WO2013119104A1

WO2013119104A1 - Microbial growth chamber

Info

Publication number: WO2013119104A1
Application number: PCT/NL2012/050073
Authority: WO
Inventors: Martin Hessing; Colin John Ingham
Original assignee: The Microdish Company B.V.
Priority date: 2012-02-10
Filing date: 2012-02-10
Publication date: 2013-08-15
Also published as: WO2013119122A1

Abstract

Described is a device (1), comprising a first section of porous material (20) and a second section of porous material (30) and a first chamber (40), defining a first chamber space (41) within the first chamber (40) and at least one outer space (421, 422) outside the first chamber (40), the first section of porous material (20) having an outer surface (201) being in fluid contact with outer space (421), and an inner surface (202) being in fluid contact with the first chamber space (41), allowing fluid communication between the first chamber space (41) and the outer space (42), the second section of porous material (30) having at least an outer surface (301) being in contact with outer space (422), the second section of porous material (30) being free of fluid contact with the first chamber space (41), as well as a method for assessment of the effect of medium on microbial growth using the said device.

Description

Microbial growth chamber

The present invention relates to a device, comprising a first section of porous material and a second section of porous material and a first chamber, defining a first chamber space within the first chamber and at least one outer space outside the first chamber, the first section of porous material having an outer surface being in fluid contact with outer space, and an inner surface being in fluid contact with the first chamber space, allowing fluid communication between the first chamber space and the outer space, the second section of porous material having at least an outer surface being in contact with outer space, and to a method for assessment of the effect of medium on microbial growth using the said device. Herein, the term 'microbes' is deemed to encompass any microorganism, such as bacteria, viruses and bacteriophages, yeast, fungi, archaea, protists, algae, phyto and zooplankton, planarian etc. Furthermore, the term microbes is also deemed to include genetically modified and otherwise constructed self-replicating entities designed to the basic properties of microbes from the environment. The term 'microbial' is to be understood accordingly.

Such devices are known in the art, e.g. from US7, 01 1 ,957, disclosing a growth chamber, comprising an O-ring, whereto on both sides a porous membrane is attached, therewith forming an incubation chamber in the O-ring. Such chambers are used to allow microbial growth in the natural environment of the microbes. Thereto, a membrane is attached to one side of the O-ring, and the space within the ring is filled with a microbial sample, which sample may also contain added agar. Once filled, the second membrane is attached to the other side of the O-ring and the thus closed chamber is then incubated in the natural environment of the microbes in the sample. Both membranes are identical and have a pore size of 0.03um, therewith being impermeable for microbes, but permeable for at least non-polymeric compounds. Such a chamber is used to grow microbes under natural conditions, which is necessary for microbes that cannot be grown under laboratory conditions. Nutrients, present in the said natural environment can enter the chamber and the microbes present therein can grow. After a certain incubation time, the chamber is taken from the natural habitat and analysed in the laboratory. Similar devices were described by Gavrish et al. , (J. Micr. Meth. 72 (2008) 257-262), where one of the membranes had a pore size of 0.2-0.6 urn, i.e. permeable for both nutrients and microbes, whereas the other membrane having a pore size of 0.03 urn was impermeable for microbes. Such devices were used as microbial traps. To this end, the devices were laid with the large porosity membrane side downward in contact with soil. Micro-organisms could enter the chamber from the soil, and could be further analysed after a certain incubation time at natural conditions.

The devices of the art have however several disadvantages. Importantly, in case a microbe is to be entrapped in the chamber, such as described in US7, 01 1 ,957, the microbial sample has to be taken from the natural environment, and the growth chamber has to be loaded with the sample away from the said natural environments in order to get the sample in the chamber and to seal the said chamber. This may cause stress or death to at least some of the microbes in the sample, so that the sample does not reflect the situation as it was in the natural environment. Further, it is not possible to use such a chamber for microbes, that cannot be sampled, e.g. from difficultly accessible habitats, such as the deep sea. Also, the known devices are not suitable to assess the influence of the natural environment on the entrapped sample, or vice versa, as a proper control is missing, or must be done in a separate chamber, which impairs the reliability of the assessment.

The present invention intends to overcome one or more of the above problems, and is thereto characterized in that the second section of porous material is free of fluid contact with the first chamber space. In contrast with the devices known in the art, the device of the invention comprises a first section of porous material allowing fluid contact between the chamber and the environment outside the device (herein also Outer space'), but a second section of porous material is present, being in contact with the environment outside the device, but free of fluid contact with the space inside the chamber. This allows a convenient assessment of the effect of the contents of the chamber to a particular natural environment, by comparing the microbial colonisation of both first and second sections of porous material of the device, said colonisation taking place on the outer surfaces of the first and second sections. The chamber can be loaded with a particular medium, optionally comprising microbes, or can be a complete sample, taken from a natural habitat. The device can subsequently be incubated in an environment, such as a natural environment, or a controlled environment such as a growth medium of known composition, optionally comprising microbes.

The pore sizes of the first and second section of porous material can be chosen such that (non-polymeric) biomolecules and other compounds are capable to move from the environment outside the chamber into the chamber and vice versa, while microbes cannot pass the said sections. Optionally, the selectivity of the porous membrane may be altered by the chemical nature of the surface as well as pore size. For example, alterations in surface charge or hydrophobicity or the attachment of specific molecules (e.g. antibodies) within the pores of the porous material may be used to alter what molecules or microorganisms can pass through. The sample entrapped in the chamber can e.g. contain microbes, either grown in the laboratory, or derived from a sample taken from a natural habitat. With the device, the effect of the presence of such microbes on the environment can conveniently be assessed by incubation of the device in the environment. As the second section is not in fluid contact with the contents of the chamber, the second section serves as a control where microbes, that will be colonised by microbes present in the environment, without subjected to the effect of the contents of the chamber. On the first section however, microbial colonisation is influenced by the contents of the chamber, as fluid contact between the chamber and the environment is possible over the first section of porous material. The device according to the invention for the first time makes it possible to assess the effect of the contents of a particular medium, or the presence of a particular microbe or combination of microbes to the microbial activity in the environment.

The second section of porous material can be integral with the first section of porous material, the second section comprising an additional impermeable layer between the porous material and the chamber. Also, the first and second sections of porous material can be separate, and can be arranged as the devices of the prior art, wherein the fluid contact between the chamber and the second section porous material is made impossible e.g. by arrangement of an impermeable layer or wall between the chamber and the second section of porous material.

