixing 3-4 times by pipetting up and down. The sample extractor was closed immediately to begin an integration period of ten seconds, and the readings in RLU were recorded from the digital reading. These values of RLU were considered of absolute order to obtain 100% of the material retained (control).

# The results are summarized as follows: TABLE: EFFECTIVENESS (%) OF VARIOUS MEMBRANES IN THE FILTRATION / RETENTION OF BACTERIA AND YEAST Type of S. pyogenes E. Coli S. cerevisiae Membrane% retenc. % filt. % retens% filt. (Yeast)% retenc. % filt. Membrane 1 100 0 100 0 100 0 Membrane 2 ND ND 94.1 5.9 99.8 0.2 Membrane 3 ND ND 7.6 92.4 95.4 4.6 Membrane 4 81.1 18.9 ND ND 99.9 0.1 Membrane 5 66.2 33.8 ND ND 99.9 0.1 Membrane 6 79.5 20.5 ND ND 99.9 0.1 Membrane 7 76.2 23.8 ND ND 99.9 0.1 Membrane 8 0.1 99.9 ND ND 69.9 30.1 Membrane 9 25.5 74.5 28.9 71.1 99.3 0.7 ND: Not done Example 5: Detection of Bacterial ATP from the Liquid Waste Sample of Gasoline Prior to taking samples of a liquid waste from the gasoline, the sample of the liquid waste of the gasoline is left unchanged until clear zones of the separated phases appeared. A total of up to three phases with two interfaces could appear in the liquid waste sample of gasoline. The specimens are collected from each phase using a pasteur pipette and placed in an eppendorf tube. 50 μl of the sample from each eppendorf tube that represents the respective phases of the waste gasoline liquids are placed in the RV filter. Four drops of SRA are added. The FiltravetteR is placed on the plastic pedestal with the paper for transfer by absorbency underneath, and the solution is filtered using the positive pressure device.

This step removes all of the ATP from the somatic cells and subsequently allows them to be absorbed by the paper for transfer by absorption while retaining all the bacterial cells on the filter paper. Six are added more drops of SRA in the FiltravetteR and the filtration step is repeated using a positive pressure device. The filtravetteR is placed in the microluminometer, and 50 μl of BRA are added to release the bacterial ATP, and the liquids are completely mixed using the tip of the pipette. Add 50 μl of reconstituted luciferin-luciferase and mix two to three times, using the tip of the pipette. The extracted sample is closed to begin the integration period of ten seconds, and the readings in Relative Illumination Units (RLU's) are recorded from the digital reading of the microluminometer. Using the method described above, a sample of liquid gasoline waste is tested, which has three different phases (1. Upper translucent oil phase, 2. Medium brown phase, and 3. Intense brown, lower viscous phase. The phases listed above are tested, including two additional "interfaces" present between the three primary or main phases.The results are summarized as follows: # * A quantity of liquid from the specimen of each phase was scraped over the Tryticase Soy Agar (TSA; Difco). The plates were then placed for 24-48 hours at 37 ° C. Bacterial colonies were observed on the plate representing a two-phase sample with a count of 5.5 x 103 units forming the colony / ml. The other phases produced no visible colony on their respective plates. 10 Example 6: Competitive Test on Tap Water A tap water sample was tested using the conventional heterotrophic plate (HPC) plate counting method, widely used to check the drinking water and comparing the results with the method of the invention of using a large volumetric concentration apparatus (FIG 3, # 7) to collect and concentrate the water obtained from two different sites. The water sample was collected in the plastic device. Four drops of SRA were then added to the plastic device and processed according to the Steps in Example 2.

The result of these two sampling periods shows that the invention will be capable of detecting the bacteria which will be below 100 CFU / ml, probably also below 10 CFU / ml.

Example 7: Determination of the Effectiveness of the Immobilization of Luciferin-Luciferase on a Membrane A plastic device similar to that of Figure 10 with luciferin-luciferase on a glass membrane is used. A 10 μl sample of a TP standard is added. The value of Relative Lighting Units of the ATP standard is referred to as a liquid phase LL system. The results show a correlation between the LL immobilized on a plastic device and a liquid phase system Ll. The information on three groups of membrane devices is tested to determine if there is a direct correlation between the amount of ATP present and the light released using a microluminometer.

