MXPA97009031A - Methods and apparatus for filtration and cult - Google Patents

Abstract

The present invention relates to a method and apparatus for filtering microorganisms from a sample, and to growing the microorganisms using a membrane filter in a filter holder, the filter holder being supported on an absorbent support, which is maintained in a compressible-expandable state against the membrane filter, such that any expansion of the membrane filter when moistened by a sample, does not cause trapped air bubbles to separate it from its support and prevent the nutrients from the medium of culture supplied to the support reach the microorganisms of the membrane filter

Description

METHODS AND APPARATUS FOR FILTRATION AND CULTIVATION DESCRIPTION OF THE INVENTION The present invention relates to methods and apparatus for the filtration of microorganisms from a sample and to the culture of the microorganisms in itself on the filter used in the filtration. As described in EPO Patent 150775, it is known how to capture microorganisms for cultivation, by membrane filtration of a sample through a sterile membrane placed in a fastener. After filtration, the membrane can be removed and deposited on a gelatinous culture medium contained in a Petri dish. Then, the Petri dish can be incubated at a suitable temperature for the time necessary for the microorganism to be able to develop and multiply in a sufficient manner to form colonies visible to the naked eye, to allow its identification and make counts. Even slightly contaminated samples can be evaluated, since the microorganisms are concentrated in the membrane and it is possible to filter a significant volume of sample to collect a significant number of microorganisms. However, REF: 26263 disadvantages that arise from this method are, first, that the colonies can deplete nutrients from the local area of the agar medium, which, in circumstances where there is a large number of bacteria in the membrane, can cause the suppression of the growth of individual colonies. Also, care must be taken to place the membrane filter on the culture medium to avoid trapping any air bubbles between the culture medium and the membrane, which avoids contact between the culture medium and a portion of the surface. of the membrane, thus hindering the diffusion of the culture medium and inhibiting the growth of microorganisms. In an alternative procedure, it has been proposed to place the membrane containing the filtered bacteria on a wick of filter paper, to drive the nutrient medium towards the membrane. However, the amount of medium on the wick has to be carefully controlled in order that the membrane filter does not get too wet and a confluent growth is observed instead of the growth of individual colonies. Likewise, these techniques generally involve the transfer of the membrane from the filtration apparatus to the culture operation and this additional step has the potential to induce contamination, since the membrane is exposed to air. This also means that the culture can not start until the samples have traveled from the sampling site to the laboratory, which can take several hours. As described further in the patent EP-B0150775, it has been proposed to remsome of these drawbacks by the use of an apparatus consisting of a sterilized box with circular elements tucked in one another and a removable lid. The box includes an inlet and an outlet disposed on both sides of a fastener on which there is an absorbent pad and a filtration membrane, fastened at the periphery of the fastener by one of the circular elements. After filtration, a culture medium is introduced through the outlet, ie, countercurrent to the filtration operation, to saturate the absorbent pad. The box can be placed in an incubator to allow the collected microorganisms to grow. This avoids the need for any transfer operation after filtration and before cultivation, and allows the samples to be taken directly at the site where the liquid was collected. However, as established in Patent EP-B0150775, this method still has several drawbacks. In particular, the diameter of the membrane increases when the membrane becomes wet and, since the membrane and the absorbent pad which is attached on its periphery to the sterilized box are kept dry before being used, by filtering the sample, moisture in the pad and the membrane can cause the membrane filter to peel off the absorbent pad. This will avoid the contact between the membrane and the pad saturated with the culture medium and, therefore, alter the development of the microorganisms when they are incubated. Patent EP-B0150775 proposes an elaborate solution for this problem, which involves sealing a membrane filter through the bottom of a tubular bra, in such a way that the bra can be pressurized with air to bulge the membrane outward before coupling it against the surface of a gelled nutrient medium in a cup. This reintroduces the danger of contamination of the system during the required handling. Any contamination by microorganisms at the interface between the nutrient medium and the filter membrane is likely to interfere with the growth of the microorganisms on the opposite side of the membrane filter. In addition, the apparatus illustrated in Patent EP-B0150775 is undoubtedly elaborate and problematic to manufacture and use. Now we observe that the problem indicated in Patent EP-B0150775 is likely to have a substantially simpler solution, which avoids reintroducing the drawbacks inherent in the prior art. Accordingly, the present invention now provides a method for filtering microorganisms from a sample and culturing said microorganisms, which comprises filtering a sample containing microorganisms through a membrane filter in a filter holder, wherein the Filter holder is supported on an absorbent support; and then, supplying culture medium to the microorganisms of the membrane filter in the filter holder, by absorbing the medium in the absorbent support, characterized in that the absorbent support is maintained in a compressible-expandable state against the membrane filter. Inasmuch as the absorbent support is being compressed against the membrane filter, in case of expansion of the membrane filter, any tendency of it to expand beyond the absorbent support is counteracted because the absorbent support is able to expand and maintain contact with the membrane filter. Therefore, there is no need to remove the membrane filter from the absorbent support after the filtration operation and to attach the surface of the membrane filter against a gel of nutrient medium. Instead, the sterile nutrient medium can be applied directly to the compressed absorbent support in the filter holder. The absorbent support is preferably a crosslinked foam. Suitable polymeric foams available are made from a wide variety of plastic materials such as polyethers, polyesters, polypropylene, polyvinyl chloride and polyurethanes. Preferred polymeric foams have 50 to 200 ppi (pores per inch) (equivalent to 20 to 80 pores per centimeter), for example about 100 ppi (equivalent to 40 pores per centimeter). Suitably, the absorbent support has the form of a cross-linked foam block having a non-compressed thickness of 0.5 to 3 cm, for example of approximately 1 cm, and has a main surface on which the membrane filter can be directly placed. , or more preferably, with a layer of a wicking material such as filter paper between the membrane filter and the absorbent support.

