The invention relates to a device for collecting micro-organisms from a liquid in a filter, a method for collecting micro-organisms from a liquid and a method for identifying and quantifying micro-organisms.

Such a device is generally known, such as for instance at different certified or non- certified agencies which are employed in the Netherlands, on the basis of the Drinking Water Decree, to determine the quantity of micro-organisms, and legionella bacteria in particular, in a determined quantity of liquid, in particular water. Companies and organizations legally obliged to prevent legionella must have their water supply system tested periodically by an external service provider. This service provider takes on-site samples of the water which are subsequently propagated in the laboratory using a bacterial culture and then analysed by means of counting. In this way it takes a minimum of a week before the result of such a test is known. Many litres of water must moreover be transported, this entailing high energy and transport costs.

At present there is one series of products on the market which enables people to test water samples they have taken themselves for the presence of the legionella bacteria. These are Hydrosense® tests (www.hydrosense.biz) of Albagaia Limited. Performing these tests takes a great deal of time. Filtering takes 20-30 minutes and the subsequent analysis takes about 25 minutes per sample. In addition, the analysis is based on an immunochromatographic assay with which companies do not comply with the Drinking Water Decree. Furthermore, this test is not quantitative.

A test is also described in the German patent DE 10 2007 028 207 B3 for quantifying legionella bacteria with a high sensitivity using DNA analysis, made possible by amplification with the polymerase chain reaction (PCR). A considerable volume of 20 litres, preferably 50 litres, of water is however necessary to perform this test.

Described in the French patent application FR 2857606 is the filtering of roughly 1 litre of water containing legionella bacteria by upward pressure, wherein these bacteria are collected on a filter. A drawback hereof is that a great pressure appears to be necessary to press all liquid through the filter and that the liquid with bacteria comes into contact with the holder, whereby contamination cannot be precluded in the case of reuse.

The object of the invention is to design a device for efficient collection of microorganisms such as the legionella bacteria from water, wherein the collected micro-organisms take up the smallest possible volume in order to minimize transport costs to the testing location. This device has to be user-friendly and environmentally-friendly and it must be possible to use it many times. It must further be possible to analyse in reliable manner the micro-organisms collected in this way.

The present invention has for its object to provide a device of the type stated in the preamble, comprising

- a bottle (1) with balloon holder (3) and, at least during use, balloon (4);

- a filter holder suitable for placing at least during use on the balloon holder (3) and comprising a filter (8), the pore size of which is such that the micro-organisms are stopped;

- a cover (19) fitting onto the bottle for clamping the filter holder on the balloon (4) during operation and comprising an outlet for the liquid (21), a connection point (20) for gas supply and a pressure-relief valve (14); and

- optionally a number of O-rings.

The gas supply to the device is preferably provided by a gas bottle such as a gas cartridge. The device according to the invention more preferably comprises a filter holder having on the underside a lower filter shell part (5) with thickened portions fitting into the recesses of the balloon holder, the filter (8) with filter plate (6), and on the upper side an upper filter shell part (9). The different components of the filter holder can also be attached to each other, this being the case particularly for the filter plate (6) and the upper filter shell part (9). All components of the filter holder are preferably manufactured from a polymer, more preferably from ABS (acrylonitrile butadiene styrene) polymer. With particular preference the balloon holder (3) comprises a ring with recesses (3') and is provided with a first edge (3") lying substantially perpendicularly of the ring and having a dimension such that it fits into the upper side of the bottle and wherein the inner ring of the ring is reinforced with a second edge (3 "') with a dimension such that the lower filter shell part (5) fits therein. The first edge is particularly provided with an O-ring (2).

The balloon holder is preferably a synthetic polymer, more preferably of nylon. Any gas can in principle be used, although C0 ₂ is recommended since use thereof is simple and it is freely available on the market. The volume of the bottle preferably amounts to 1000-2000 ml, more preferably 1500-1800 ml, in particular 1650-1700 ml, more particularly 1680 ml. The volume of the balloon preferably amounts to 400-600 ml, more preferably 450-550 ml, in particular 520 ml. The ratio of the volume of the balloon and the volume of the bottle must however always be such here that sufficient pressure can be developed. This ratio preferably lies between 0.20 and 0.60, more preferably between 0.25 and 0.35, and is in particular 0.30. It will be evident to a person with ordinary skill in the art that the neck of the balloon may not be so narrow that it is squeezed together by the pressure to be developed.

The filter holder particularly has a thickness of 10 mm to 25 mm, preferably of 15 mm to 20 mm. The filter plate (6) preferably has a pore size of 0.1 mm to 5 mm. The filter (8) preferably has a pore size of 1 μ - 3 μ, in particular about 2 μ. The maximum thickness of the filter holder is particularly determined by the opening of the letterbox which can receive a package for mailing. The pore size of the filter must be such that the micro-organisms for analysis are stopped.