Table 1. Counts of three L.L. using an intermediate-range ATP control solution. (Counts of the ATP -10000 Solution) Counts of three membrane devices using a high-range ATP control solution. (ATP Solution Counts of -20000) Example 8: Detection of Bacterial Water Content During a Sequential Purification Step This example involves a procedure for testing the bacterial content of ultrapure water samples used in the manufacturing process of silicon microcircuits during various stages of purification. The samples were taken from the materials collected from each of the sequential purification steps. For each test, a membrane device was inserted into a support apparatus which served to compress the glass filter membrane between the two "o" rings, allowing relatively large sample volumes to be removed by means of the filter when applies a negative pressure. The membrane device was removed, 10 μl of the bacterial release agent is added, and the device is closed and inserted into the tensile carriage of the th (th) luminometer. The results are tabulated in the following table and clearly indicate the progressive depletion of the bacterial content in each step of sequential purification.

# § All of the above examples and tests can be made using the version of the membrane device of the invention. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood within the scope of the appended claims that the invention may be protected in a manner other than that specifically described.

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.

Having described the invention as above, property is claimed as contained in the following

Claims

1. A method for determining the presence and quantity of a substance to be analyzed, characterized in that it comprises the steps of: a) collecting a sample of the substance to be analyzed; b) place the sample on a disposable test device with filter media 10 permeable, the disposable test device is a device with membrane, the device with membrane comprises: a support with two articulated sides having an upper section and a lower section, a 15 absorbent pad placed on the upper part of an internal side of the upper section, a filter membrane, on the upper part on the absorbent pad, a rigid layer that retains the membrane in its 20 place; the rigid layer placed on the filter membrane; a rigid layer placed on the filter membrane; a hole in the lower section of the membrane device; a disc with membrane fitted or adjusted in the 25 hole of the device with membrane; a lighting solution immobilized on the membrane disc; a transparent window on an outer side of the lower section under the membrane disc; wherein the sample 5 is applied through a hole in the rigid layer on the surface of the filter membrane; c) Apply a release agent of somatic cells to the surface of the membrane 10 filtering; d) applying a bacterial release agent to the surface of the disc with membrane placed on the inner side of the lower section of the membrane device; 15 e) compressing the upper section and the lower section of the membrane device; f) sliding the membrane device in a photometer by means of a traction carriage; and g) measuring the emission of the light resulting from the luminescent reaction with a photometer comprising a photodetector means and a light-tight chamber for the disposable test device and a means for measuring the light passing through the wall transparent of the device with membrane; and • h) causing the photometer means to output a signal indicative of the presence and amount of the substance to be analyzed.

2. A method according to claim 1, characterized in that steps (c) to (g) are carried out within the time interval of 5 minutes.

3. A method according to claim 2, characterized in that steps (c) to (g) are carried out within the time interval of 2 minutes.

4. A method according to claim 1, characterized in that the means of the disposable collection apparatus is comprised of a soft absorbent.

5. A method according to claim 1, characterized in that the means of the disposable collection apparatus consists of an absorbent sponge.

* 6. A method according to claim 1, characterized in that the means of the collection apparatus is comprised of a soft absorber and an axis.

7. A method according to claim 1, characterized in that the fluid for the collection contains a detergent.

8. A method according to claim 1, characterized in that the fluid for the collection contains a salt.

9. A method of compliance with 15 claim 1, characterized in that the substance to be analyzed contains adenosine triphosphate (ATP) and the emission of light correlates with the concentration of ATP.

10. A method according to claim 1, characterized in that a large volumetric concentration apparatus is used to concentrate the fluid in the disposable test device as part of step (c). 25

11. A method according to claim 1, characterized in that the filter medium is a hydrophilic permeable membrane.

12. A method according to claim 1, characterized in that all substances emitting light are retained within a disposable test device during the operation of step (f).

13. A method according to claim 1, characterized in that the device of the membrane is comprised of a material selected from the group consisting of plastic, cardboard, or paper.

14. A method according to claim 1, characterized in that the absorbent pad is comprised of cellulose.

15. A method according to claim 1, characterized in that the absorbent pad is selected from the group consisting of cotton, corn beard, and glass fiber.

16. A method according to claim 1, characterized in that the filter membrane is comprised of a glass filter.

17. A method according to claim 1, characterized in that the illumination solution is a luciferin-luciferase solution.

18. A method according to claim 1, characterized in that the luciferin-luciferase solution comprises luciferin, luciferase, and magnesium.

19. A method according to claim 18, characterized in that the luciferin-luciferase solution further comprises the chemicals selected from the group consisting of trehalose, di thiothreito, a buffer solution of HEPES, and combinations thereof.

20. A method according to claim 1, characterized in that the release agent of the somatic cells is added by dripping onto the surface of the filter membrane in order to avoid # the flooding of the membrane device and the washing of the cells outside the membrane. the filtering membrane.

21. A method of compliance with 5 claim 1, characterized in that the photodetector means is placed directly under the disc of the membrane, the disc of the membrane is turned downwards on the luminometer.