Preferably, the absorbent support is compressed to have a thickness of 7/8 to 1/10 of its uncompressed thickness. The degree of compression needs to be uniform throughout the area of the absorbent support. For example, if the compression is applied at the edges of the absorbent support, the compression of the center of the absorbent support could be significantly less. Preferably, between the membrane filter and the crosslinked foam absorbent support, there is an intermediate compression distribution support layer, which transmits the compression forces applied to the edges of the membrane filter assembly, compression distribution layer and cross-linked foam absorbent support, in such a way that the center of the assembly is also under compression. Such a compression distribution layer, conveniently, may be a sheet of filter paper. Other suitable materials can be used. These should preferably be flexible and porous. Preferably, such compression distribution material also serves as a wick for taking culture medium from the absorbent support and supplying it to the membrane filter. This should not obstruct the expansion of the absorbent support to equalize the expansion of the membrane filter. It should be selected in such a way that it expands as much as the membrane filter when it gets wet. A liquid sample to be filtered can be placed in a sample chamber above the membrane filter and can be passed through the membrane filter by suitable mechanisms, such as a vacuum pump or a syringe. The filtered liquid will be removed by an outlet in the filter holder. Preferably, the culture medium is supplied to the absorbent support for the membrane filter through this same outlet. To facilitate this, the device can be inverted at this stage. Preferably, the culture medium is initially contained in a sealed container, in which it can be maintained under sterile conditions and the culture medium container is connected to the outlet of the filter holder before breaking the seal to release the medium from the filter holder. cultivation towards the absorbent support. The present invention includes an apparatus for use in the filtration and culture of microorganisms, comprising a filter holder defining a flow path for the fluid to be filtered, a filter element in said flow path comprising a filter of membrane supported on an absorbent support, and elements for compressing the absorbent support against the membrane filter.