The skilled person is aware of suitable materials for the first and second sections of porous material. Membranes of the envisaged porosity are available, e.g. from Osmonics Inc. , USA, GE Osmonics Labstore, USA, Millipore, USA. Additionally, suitable membranes may be custom fabricated or commercially manufactured from a variety of materials such as glass, silicon nitride, fibrous materials including cellulose nanofibres, plastics and other polymers, ceramics (including porous metal oxides), metal foams and membranes derived from photoresists via photolithography.

In a preferred embodiment, the first chamber of the device according to the invention comprises a first chamber opening, allowing fluid to enter the chamber. The presence of an opening allows easy loading of the chamber with a sample, without the need to load the sample during assembly of the device. Further, this embodiment enables loading of a sample in situ, i.e. at the location of the envisaged environment, in particular a natural environment. There is no need to take an environmental sample and to take it away from the environment for loading the chamber of the device.

Preferably, the chamber opening is sealable, so that after loading the chamber is closed, allowing incubation to take place without material flow though the chamber opening, but over the sections of porous material. Once the opening is sealed after loading of the sample, the device can also be removed from the environment where the sample was taken, and be moved to a second environment, to assess the effect of the sample of the first environment, entrapped in the chamber, to the second environment.

In an attractive embodiment, the first chamber, at the chamber opening, comprises a polymer, that swells and/or otherwise becomes a more significant barrier upon contact with the fluid entering the chamber, the swelling resulting in sealing of the chamber. In this embodiment, the chamber can be loaded with a sample by e.g. submersing the device in a first liquid or humid environment, allow the first environment to flow into the chamber, where after the chamber opening is sealed by swelling of the polymer e.g. as an effect of contact of the polymer with the environment, or as a result of passing of time. Examples of suitable polymers or materials include gums, gels and natural or synthetic sponges which grow in volume upon hydration whilst being sufficiently impermeable to seal the first chamber. Preferably, the polymer swells upon contact with water, as the samples to be entrapped will usually comprise aqueous media.

In an attractive embodiment, the first chamber comprises at least one impermeable wall, having an inner surface facing towards the chamber space and an outer surface, the second section of porous material being attached to the said outer surface of the impermeable wall. In this arrangement, the chamber is separated from the second section of porous material by a wall, therewith making it impossible for any medium or compound to move from the chamber over the second section of porous material to the outer surface thereof to the outer environment.

In another attractive embodiment, the first chamber of the device comprises a second chamber opening, allowing fluid to flow though the chamber. In this embodiment, the device can be designed as a flow cell, wherein the effect of the medium flowing through the chamber on any second medium can be assessed by contacting the outside of the flow chamber with the said second medium. The first medium flowing through the flow cell may comprise a compound attracting microbial growth. Accordingly, the first and second sections of porous material will be susceptible to microbial colonisation from microbes from the second medium, and the effect of the first medium can be conveniently assessed by comparing the colonisation by microbes from the second medium of the first section with the colonisation of the second section. To this end, the device preferably comprises flow enhancing means, such as e.g. venturi elements or other methods of converting fluid flow present into the natural environment into directed flow or active fluid transport including pumps and turbines.

In another embodiment, the device according to the invention further comprises a second chamber having a second chamber space, the second chamber space being in fluid contact with the outer surface of the first section of porous material, allowing fluid communication between the first chamber and the second chamber space, the second chamber comprising a third porous material the third section of porous material having an outer surface being in fluid contact with outer space, and an inner surface being in fluid contact with the second chamber space, allowing fluid communication between the second chamber space and the outer space; and a third chamber having a third chamber space, the third chamber space being in fluid contact with the outer surface of the second section of porous material, the third chamber comprising a fourth porous material the fourth section of porous material having an outer surface being in fluid contact with outer space, and an inner surface being in fluid contact with the third chamber space, allowing fluid communication between the third chamber space and the outer space.

In this arrangement the first chamber is now flanked by two additional chambers, one of which, here the second chamber, is in fluid contact with the first chamber through the first section of porous material, whereas fluid contact between the third and first chambers is avoided. Both second and third chambers comprise a section of porous material allowing fluid contact with the respective chamber and the outer environment.

With this device it is possible to load a first medium or sample in the first chamber, and allow the second and third chamber to be loaded with a second sample or medium. Said medium can e.g. comprise plankton. The device can then be incubated in an envisaged environment (outer space), to check the effect of the first medium on the plankton in the first and second chamber, to be assessed by microbial colonisation of the third and fourth sections of porous material. The third section allows fluid contact between the environment and the second chamber, which second chamber is in fluid contact with the first chamber, whereas the fourth section allows fluid contact between the environment and the third chamber, but there is no fluid contact between the third chamber and the first chamber. In case of plankton being entrapped in the second and third chamber, the plankton in the second chamber is affected by the first medium in the first chamber, whereas the plankton in the third chamber is not. This difference can be used to deduce which microorganisms (present in the second chamber but absent or at least less numerous in the third chamber) are influenced by metabolites or other compounds (produced directly or indirectly) by microorganisms in the first chamber. In the above-described multiple chamber device, the first, second and third chambers preferably each comprise a first chamber opening, allowing fluid to enter the respective chamber. Again, the openings in the chambers allow convenient loading of the chambers

One or more of said first chamber openings are preferably sealable in order to avoid exchange between the environment and the content of the chamber through the said chamber openings.

In an attractive embodiment of the above-described multiple chamber device, more than one of the first chamber openings are differentially sealable. This allows sealing of the chambers at different time points and at different location of the device, i.e. loading of the first chamber can take place when the device is held in a first environment, whereas the second and third chambers are sealed at a later stage, e.g. in another environment. Differential loading can be achieved by having a slower rate of sealing the second and third chambers after immersion of the device than the first. Alternatively, differences in the two environments (e.g. salinity, temperature) can be used to seal the first chamber in the first environment and the subsequent chambers in a subsequent environment. This could be achieved, for example, using polymers (such as hydrogels) with different sensitivity to salinity in terms of swelling.

In order to be able to make a sound assessment of the microbial growth, the porous materials of the first and second sections of porous material in the device according to the invention are the same.

As discussed above, the porous materials of at least the first and second sections of porous material are impermeable for microbes. Preferably, the porous materials of at least the first section, but preferably of both the first and second section of porous material (and if present of the third and fourth section as well) is/are at least permeable for non-polymeric molecules. Polymeric macromolecules such as DNA, glycoproteins, proteins and may be too large to pass the porous sections, whereas smaller biomolecules and other chemical compounds, such as nutrients can freely pass the porous materials. The choice of porosity of the porous materials, as well as the charge and/or hydrophobicity and/or presence of binding agents such as antibodies determines what molecules can still pass the porous materials and what molecules cannot.