22. A method for determining the presence and quantity of a substance to be analyzed, characterized in that it comprises the steps of: a) collecting a sample of the substance to be analyzed; 15 b) placing the sample on a disposable test device with permeable filter media, the disposable test device which is a membrane device, the membrane device comprises: a support with two sides 20 articulated having an upper section and a lower section, an absorbent pad placed on the upper part of an internal side of the upper section, a filter membrane, on top of the Absorbent pad, a somatic cell releasing agent fixed to the surface of the filter membrane; a rigid layer that holds the membrane in place; a hole in the rigid layer placed on the filtering membrane; a 5 hole in the lower section of the membrane device; a hole in the lower section of the membrane device; a membrane disc equipped or fitted in the orifice of the membrane device; a solution 10 illumination immobilized on the membrane disc; a transparent window in the lower section under the membrane disc; where the sample is applied through a hole in the rigid layer on the surface of the membrane 15 filtering; c) applying a bacterial release agent to the surface of the disc with membrane placed on the inner side of the lower section of the membrane device; 20 d) compress the upper section and the lower section of the membrane device; e) sliding the membrane device in a photometer by means of a traction carriage; and • f) measure the emission of light resulting from the

A luminescent reaction with a photometer comprising a photodetector means and a light-tight chamber for the disposable test device and a means for measuring the light passing through the transparent wall of the membrane device 5; and g) causing the photometer means to output a signal indicative of the presence and amount of the substance to be analyzed. 10 23. A membrane device for placing a sample in a luminometer to obtain a bacterial count, the membrane device comprises: a support with two articulated sides having an upper section and a lower section, a pad 15 absorbent placed on top of an internal side of the upper section, a filter membrane, on top of the filter pad, a rigid layer that holds the filter element in place; a rigid layer placed on the membrane 20 filtering; a hole in the rigid layer placed on the filtering membrane; a hole in the lower section of the membrane device; a disc with membrane equipped in the hole of the device with membrane, a lighting solution immobilized on the disc with 25 membrane; and a transparent window in the lower section under the membrane disc; where the sample is applied through a hole in the rigid layer on the surface of the filter membrane.

24. A method according to claim 22, characterized in that it also comprises the somatic cell release agent that is incorporated in the surface of the filter membrane.

25. A method for determining the presence and quantity of a substance to be analyzed, characterized in that it comprises the steps of: a) collecting the substance to be analyzed with a disposable collection apparatus means; 15 b) add the fluid for the collection to the medium of the collection apparatus; c) placing the fluid for the collection in a disposable test device with permeable filter media at its lower end, 20 an upper open end, and transparent side walls; d) applying pressure to the disposable test device to force the fluid to pass through the permeable filter medium and retain the substance to be analyzed on the permeable filter medium; e) add a reagent to the disposable test device, which establishes a 5 luminescent reaction in the test device; f) measuring the emission of light resulting from the luminescent reaction with a photometer comprising a photodetector medium and a light-tight camera 10 for the disposable test device and a means for measuring the light passing through the transparent side wall of the test device; and g) causing the photometer means to output a signal indicative of the presence and amount of the substance to be analyzed.

26. The method according to claim 25, characterized in that steps (c) a 20 (g) are carried out within the time interval of 5 minutes.

27. The method according to claim 25, characterized in that the disposable collection apparatus is comprised of a soft or soft absorbent.

28. The method according to claim 25, characterized in that the disposable collection apparatus means consists of a sponge absorbent.

29. The method according to claim 10, characterized in that the means of the disposable collection apparatus is comprised of a soft or soft absorbent and an axis.

30. The method according to claim 25, characterized in that the collection fluid contains a detergent.

31. The method according to claim 25, characterized in that the fluid of 20 collection contains a salt.

32. The method according to claim 31, characterized in that a washing fluid is added to the fluid for collection, salting, 25 in the disposable test device in step (c) and the washing solution is comprised of a saline solution containing detergents, and the washing solution lyses the somatic cells and does not lyse the microorganisms.

33. The method according to claim 25, characterized in that after carrying out step (d), the additional steps of adding a washing solution and applying 10 pressure to the disposable test device, wherein the pressure forces the wash solution to pass through the filter medium.

34. The method according to claim 25, characterized in that a bacteriological reagent is added to the disposable test device in step (e).

35. The method according to claim 25, characterized in that the substance to be analyzed contains adenosine triphosphate (ATP) and the emission of light correlates with the concentration of ATP.

36. The method according to claim 25, characterized in that the luminescent reaction of step (e) is established by adding luciferin-luciferase.

37. The method according to claim 25, characterized in that the luminescent reaction is established by adding isoluminol.