Preferably, the filter holder can comprise a first and a second telescopic adjustment section, wherein the first section has a floor containing an outlet for the filtered fluid and a peripheral wall, therefore, the membrane filter and the Absorbent support are received in said first section on the floor, the membrane filter extending beyond said floor, and the second section of the filter holder is received in the first section and compresses the membrane filter and the absorbent support against said floor . The exit on the floor of the first section of the filter holder may have a bridge with a porous support surface underlying the absorbent support of the membrane filter. Suitably, the floor outlet of the first section of the filter holder is adapted to be connected to a syringe by a normal syringe coupler. The apparatus further includes a test kit comprising an apparatus as described above together with a container of sterile culture medium to be introduced into the absorbent support. Preferably, the culture medium container is adapted to be coupled with the outlet of the culture medium introduced therethrough to the absorbent support. Preferably, elements are provided at the outlet of the first section of the filter holder to cooperate with the seal of the culture medium vessel, to break the seal and release the culture medium after a connection between the vessel has been established. of culture medium and the filter holder. The present invention will be further described and illustrated with reference to the accompanying drawings, in which: Figure 1 is a schematic longitudinal section through the apparatus in accordance with the present invention; Figure 2 is a view similar to that of Figure 1 of the apparatus in use during the filtration of a sample; Figure 3 is a similar view of the apparatus during the introduction of the culture medium onto the absorbent support thereof; Figure 4 shows a second embodiment according to the present invention, in a perspective view, whose body and lid portions are cut along its diameter. Figure 5 is a similar view but with both the lid and the body portion cut through its diameter.

As shown in Figure 1, a typical apparatus in accordance with the present invention comprises a filter holder 10 having a first section 12 and a second section 14 coupled thereto. Both have a preferably circular cross section with respect to the plane of the drawing. The first section 12 has the shape of a tray having at its base a centrally positioned outlet 16 and having a protruding peripheral wall 22, which is received in the second cylindrical section 14, is sealed thereto by means of a gasket 18. A cover 20 for the second section 14 is provided. Within the filter holder is positioned a first layer of support material, such as a plastic sheet 24. Alternatively, the neck of the outlet 16 may have a bridge. support material located at outlet 16, such as a porous plastic plug or a piece of radial arms. On the support canvas 24 there is a reticulated absorbent foam pad 26, which acts as a support for the membrane filter, as will be described later. Above the reticulated foam 26 there is a layer of filter paper 28 and above it there is a membrane filter 30. The assembly of the cross-linked foam 26, filter paper wick 28 and membrane filter 30, is comprised of the lower end of the filter. the second section 14 of the filter holder 10. The cross-linked foam pad, for example, may be 1 cm thick without compression and at the edge of the assembly may be compressed to about 3 mm. The compression is distributed by the center of the reticulated foam pad mainly by the membrane filter, but also by the filter paper layer 28, so that in the center the compression would typically be a little less than that of the edges; for example, about 5 mm. The amount of compression applied will generally not be critical. As long as there is some compression, then if there is expansion of the membrane filter during use, the foam pad 26 can expand to accommodate and maintain contact between the filter paper 28 and the membrane filter 30. To be more complete, the Figure 1 also illustrates the presence of a container 32 in the form of a bulb of liquid culture medium 34, attached to outlet 16 by a suitable connector which is shown schematically with the number 36. However, during use the bulb 32 normally will not be attached to the filter holder 10 at this stage. In the place of a flexible bulb containing the culture medium, a sealed vial or closed syringe could be used appropriately. However, preferably, any form of container that is used will be sealed by a seal 38, which can be broken after the container has been connected to outlet 16 in an airtight manner. When the container 32 is a flexible bulb, the seal can be adapted to simply explode by applying external pressure to the bulb. Alternatively, the connection between the outlet 16 and the connector 36 can be designed in such a way that when the connection is completed, some mechanical means pierce the seal 38. For this purpose, for example, a bayonet-type lock can be provided between the container 32 and outlet 16, such that after they have been engaged, the container 32 can be rotated to a position in which it is pushed towards the filter holder 10, causing the seal 38 to be pierced by a bishop or other perforator element located in the connector. The use of the apparatus of Figure 1 is illustrated in Figures 2 and 3. In a first phase, a liquid sample 40 is introduced to the filter holder 10 and the lid 20 is placed to prevent air contamination. Using a syringe 42, the liquid sample is forced through the filter assembly 26, 28, 30, depositing the microorganisms from the liquid sample on the upper surface of the membrane filter 30. After, the apparatus is inverted in the manner shown in Figure 3 and the container 32 of the culture medium is put in position and the seal 38 is punctured to spill the culture medium 34 on the absorbent foam 26, from where it is delivered, by means of a wicking action by the filter paper layer 28, towards the membrane filter 30. Then, the apparatus can be incubated at a suitable temperature to produce the growth of the microorganisms. The described apparatus is simple in construction and can be used in the field. For example, a sample of water taken from a river, spring, storage tank or other source may be placed directly in the filter holder 10 and filtered at the time of sampling. The culture medium can be applied immediately, thus initiating the culture process. It may take several hours for the sample to be taken to a lab in which additional work is done, but the transportation time is not wasted. Instead, it is used to grow microorganisms. The alternative modality shown (omitting some of its characteristics for the sake of clarity) in Figures 4 and 5, is similar in functionality, although different in form. It comprises a filter holder 100 having a lid portion 114 and a body portion 112. The body portion 112 is an integral plastic mold having a circular base 50 from which a generally circular wall 52 emerges that defines a wall. A generally circular projection 54 extends radially outwardly from the upper portion of the well and from there extends a cylindrical wall 56. A radially inward portion of the upper surface of the projection 54 forms a projection 58 for receiving the edge of the well. a filter assembly (not shown) and the walls 60 projecting from the base 50 provide support for the more central portions of the filter assembly, but leave a central channel 61 between their ends 63. The upper surfaces of the walls 60 and the flange 58 of projection 54, define a floor on which the filter assembly is received. Bayonet adjustment lugs 62 project radially outwardly, at intervals from the edge of the projection 54. The lid portion 114 is an integral plastic molded part comprising an upper circular plate 64 from which a cylindrical wall 66 comes down. , ending in a circular projection 68 extending radially, which has an additional cylindrical wall 70. Within the cylindrical wall 66 there is a cylindrical wall of smaller diameter 72, separated therefrom by an annular void space 74 adapted to receive a wall 56 of body portion 112. Lugs are provided to cooperate with bayonet lugs 62 within the interior of wall 70. A complete opening 76 pierces both walls 66 and 72. A shield wall 78 is provided on the wall side. below the projection 68, with an approximately opposite opening 76. A circular cross-section bore 80 pierces the wall 52 of the body portion 112 from below and in parallel to the s encourage 54 and a similar bore 82 is provided directly in the opposite position. During the use of the apparatus, a filter assembly comprising the canvas 24, the cross-linked foam 26, the filter paper 28 and the membrane filter 30 described above in relation to FIGS. 1 to 3, is placed in the flange 58 within the wall 56 and then a cylindrical bottle containing culture medium is placed in the channel 61. When the position lid 114 is adjusted, the bottom of the wall 72 presses on the edge of the filter assembly in the manner described in relation to Figures 1 to 3.