The pore size of the porous materials of at least the first and second sections (and if present of the third and fourth section as well) of porous material is from 0.025um to 0.03 urn. This pore size makes the material impermeable for microbes, but renders it permeable for nutrients and smaller macromolecules such as proteins. Examples of such suitable materials include polycarbonate, 0.22 micron diameter pore size, filters which exclude all except the smallest microorganisms but permit many macromolecules but not necessarily polymers to pass. Other examples are 2 to 5 micron diameter polycarbonate or nylon membranes, which allow the passage of most bacteria and archaea but exclude most protozoa and many other eukaryotic microorganisms.

The invention further relates to a method for assessment of the effect of medium on microbial growth using the device of the invention, comprising the steps of:

a) allowing a first medium to enter the first chamber space

b) sealing the first chamber,

c) incubating the sealed chamber in a second medium,

d) allowing microbes from the second medium to settle and grow on the outer surfaces of the first and second sections of porous material,

e) analyse growth of microbes on the first and second sections of porous material, and assess the difference in growth on the first and second sections of porous material. This method enables a reliable assessment of microbial growth in e.g. a natural environment, but in fact in any envisaged medium, as a function of another medium or contents therein, entrapped in the chamber of the device.

The analysis of microorganisms can be via sequencing representative nucleic acids (DNA or RNA) such as the genes encoding ribosomal RNA, other specific genes or intergenic regions or other non-coding sequences. Random sequencing or total genome sequencing (genomics) or the sequencing may also be used to assess microbial diversity. Analysis of other macromolecules such as proteins (proteomics), lipids, cell wall components, carbohydrates, metabolites (metabolomics). Further, microorganisms may be identified by cellular characteristics (phenotyping) including growth and the catalysis of biochemical reactions.

In a preferred embodiment, the composition of the first medium is known, so that the effect of a known composition can be assessed. The method encompasses assessment of the effect of one or more one or more factors or a mixture thereof, present in a first medium, on growth of microbes present in a second medium, wherein the first medium is allowed to enter the first chamber space of the device of the invention, and the chamber after sealing is incubated in the second medium. Both the first and second medium can be different natural environments as discussed above.

The factors are preferably chosen from chemical compounds, microbes or a mixture of two or more thereof. In a particular embodiment of the method of the invention, the factor(s) is/are hindered from equilibration over the first section of porous material with the second medium during the incubation step. This can be effected by limiting the incubation time of the device in the second medium, not allowing an equilibrium to settle. As soon as equilibrium is arrived, the effect of the respective compound is minimised, and colonisation by microbes will not anymore be effected by the said compound.

The invention also relates to an assembly comprising a plurality of devices as described above. Such an assembly can be designed by arranging multiple chambers next to one another, sharing wall elements. Such assembly can conveniently be used when different assessments have to be made under the same conditions of incubation time and contact with outer environment.

In an attractive embodiment, at least one chamber of an assembly of the invention is free of a second section of porous material. Such a chamber of the assembly does not necessarily contain a second section of porous material. Such a section can be present in another chamber of the assembly, so that assessment of microbial growth on the second section of porous material can be assessed on the said other chamber. The results of this assessment can also be valid for the chamber without the second section of porous material, as both chambers are in close vicinity to one another and incubated in the same outer environment for the same incubation time. This is particularly true if both chambers comprise the same medium and other ingredients such as microbes. Correspondingly, the assembly may contain at least one chamber being free of a first section of porous material. Such a chamber would function as a control chamber, as it cooperates with the second section of porous material not allowing fluid communication between the said chamber and the outer space. In fact, in the assembly of the invention, the function of the first and second sections of porous material are now divided over different chambers. In a very attractive embodiment, the assembly of the invention comprises a fluid passage having a fluid inlet and a fluid outlet, the outer surfaces of the first sections of the porous material of the plurality of chambers being in fluid contact with the fluid passage, allowing fluid communication between the plurality of chambers and the fluid passage. This design is very useful as flow cell device. The multiple chambers are in fluid contact with the passage through the first sections of the porous material. A test fluid, such as a polluted liquid can be passed trough the passage, and the microbial growth can be assessed for each chamber. Each chamber can comprise a different medium or can be of different microbial content or both. For each chamber, the microbial growth can be assessed, either within the chamber, or on the outer surface of the first section of porous material that is in contact with the fluid in the passage. One or more of the chambers can, instead of a first porous section, cooperate with a second section of porous material, not allowing fluid communication between said chamber and the passage. Such chamber would function as a control for those chambers, allowing fluid communication between the passage and the said chambers. In another attractive embodiment, the assembly does not contain a second section of porous material, in case such controls are not necessary to be made. Attractively, the fluid passage comprises a passage chamber. In another embodiment, the inner surfaces of the first sections of the porous material of the plurality of chambers are arranged in a single plane. The said plane can define a portion of the said passage chamber.

Attractively, the assembly according to the invention comprises a sheet of impermeable material comprising perforations of equal size, which sheet is mounted on an inner surface of a sheet of porous material having an inner surface and an outer surface and a porosity allowing fluid passage over the said sheet of porous material, the outer surface of the said sheet of porous material being in contact with the passage, wherein the perforations define the plurality of chambers. Chambers are defined by the perforations of the impermeable sheet and the inner surface of the porous material mounted to the said impermeable sheet. The chambers can be open to one side, allowing easy access for loading a medium in the chambers. One ore more of the chambers can also, instead, or in addition to, the first section of porous material, be limited by a second section of porous material, not allowing fluid communication between the chamber and the passage therethrough. This assembly enables a convenient high throughput assessment of microbial growth or any other effect imposed by the fluid flowing through the passage on the content of the chambers and/or vice versa.

In use, the chambers are loaded with medium of interest, and a fluid of interest is passed through the passage, allowing fluid communication between the passing fluid and the fluid present in the chambers. The fluid in the chambers can vary from one to another, to allow a convenient high throughput assessment. If desired, the passage of the assembly of the invention can also be shut off to allow a static situation within the passage for fluid communication purposes.