38. The method according to claim 25, characterized in that a large volumetric concentration apparatus is used to concentrate the fluid in the disposable test device.

39. The method according to claim 25, characterized in that the filter medium is a hydrophilic permeable membrane.

40. The method according to claim 25, characterized in that all the substances that emit light are retained within the disposable test device while the step (f) is carried out.

41. A method for determining the presence and quantity of a substance to be analyzed, characterized in that it comprises: a) collecting the substance to be analyzed with a disposable collection apparatus means; b) add the collection fluid to the middle of the collection apparatus; c) placing the collection fluid in a disposable test device with permeable filter media at its lower end, an open top, and transparent side walls; d) applying pressure to the disposable test device to force the fluid to pass through the permeable filter medium and retain the substance to be analyzed on the permeable filter medium; e) adding a detection reagent within the disposable test device, the detection reagent is attached to the substance to be analyzed; f) adding a wash solution to the disposable test device and applying pressure to the disposable test device to force the fluid to pass through the permeable filter medium; g) establishing a luminescent reaction in the test device; 5 h) measuring the emission of the light resulting from the luminescent reaction with a photodetector means comprising a light-tight chamber for the disposable test device and a means for measuring the light passing through the transparent side wall. of the test device; and i) causing the photometer means to output a signal indicative of the presence and quantity of the substance to be analyzed.

42. The method according to claim 41, characterized in that steps (c) to (i) are carried out within the time interval of 30 minutes.

43. The method according to claim 41, characterized in that the disposable collection apparatus is comprised of a soft or soft absorbent. 25

44. The method according to claim 41, characterized in that the means of the disposable collection apparatus consists of a sponge absorbent.

45. The method according to claim 41, characterized in that the means of the disposable collection apparatus is comprised of a soft or soft absorbent and an axis.

46. The method according to claim 41, characterized in that the fluid for the collection contains a detergent. 15

47. The method according to claim 41, characterized in that the fluid for the collection contains a salt.

48. The method of compliance with 20 claim 41, characterized in that a washing fluid is added to the fluid for collection in the disposable test device in step (c), and the washing fluid is comprised of a saline solution containing detergents. 25 *

49. The method according to claim 41, characterized in that the particles coated with a specific antibody that binds to the substance to be analyzed, are added to the 5 disposable test device in step (c) with the collection fluid.

50. The method according to claim 41, characterized in that the particles 10 coated with an antigen or specific antibody that binds to the substance to be analyzed, are added to the disposable test device in step (c), with the collection fluid.

The method according to claim 41, characterized in that after carrying out step (d), the additional steps of adding a washing solution and applying the pressure to the disposable test device are carried out where 20 the pressure forces the washing solution to pass through the filter media.

52. The method according to claim 41, characterized in that it also comprises To add capture particles coated with a binder, such that the binder is bound to the substance to be analyzed or to the fragment of the substance to be analyzed, the binder is selected from the group consisting of antibodies, antibodies enhanced with ATP, antigens, lectins, DNA fragments, viruses, and combinations thereof.

53. The method according to claim 41, characterized in that the reaction The luminescent of step (f) is established by the addition of a chemiluminescent substrate.

54. The method according to claim 41, characterized in that step (d) is 15 removed so that the collection fluid and the detection reagent are placed together in the disposable test device.

55. The method according to claim 41, characterized in that a large volumetric concentration apparatus is used to concentrate the fluid in the disposable test device as part of step (c).

# 56. The method according to claim 41, characterized in that an air sample can be tested for bacterial agents by placing the collection device on an impactor 5 or vacuum, allowing air to be extracted through the collection device.

57. The method according to claim 1, characterized in that a sample of 10 air can be tested to verify the presence of bacterial agents by placing the collection device on an impactor or vacuum, allowing air to be extracted through the membrane device. 15

58. The method according to claim 41, characterized in that the particles coated with a specific antigen are added with the fluid for collection to the disposable test device in step (c), the antigen is bound to a 20 antibody specific for the virus.

59. The method according to claim 1, characterized in that the particles coated with a specific antigen are added with When the fluid from the sample to the disposable test device, the antigen binds to an antibody specific for the virus.

60. The method according to claim 1, characterized in that it also comprises the agent for the release of bacterial cells incorporated in the surface of the filter membrane.

61. The method according to claim 52, characterized in that it further comprises the addition of enzyme-labeled antibodies to the capture particles and the collected material, in such a way that they are all reacted simultaneously within the test device, and the enzymes of the antibodies labeled with the enzyme are selected from the group consisting of peroxidase, a phosphatase, an oxidase, a luciferase, and combinations thereof.