The bayonet lugs 62 secure the lid portion to the body portion but allow some torsional movement in relation to one another, whereby the opening 76 can be aligned and misaligned with a similar opening 77 in the wall 56, and placing the shield wall 78 in the locking and unlocking position relative to the bore 82. Finally, a chisel element (not shown) is inserted into the bore 80 to engage with the vial of culture medium. The chisel may have a cylindrical body portion and a pointed end that engages the bottle. The pointed end can be of any desired shape. The cylindrical portion can be adjusted by pressure in the hole 80 or it can be screwed with the hole. A sample to be investigated is introduced through the openings 76, 77, for example from a syringe, and is passed through the filter assembly to exit through the bore 82. Then, the cap and body portions are rotated to closing the opening 76 and placing the wall 78 in a locking position to close the bore 82. Subsequently, the chisel is pressed (for example, by screwing it if it has rope) to break the seal of the vial of culture medium. The culture medium impregnates the foam of the filter assembly and begins the culture of any trapped microorganism. The apparatus can be inverted during cultivation, after which the microorganisms can be observed through the lid portion and, if desired, the apparatus can be disassembled to harvest the cultured microorganisms. The possibility of closing inlet and outlet holes 76, 77 and 82 by rotation of the lid provides a convenient way to exclude contamination during the incubation of the crop, while at the same time allowing access to a little air. Likewise, the apparatus is adapted to be prepared in advance and to be packed fully assembled in a sterile manner, to be stored before use. The use of the apparatus can be illustrated by the following examples: EXAMPLE 1 100 ml of water inoculated with E. coli was filtered through the unit described above with reference to Figures 1 to 3. A number of units were prepared in this way . Each unit was inverted and, at various volumes and concentrations of nutrient broth, the foam base of each unit was allowed to soak. The units were incubated at 37 ° C overnight and EXAMPLE 2 100 ml of river water was diluted in dechlorinated tap water and filtered through a number of units as described above. Several amounts and concentrations of lauryl broth (Oxoid) were added to the inverted units, to soak the foam support of each unit. It was incubated overnight at 37 ° C and the yellow colonies were presumptively recorded as coliforms. The membrane filter of a unit was removed and placed in solidified medium as a control. The microorganism accounts were the following: EXAMPLE 3 Volumes of 100 ml of water samples inoculated with E. coli were filtered through units as described above. Each unit was inverted and 2 ml of lauryl sulfate broth was added with a force of 1.3X, containing the BCIG chromogen. The broth was prepared in accordance with Sartory and Howard, but 39 mg of BCIG / 100 ml was used, instead of 20 mg / 100 ml. The control membranes were transferred after filtration to MLGA medium (Sartory et al.). Incubation was carried out for 4 hours at 30 ° C and for 14 hours at 37 ° C, after which the green and yellow colonies were counted: In summary, it can be concluded that there are no statistically significant differences between the number of microorganisms recovered in the agar plate with respect to those recovered in the foam pad system. In this way, it is verified that the foam pad system is comparable to the agar plates even when using more complex growth media, for example the inclusion of chromogens, in the system. Since the invention has been described with reference to the specific embodiment illustrated, numerous variations and modifications thereof can be made without departing from the scope of the present invention. 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 to which it relates. Having described the invention as an antecedent, what is contained in the following is claimed as property.