The invention also relates to a method for assessment of the effect of medium on microbial growth using the flow chamber device of the invention as described above, comprising the steps of:

a) allowing a first medium to flow through the first chamber space, the outer surfaces of the first and second sections of porous material being in contact with a second medium, b) allowing microbes from the second medium to settle and grow on the outer surfaces of the first and second sections of porous material,

c) analyse growth of microbes on the first and second sections of porous material, and assess the difference in growth on the first and second sections of porous material.

The invention also relates to a method for assessment of the effect of medium on microbial growth using the multi-chamber device of the invention as described above, comprising the steps of:

a) allowing a first medium to enter the first chamber space,

b) sealing the first chamber,

c) allowing a second medium to enter the second and third chamber spaces, d) sealing the second and third chambers,

e) incubating the device in a third medium,

f) allowing microbes from the third medium to settle and grow on the outer surfaces of the third and fourth sections of porous material,

g) analyse growth of microbes on the third and fourth sections of porous

material, and assess the difference in growth on the third and fourth sections of porous material.

The second medium preferably comprises microbes, such as plankton, to check the effect of the medium entrapped in the first chamber on growth of such microbes, and the effect thereof on the effect of microbial growth in a particular environment.

The invention is now further exemplified by the appended figures and examples which are by no means limiting the invention.

Figure 1 shows a single chamber embodiment of a device according to the invention,

Figure 2 shows a flow cell embodiment of a device according to the invention, Figure 3 shows a multi chamber embodiment of a device according to the invention, and Figures 4-6 show different embodiments of an assembly comprising a plurality of chambers used as flow cell.

In figure 1 , a device (1 ) according to the invention is shown having a single chamber 40, defining chamber space 41 . The top of the chamber is limited by a first section of porous material 20, having an outer surface 201 , and an inner surface 202. This first section of porous material 20 allows fluid communication between the chamber space 41 , over the first section of porous material 20, with an outer space 421 . The porous material can be chosen such, that microorganisms, in particular bacteria and other single or cell organisms cannot pass, but nutrients and other compound present in the chamber 40 and or the outer space 421 can pass. The chamber is further limited by a continuous vertical wall 401 and a bottom wall 402, impermeable for both microorganism and nutrients or other compounds. Bottom wall 402 has an inner surface 4021 , facing towards the chamber space 41 , and an outer surface 4022 to which a second section of porous material 30 is attached. Said second section of porous material 30 has an outer surface 301 , being in contact with outer space 422. Outer space 421 and outer space 422 can be the same space. There is no fluid communication possible between the chamber space 41 and the outer space 422 over the second section of porous material 30, as the impermeability of the bottom wall 30 does not allow fluid contact of the outer space with the chamber space or vice versa.

Vertical wall 401 comprises an opening 431 , allowing medium entry from the outer space 441 , 422 into the chamber space 41 . Around opening 44, a swellable polymers is arranged, rendering the chamber sealable upon contact of the device with a medium, such as an aqueous medium.

In a use of device 1 , a first medium is allowed to enter the first chamber space 41 . This can be achieved e.g. by removing the first section of porous material and allowing the medium to enter the chamber, and closing the chamber 40 by placing the first section of porous material on the vertical wall 401 . If opening 431 is present, the medium can enter via the said opening 431 , whereafter the opening is preferably sealed. No displacement of the first section of porous material is needed in that case. The sealed chamber can be relocated and be brought into contact with a second medium in the outer space 421 and 422, allowing microbes from the second medium to settle and grow on the outer surfaces 201 , 301 of the first and second sections of porous material 20, 30 respectively. The growth of microbes on the first and second sections of porous material can be analysed, and the difference in growth on the first and second sections of porous material can be assessed.

In figure 2, a similar device as depicted in figure 1 is shown. Only differing features are indicated. The same features as present in the device of figure 1 are indicated by the same reference numbers. By the presence of a second opening 432 in the vertical wall 401 , opposite to the opening 431 , a flow cell arrangement is obtained. Flow enhancing means 45, such as venturi elements, are present in the chamber 40. In a use of the device of figure 2, a first medium is allowed to flow through the first chamber space 41 , entering the said chamber space through the first opening 431 and leaving the chamber by the second opening 432, or vice versa. The outer surfaces 201 , 301 of the first and second sections 20, 30, respectively, of porous material are in contact with a second medium, constituting outer space 421 , 422. Microbes from the second medium are allowed to settle and grow on the outer surfaces 201 , 301 of the first and second sections 20, 30 of porous material, and the growth of microbes on the first and second sections (20, 30) of porous material is analysed, and the difference in growth on the first and second sections (20, 30) of porous material are assessed.

In figure 3, a multi chamber device 10 is shown, comprising a first central chamber 40, having the same configuration as the device 1 of figure 1 . The same features as present in the device of figure 1 are indicated by the same reference numbers. Above the said chamber 40, a second chamber 50 is arranged defining a second chamber space 51 , and fluid communication between the first chamber space 41 and second chamber space 51 is possible through the first section 20 of porous material. The second chamber has an optionally sealable opening 531 . The second chamber comprises a third section 70 of porous material, having an inner surface 702 facing to second chamber space 51 , and an outer surface 701 , facing outer space 423. The third section of porous material allows fluid exchange between the second chamber space 51 and the outer space 423.

Below chamber 40, a third chamber 60 is arranged defining a third chamber space 61 , and fluid communication between the first chamber space 41 and third chamber space 61 via a second section 30 of porous material is not possible by the presence of the bottom wall 402 of the first chamber. The third chamber has an optionally sealable opening 631 . The third chamber comprises a fourth section 80 of porous material, having an inner surface 802 facing to third chamber space 61 , and an outer surface 801 , facing outer space 424. The fourth section of porous material allows fluid exchange between the third chamber space 61 and the outer space 424.

In a use of the device 10, a first medium is allowed to enter the first chamber space 41 , wherafter the first chamber can be sealed. A second medium is allowed to enter the second and third chamber spaces 51 , 61 , whereafter the second and third chambers 50, 60 are sealed. The device 10 is incubated in a third medium, which can be identical to the second medium. Microbes from the third medium are allowed to settle and grow on the outer surfaces 701 , 801 of the third and fourth sections 70, 80 of porous material, and the growth of microbes on the third and fourth sections 70, 80 of porous material are analysed, and the difference in growth on the third and fourth sections (70, 80) of porous material is assessed.

In figure 4 an assembly according to the invention comprises a housing 7, comprising inlet channel 732 with an inlet 731 and an outlet channel 733 with an outlet 735. Inlet 731 is connected to a feed tube 734 via connector 736, whereas outlet 735 is connected with outlet tube 738 via connector 737. A fluid passage, in particular a passage chamber, is denoted by 721 . Passage chamber 721 is connected to inlet channel 732 and outlet channel 735 and made fluid-tight by the use of rubber washers 739. The passage chamber 721 is in contact a sheet 800 of porous material, such as porous aluminium oxide (20 to 200 nm diameter pores), facing with an outer surface 801 thereof towards the passage chamber 721 . The outer surface 801 is in fluid contact with the passage chamber 721 , and defines the upper boundary thereof. At the opposite surface, i.e. inner surface 802, porous sheet 800 is mounted on a perforated sheet 71 of impermeable material such as glass. With 'impermeable' is meant that no passage of contents of fluid through the said material is possible. Each perforation 710 defines a chamber, also being denoted by reference number 710, the bottom thereof being defined by the inner surface 802 of the sheet 800 of porous material. The inner surface 802 is therefore in fluid contact with the spaces of chambers 710 at the location where the said inner surface 802 defines the respective bottoms of the said chambers 710 with dimensions of e.g. a few mm across. This allows fluid communication between the passage chamber 721 and the chambers 710. So the sheet 800 constitutes a first section of porous material for each chamber as defined herein. Passage chamber 721 may be regarded as an equivalent of the outer space as defined herein and as shown in the previous figures. The perforations 710 are separated from one another by the sheet 71 of impermeable material, shown by position 72. Below this position 72 between the chambers 710, the porous material does not allow fluid communication between the chambers 710 and the passage chamber 721 . This portion of the porous material can be regarded as a second section of porous material as defined herein, as this portion is free of fluid contact with the chambers. However, the assembly can be free of a second section. On the other hand, some of the perforations can be made in the impermeable sheet 800 such, that only an intrusion is formed in the said sheet, and not a hole. Such intrusion would have a bottom section of the said impermeable material, and therewith, no contact with the porous material mounted thereon is possible. Again, the porous material at the location of such bottom section is free of fluid contact with such a chamber and can therefore be regarded as second section of porous material as defined herein. It is also possible to arrange a chamber such, that this chamber is in fluid contact with the inner surface 802 of the porous material 800, but not allowing fluid communication with the passing chamber 721 , e.g. as is the case for chamber 71 1 . The presence of such chambers 71 1 is however optionally. The chambers 710 of the assembly are closed by a cap element 780, fixed to the housing but removable if desired. The chambers can be protected by an additional impermeable sheet or layer 73, such as glass. The said additional impermeable layer 73 may be transparent or otherwise permit imaging through the material to the chambers 710 beneath.

A source of liquid, such as water from a natural source or a polluted environment, is piped from a distant location through the device from feed tube 734 via inlet channel 732 into the flow chamber 721 and then out via outlet channel 733 and outlet tube 738 to a second distant location.

In use, a fluid is passed from inlet tube 734 via inlet 731 and inlet channel 732 to passage chamber 721 . The said passing fluid is in fluid communication with any fluid or medium present in chambers 710 via the sheet 800 of porous material. During passage through the passage chamber 721 , fluid communication, e.g. interaction or exchange can take place between the passing fluid and the fluid medium in the chambers 710. The pore size can be chosen such, that passage of nutrients and other small molecules are allowed, but that passage of microbes is not possible. Such fluid communication is not possible between the passage chamber 721 and chamber 71 1 . Each of the chambers 710 can be loaded with different medium or having different content, and the influence of the passing fluid on the composition of the media or vice versa can be assessed by allowing microbes to grow on either side of the porous material, i.e. on the inner surface 201 or the outer surface 202, whatever is applicable. The fluid leaves the passage chamber 721 via outlet channel 733 and outlet tube 738.

The device as indicated in Figure 4 can be used to culture and isolate new microorganisms sensitive to a fluid source passing through the device including such factors as nutrients, gases, pH or temperature of this fluid.

In figure 5 an assembly according to the invention comprises is as described for Figure 4 with the following changes. The same reference numbers are used for the same features, but not always indicated.

As second sheet 900 of impermeable material, such as glass, may line the opposite surface of the passage chamber 721 with an upper surface 901 facing the flow chamber and a lower surface 902 facing the housing 7. However, this second impermeable material may also be part of the housing, if the housing is made of material, suitable to be impermeable. The passage chamber 721 is, via sheet 800 of porous material, in fluid communication with an outer environment 1000, for example a location in the sea in which the device is immersed. A portion 803 of sheet 800 of porous material may be masked by an additional sheet of impermeable material 73, so that this portion 83 cannot be in contact with the outer environment 1000. A second source of fluid, such as water from another source is piped from a distant location 1001 through the device from inlet tube 734 via inlet channel 732 into the flow chamber 721 and then out via outlet channel 733 and outlet tube 738 to a second distant location 1002. A distant location means for example from 5 cm to 10 metres from the main body of the device.

Isolation of microorganisms sensitive to two types of environment can be performed using the version of the device of figure 5, as will be exemplified in example 7.

In another version of the device (Figure 6) most aspects are the same as described in figures 4 and 5. The same reference numbers are used for the same features, but not always indicated. However, there is a second sheet 1 100 of porous material, such as porous aluminium oxide with an upper surface1 101 facing the passage chamber 721 , and a second surface 1 102 facing the housing 7. This feature replaces the sheet 900 of impermeable material illustrated in Figure 5, and is to be regarded as the second section of porous material as defined herein, as it is not in contact with an outer environment, whereas sheet 800 is in contact with outer environment via the outer surface 802 thereof, i.e. constituting a first section of porous material as defined herein.

EXAM PLE 1

Isolation of previously uncultivated microorganisms that grow on compounds liberated from asphalt or ones, which are dependent on microorganisms that grow on asphalt.

This experiment describes a method for isolating microorganisms that are dependent (directly or indirectly) on the degradation of asphalt. Such microorganisms are likely to be valuable in industrial bioconversions and in the degradation (removal) unwanted materials.

The chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with the poorly soluble material asphalt as growth substrate. The main body of the chamber is fabricated from stainless steel, with the dimensions of the device being 2 cm across. The porous membranes are clamped into the device using silica seals allowing them to be removed later for microbial analysis. The porous membrane chosen is porous aluminium oxide with a pore size of 0.2 microns (as available from companies General Electric, Synkera and SPI Supplies) allowing nutrients including proteins to pass the membrane but retaining microorganisms including bacteria. The stainless steel device with silicon rubber and porous aluminium oxide is designed to be highly resistant to corrosion in salt water. The device is lowered into the sea using a fine cable and is protected by an open cage of stainless steel (mesh diameter 2 mm) to protect from fish and other macroscopic sea life. The device is implanted in the sea attached to a marker buoy. The chamber fills with seawater and the chamber opening seals within 2 h. by using a swellable hydrogel ring (e.g. polyvinyl copolymers), attached around the chamber opening. The device is then incubated in situ for from 1 to 60 days. Some of the microorganisms (total) trapped within the growth chamber grow - some directly on the substrate (group A), some growing dependent on the growth of group A (group B), and some independently of group A or the asphalt. However, groups A, B and C are currently indistinguishable from each other as a mixture so will be referred to as group A/B/C. Further, the two outer surfaces of the first and second sections of the porous material will also support growth of microorganisms. The surface of the second section, i.e. the outer surface with no direct connection with the central chamber (301 ) will accumulate microorganisms from the environment (group D) most of which cannot degrade or grow on asphalt. The surface of the first section, i.e. the outer porous surface with connection with the chamber will accumulate not only group D but also microorganisms that are directly or indirectly influenced by asphalt degradation (group E). Sequencing whole genomes, specific genes (such as the rRNA encoding genes), proteomics or other analytical method is used to identify groups A/B/C, D and E. In the subsequent analysis, by subtracting group D from group E (group F) it is possible to increase the chance that microorganisms within group F are identified which are interacting with groups A and B, i.e. those unique to group E. For example, group D (determined by sequencing subcloned fragments of DNA encoding 16S rRNA genes) comprises primarily of SAR bacteria, Ralstonia spp. , Rhodospirillum spp. and Sinorhizobium spp. Group E comprises SAR bacteria, Ralstonia spp. , Rhodospirillum spp. , Chromatobacterium spp and Burkholderia spp. The organisms present in E not D (group F) are Chromatobacterium spp and Burkholderia spp. and these therefore are candidate species for growth on asphalt. The group of bacteria Sinorhizobium spp. are present in D not E and are candidates for microorganisms that have their growth inhibited by asphalt.

EXAM PLE 2

Screening for microorganisms from deep sea water that are stimulated in growth by microorganisms from shallow sea water.

Microorganisms grow at all depths in the sea. Due to motility, transport by larger organisms and currents microorganisms from different depths can recirculate and interact. However, no good method exists for determining such interactions. The chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with microorganisms. To do this, the device is implanted in the sea attached to a marker buoy at a depth of 10 m for 2 hours. The chamber fills with seawater and the chamber opening seals trapping microorganisms (group G) from 10 m depth due to the swelling of the hydrogel (e.g. polyvinyl copolymers) to block the 2 mm diameter aperture within 2h. The device is then lowered to a depth of 50 m and incubated for 1 to 60 days. The two outer surfaces of the first and second section of porous material device will also support growth of microorganisms. The outer surface with no connection with the central chamber, i.e. of the second section of porous material, will accumulate (by growth and attachment) microorganisms from the environment (100 m depth, group H). The outer porous surface with connection with the central compartment, i.e. of the first section of porous material, will accumulate group H but also microorganisms that are directly or indirectly influenced by group G (group I). Sequencing whole genomes, specific genes (such as the rRNA encoding genes), proteomics or other analytical method is used to identify groups G, H and I . In the subsequent analysis, by subtracting group G from group H (group J) it is possible to increase the chance that microorganisms within group J are identified which are from 100 m depth and are likely interacting with the microorganisms from 10 m depth. EXAM PLE 3

Screening for microorganisms sensitive to liquid flow in a natural environment. In this example, it is shown that using a device according to the invention, designed as flow chamber such as shown in figure 2, the effect of fluid flow on microorganisms growing on a surface can be measured, for example in studying fouling. In nature, it is difficult to manipulate or assess the effect of liquid flow and also difficult to separate the physical effects of liquid flow (e.g. shearing) on the provision of nutrients. The device of the invention offers novel solutions to this problem.

The central chamber of the device of the invention has two apertures, such as shown in figure 2. The device is placed in a liquid environment so that liquid flows through the central chamber. By manipulating the dimensions of the central chamber or adding a flow enhancing element a fluid flow is created that is more rapid than that flowing over the outside of the device. As discussed above, one of the surfaces allows fluid communication between the chamber and the outer environment (corresponding to surface 201 shown in Figure 1 ), whereas the other surface is only in contact with the outer environment (corresponding to surface 301 shown in Figure 1 ). Microorganisms growing as a biofilm or microcolonies on the outer surface that is not in fluid contact with the chamber, (group K) will only experience fluid flow and nutrients delivered from the environment (422 in Figure 1 ). Microorganisms growing on the surface allowing fluid communication between the chamber and the outer environment (group L) will experience both the environment and the accelerated flow through the central chamber. By comparing the populations of cells, proteins, nucleic acids or other parameters on the two surfaces (groups K and L) the effect of the flow of liquid through chamber 41 can be assessed.

EXAM PLE 4

Screening for microorganisms, which generate compounds or extracellular enzymes that can be used to prevent fouling of ships. There is a need to isolate microorganisms that reduce the fouling of ships or other surfaces. However, such organisms are likely not to be isolated by adhering to the surfaces they are designed to protect. Furthermore, it is not easy to screen for such microorganisms under the conditions under which fouling occurs. The device of the invention offers a more effective route to this goal.

The central chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with microorganisms into the central chamber (group M) and attached to the hull of the ship with the outer porous surfaces being equally exposed to the outer environment, with the chamber opening of the chamber sealing after 2 h due to the swelling of the hydrogel to block the aperture, while the microorganisms being unable to penetrate the porous material. After a cruise of 60 days the two surfaces (+/- communication with central chamber) are assessed by degree of growth (groups N and O). Devices for which the surface communicating with the chamber show less microbial growth (less fouling or a more limited biofilm) compared to the porous surface not communicating with the chamber contain samples within the central chamber that may be of value in preventing fouling. Identification of the microbial contents of the central chamber (group M) provides leads as to microbes that prevent fouling. Dilution of such mixtures of microorganisms into new chambers and repeating the process for one or more cycles (incubation attached to the ship, identification of the contents of chamber at the end of this process) may be used to purify, isolate and/or identify the desired microorganisms further. This approach can also be used to assess the antimicrobial activity of compounds under such circumstances, by introducing in the chamber the antimicrobial compound to be tested, and to check, after the incubation, the difference in microbial growth on both outer porous surfaces of the device. To this end, the compound to be tested can be provided in a slow release vehicle to avoid premature leakage of the compound from the chamber.