Claims (9)

1. CLAIMS 1. A method for filtering microorganisms from a sample and culturing them, wherein the method comprises filtering a sample containing microorganisms through a membrane filter placed in a filter holder, with the membrane filter resting on a absorbent support, and then culture medium is supplied to the microorganisms of the membrane filter found in the filter holder, by absorbing the culture medium in the absorbent support, characterized in that the absorbent support is maintained in a compressible-expandable state against the filter of membrane.
2. 2. A method according to claim 1, characterized in that the absorbent support is a cross-linked foam.
3. A method according to any of claims 1 or 2, characterized in that the absorbent support is compressed to a thickness of about 7/8 to 1/10 of its uncompressed thickness.
4. 4. A method according to any of the preceding claims, characterized in that the culture medium is initially contained in a sealed container, which is connected to the filter holder in communication with the absorbent support before breaking the seal to release the medium of culture on the absorbent support.
5. 5. An apparatus for use in the filtration and culture of microorganisms, characterized in that it comprises a filter holder defining a flow path for the fluid to be filtered, a filter element in the flow path comprising a membrane filter supported on an absorbent support, and elements that compress the absorbent support against the membrane filter.
6. 6. An apparatus according to claim 5, characterized in that the fastener of 'filter comprises first and second sections of telescopic adjustment, wherein the first section has a floor that communicates with an outlet for the filtered fluid and a peripheral wall, the membrane filter and its absorbent support being received on the floor of the the first section, wherein the membrane filter is removed from the floor, and a portion of the second section of the filter holder is received in the first section and compresses the membrane filter and the absorbent support against said floor.
7. 7. A culture equipment comprising an apparatus according to any of claims 5 or 6, together with a container of sterile liquid culture medium for soaking the absorbent support.
8. 8. A culture equipment according to claim 7, characterized in that the apparatus is as claimed in claim 6 and the culture medium container is adapted to match the outlet of the first section of the filter holder, to allow introducing the culture medium through said outlet and soaking the absorbent support.
9. 9. A culture equipment according to claim 7, characterized in that the culture medium is contained in a closed container inside the apparatus and elements are provided in the apparatus, operated from outside thereof, to perforate the container and thus release the culture medium