EXAM PLE 5

Isolation of new groups of microorganisms growing in liquid culture, that are dependent on a particular compound or another group of microorganisms for growth. Previous versions of the device have been orientated towards isolating microorganisms growing on surfaces (e.g. as biofilms). It is also desirable to look at microorganism in suspension (such as planktonic growth). The device for this purpose is shown in Figure 3; it comprises a central chamber (40), and two outer chambers (50 and 60) rather than a single chamber of the foregoing examples.

The central chamber (40) is loaded with microorganisms (group P) or with a growth substrate such as a slowly degraded polymer such as cellulose. To load with microorganisms, the device is implanted in the sea attached to a marker buoy at a depth of 10 m for 2 hours. The chamber fills with seawater and the chamber opening seals trapping microorganisms (group P) from 10 m depth due to the swelling of the hydrogel to block the aperture, while the microorganisms being unable to penetrate the porous material. The outer chambers (50 and 60) will also be filled with water and microorganisms from the same environment; however, these will not be sealed in this step as the material intended to seal apertures 531 and 631 takes longer to seal the outer chambers. This can be achieved by choosing another sealing polymer, or by a larger aperture for the outer chambers (e.g. 4 mm diameter) so that the apertures in the outer chamber take 10 hours to seal. The entire device is then lowered to 100 m. During this time microorganisms from 100 m replace those from 10 m in the outer chambers (group Q) but not those from 10 m (central chamber) as the latter is already sealed.

The device is incubated for e.g. 20 days. The microorganisms in the central chamber influence the growth of the microorganisms in chamber 50 (via nutrients, signalling molecules, antibiotics and other molecules that can penetrate the porous material 20 allowing group Q to develop into group R) but not chamber 60 (group Q developing into group S), as between outer chamber 60 and the central chamber, no fluid communication is possible. By comparing groups R and S the influence of group P on group R can be deduced.

EXAM PLE 6

The device as indicated in Figure 4 can be used to culture and isolate new microorganisms sensitive to a fluid source including such factors as nutrients, gases, pH or temperature of this fluid. Reference numerals refer to those as used in figure 4. Microorganisms are inoculated into one or more identical chambers 710 onto surface 802 of porous material 800 and, if present, also in chamber 71 1 . Chamber(s) 710 allow the microorganisms to receive nutrients and other low molecular weight materials through the porous membrane (800) via the flow chamber (721 ). Chamber 71 1 is not connected with flow chamber 721 via a porous surface. Microorganisms growing in chamber(s) 722 are responsive to the fluid from flow chamber (721 ) and comprise group T. Microorganisms isolated from chamber 71 1 can be expected not to respond to the fluid in the flow chamber and comprise group U. Analysis can be via DNA sequencing (individual genes including those coding for rRNA or whole genomes or cloned fragments) or other analysis including proteomics and lipid analysis to give an indication of the microbial diversity within each group of microorganisms. By subtracting group U from group T gives group V, microorganisms that respond or are likely to respond to the water or other fluid from chamber 721 .

EXAM PLE 7

Isolation of microorganisms sensitive to two types of environment can be performed using the version of the device described in Figure 5. Reference numerals refer to those as used in figures 4 and 5. The device is submerged in the sea to a depth of 1 metre so that the immediate outer aqueous environment 1000 contacts the outer surface 802 of sheet 800 of porous material where it is exposed to the environment but not where it is masked by an impermeable surface (surface 803 marked by impermeable material 72). Water from another distant or separate source, for example 10 metres away (10001 ) and piped to this location via an impermeable tubing, flows through the flow chamber (721 ) from source 1001 in a path from 734 via 731 , 732, 721 , 733, 735 then out at 738 to location 1002. Microorganisms growing on surface 802 of the porous material are responsive to nutrients and other small molecules (e.g. antibiotics) from both water sources (group W). In contrast, those microorganisms growing on surface 901 of an impermeable material such as glass bordering the flow chamber 721 only experience water from one source (group X). After 10 days operation of the device in this environment the microorganisms are analysed by proteomic analysis, nucleic acid sequencing or culture. By subtracting sequences or the identity or abundance of the microorganisms comprising group X from group W (to give group Y). Group Y microorganisms are likely to include microorganism dependent on both water sources for growth.

EXAM PLE 8

This example has a similar purpose to the situation described in example 7, however, a device as described in figure 6 is used. Reference numerals refer to those as used in figures 4, 5 and 6. In this device there are two porous materials bordering the flow chamber (sheet 800 with surface 801 and sheet 1 100 with surface 1 10, respectively, therewith bordering the flow chamber 721 . These surfaces can be porous aluminium oxide. The experimental set up is otherwise the same as example 6 except that microorganisms growing on surface 801 (exposed to both environments 1000 and 1001 , group Z) are compared to microorganisms growing on surface 1 101 (exposed only to water source 1001 ; group AA). Microorganisms growing on surface 801 are in fluid contact with both the fluid from source 1001 passing through the passage chamber 721 and with the outer environment 1000, whereas the microorganisms growing on surface 1 101 are only in fluid contact with the fluid from environment 1001 passing through passage chamber 721 . Environment 1000 can e.g. be sea or lake water at the location where the device is submersed, whereas environment 1001 can be any fluid, such as another water source from elsewhere and passed through the passage chamber 721 . Deducting group AA from group Z yields group BB, microorganisms sensitive to, or requiring, both water sources.

Claims

1 . Device (1 ), comprising a first section of porous material (20) and a second section of porous material (30) and a first chamber (40), defining a first chamber space (41 ) within the first chamber (40) and at least one outer space (421 , 422) outside the first chamber (40), the first section of porous material (20) having an outer surface (201 ) being in fluid contact with outer space (421 ), and an inner surface (202) being in fluid contact with the first chamber space (41 ), allowing fluid communication between the first chamber space (41 ) and the outer space (421 ), the second section of porous material (30) having at least an outer surface (301 ) being in contact with outer space (422), the second section of porous material (30) being free of fluid contact with the first chamber space (41 ).

2. Device (1 ) according to claim 1 , wherein the first chamber (40) comprises a first chamber opening (431 ), allowing fluid to enter the chamber (40).

3. Device (1 ) according to claim 2, wherein the chamber opening (43) is sealable.

4. Device (1 ) according to claim 3, wherein the first chamber (40) at the chamber opening (43) comprises a polymer (44), swellable upon contact with the fluid entering the chamber (40), the swelling resulting in sealing of the chamber (40).

5. Device (1 ) according to any of the preceding claims, wherein the swellable polymer swells upon contact with water.

6. Device (1 ) according to any of the preceding claims, wherein the first chamber comprises at least one impermeable wall (402), having an inner surface

(4021) facing towards the chamber space (41 ) and an outer surface (4022), the second section of porous material (30) being attached to the said outer surface

(4022) of the impermeable wall (402).

7. Device (1 ) according to any of the claims 2 - 5, wherein the first chamber (40) comprises a second chamber opening (432), allowing fluid to flow though the chamber (40).

8. Device (1 ) according to claim 6, wherein the device comprises flow enhancing means (45).

9. Device (10) according to claim 5, the device further comprising

- a second chamber (50) having a second chamber space (51 ), the second chamber space (51 ) being in fluid contact with the outer surface (201 ) of the first section of porous material (20), allowing fluid communication between the first chamber (41 ) and the second chamber space (51 ), the second chamber (50) comprising a third porous material (70) the third section of porous material (70) having an outer surface (701 ) being in fluid contact with outer space (423), and an inner surface (702) being in fluid contact with the second chamber space (51 ), allowing fluid communication between the second chamber space (51 ) and the outer space (423),

- a third chamber (80) having a third chamber space (81 ), the third chamber space (51 ) being in fluid contact with the outer surface (301 ) of the second section of porous material (30), the third chamber (60) comprising a fourth porous material (80) the fourth section of porous material (80) having an outer surface (801 ) being in fluid contact with outer space (424), and an inner surface (802) being in fluid contact with the third chamber space (61), allowing fluid communication between the third chamber space (61 ) and the outer space (424).

10. Device (10) according to claim 9, wherein the first, second and third chambers (40, 50, 60) each comprise a first chamber opening (431 , 531 , 631), allowing fluid to enter the respective chamber (40, 50, 60).

1 1 . Device (10) according to claim 10, wherein one or more of the first chamber openings (40, 50, 60) are sealable.

12. Device (10) according to claim 1 1 , wherein more than one of the first chamber openings (40, 50, 60) are differentially sealable.

13. Device according to any of the preceding claims, wherein the porous materials of the first and second sections of porous material are the same.

14. Device according to any of the preceding claims, wherein porous materials of at least the first and second sections of porous material are impermeable for microbes.

15. Device according to any of the preceding claims, wherein the porous materials of at least the first section of porous material is at least permeable for non- polymeric molecules.

16. Device according to any of the preceding claims, wherein the pore size of the porous materials of at least the first and second sections of porous material is from 0.025um to 0.03 urn.

17. Assembly comprising a plurality of devices of claim 1.

18. Assembly according to claim 17, wherein at least one chamber is free of a second section of porous material.

19. Assembly according to claim 17 or 18, comprising a fluid passage(721) having a fluid inlet (731 ) and a fluid outlet (735), the outer surfaces (202) of the first sections (200) of the porous material of the plurality of chambers (710) being in fluid contact with the fluid passage (721 ) , allowing fluid communication between the plurality of chambers (710) and the fluid passage (721 ).

20. Assembly according to claim 19, wherein the fluid passage (721 ) comprises a passage chamber.

21 . Assembly according to claim 20, wherein the inner surfaces (201 ) of the first sections of porous material (200) of the plurality of chambers (71 1 ) are arranged in a single plane.

22. Assembly according to any of the claims 17 - 21 , comprising a sheet of impermeable material (71 ) comprising perforations (710) of equal size, which sheet is mounted on an inner surface (201 ) of a sheet (200) of porous material having an inner surface (201 ) and an outer surface (202) and a porosity allowing fluid passage over the said sheet (200) of porous material, the outer surface (202) of the said sheet of porous material being in contact with the passage (721 ), wherein the perforations define the plurality of chambers (710).

23. Method for assessment of the effect of medium on microbial growth using the device of any of the preceding claims, comprising the steps of:

a) allowing a first medium to enter the first chamber space (41 ), b) sealing the first chamber (40),

c) incubating the sealed chamber (40) in a second medium,

d) allowing microbes from the second medium to settle and grow on the outer surfaces (201 , 301 ) of the first and second sections (20, 30) of porous material, e) analyse growth of microbes on the first and second sections (20, 30) of porous material, and assess the difference in growth on the first and second sections

(20, 30) of porous material.

24. Method according to claim 17, wherein the composition of the first medium is known.

25. Method according to claim 17 or 18 for assessment of the effect of one or more one or more factors or a mixture thereof, present in a first medium, on growth of microbes present in a second medium, wherein the first medium is allowed to enter the first chamber space (41 ), and the chamber after sealing is incubated in the second medium.

26. Method according to claim 19, wherein the factors are chosen from chemical compounds, microbes or a mixture of two or more thereof.

27. Method according to claim 20, wherein the factor(s) is/are hindered from equilibration over the first section (20) of porous material with the second medium during the incubation step.

28. Method for assessment of the effect of medium on microbial growth using the device of claim 7 or any of the claims 8 - 16 dependent thereon, comprising the steps of:

a) allowing a first medium to flow through the first chamber space (41 ), the outer surfaces (201 , 301) of the first and second sections (20, 30) of porous material being in contact with a second medium,

b) allowing microbes from the second medium to settle and grow on the outer surfaces (201 , 301 ) of the first and second sections (20, 30) of porous material, c) analyse growth of microbes on the first and second sections (20, 30) of porous material, and assess the difference in growth on the first and second sections (20, 30) of porous material.

29. Method for assessment of the effect of medium on microbial growth using the device of claim 9 or any of the claims 10 - 16 dependent thereon, comprising the steps

a) allowing a first medium to enter the first chamber space (41), b) sealing the first chamber,

c) allowing a second medium to enter the second and third chamber spaces (51 , 61),

d) sealing the second and third chambers (50, 60)

e) incubating the device in a third medium,

d) allowing microbes from the third medium to settle and grow on the outer surfaces (701 , 801 ) of the third and fourth sections (70, 80) of porous material, e) analyse growth of microbes on the third and fourth sections (70, 80) of porous material, and assess the difference in growth on the third and fourth sections (70, 80) of porous material.

30. Method according to claim 23, wherein the second medium comprises microbes.