Preferably connected to the filter of the device according to the invention are an insert part (17) with punch (18) and press-through pin suitable for causing the gas to flow via the cover (19) out of a gas bottle such as a gas cartridge. The material from which the press- through pin, the punch and the insert part are made is not generally of critical importance. Suitable materials are alloys; hardened steel for instance for the press-through pin and the punch and brass for instance for the insert part.

Preferably arranged on the filter (8) in a determined quantity is a standard microorganism with which the application of the device according to the invention can be controlled in determining a quantity of micro-organisms in a liquid.

The present invention also provides a method for collecting micro-organisms from a liquid while applying the device according to the invention as described above, wherein

- the liquid is poured into the balloon clamped in the balloon holder of this device;

- the filter holder and the cover are screwed onto the balloon holder of the bottle;

- gas is supplied and a hose is arranged on the cover, wherein the liquid with micro- organisms is pressed through the filter plate by the pressure developed in the bottle; and

- the filter holder with filter plate with the collected micro-organisms is removed and the micro-organisms isolated.

The gas is supplied particularly using a gas cartridge. The micro-organisms legionella bacteria are preferably collected in this way. This separate filter holder provides the great advantage that it can be removed without the filter plate or filter having to be touched. The pressure developed amounts in this method to preferably 2-3.5 bar, more preferably 2.5-3 bar. It is further possible in an embodiment of the invention, instead of arranging a standard micro-organism on the filter as described above, to add to the liquid a standard micro- organism with which the application of the device according to the invention can be controlled in determining a quantity of micro-organisms in a liquid.

Another aspect of the invention is formed by a method for identifying and quantifying micro-organisms by applying a device according to the invention, wherein nucleic acid such as DNA from the collected micro-organisms is preferably amplified while applying nucleic acid amplification techniques, such as for instance the polymerase chain reaction, and analysed using for instance sequencing. The invention further provides an alternative method for identifying and quantifying micro-organisms, wherein DNA is collected and amplified in the same manner, but then analysed using hybridization with specific oligonucleotides.

However, the use of other methods for identifying and quantifying micro-organisms collected in a device according to the invention, such as for instance culture, propagation and counting of the micro-organisms, also forms part of the invention.

In a preferred embodiment the device according to the invention comprises a hose (22) which is suctioned centrally into the balloon. It is hereby generally easier to empty the whole balloon. In addition or furthermore, the gas supply in the device according to the invention is preferably controllable, for instance by applying a gas pressure controller at the connection point (20) for gas supply and/or by applying a valve (23) when a gas bottle such as a gas cartridge is used.

The device according to the invention is preferably portable and provides the option of automatically filtering a quantity of liquid at a location under all conditions with the purpose of collecting the micro-organisms present in the liquid in a filter, following which they can be identified and/or quantified. Envisaged in the first instance here is the detection and quantifying of legionella bacteria for the purpose of legionella prevention. Samples of the water are generally taken on site by a service provider and subsequently analysed using a bacterial culture in the laboratory. The device according to the invention makes it possible for companies to draw off and filter water themselves. The filter, which is situated in a closed filter holder, is generally then sent by post to the laboratory, after which the company is informed in the not too distant future of the result. In addition, the device provides a financially favourable solution for all other companies and consumers wishing to monitor their draw-off points for safety purposes and legionella prevention.

The device according to the invention is unique in that it enables companies and consumers, at a reasonable price, to monitor the water in their water supply for the presence of the legionella bacteria. Heretofore an external company has usually been brought in to take water samples which then sends these directly to a laboratory for analysis. The method according to the invention ensures that companies and consumers can themselves take the water samples in safe manner, whereby no costs need be incurred for an external intermediary plus transport. The water is after all immediately processed in the device according to the invention in uniform manner, and the filter plate with filter and collected micro-organisms is sent directly to the laboratory. The filter holder is sent unopened and preferably by post to the laboratory. If the culture method is applied, the bacteria must remain alive, wherein the filter with bacteria must be transported in cooled state. However, since the analysis which is recommended works on the basis of DNA testing following amplification thereof, only the DNA of a bacterium is in that case necessary and the bacterium itself need not be alive, nor need it be transported in cooled state. Applying the device according to the invention hereby facilitates legionella prevention for companies, and results in a considerable cost reduction. The result is also known more quickly and is better reproducible due to direct filtering following draw-off of water.

The use of the device further prevents many errors in monitoring. This is because there can be no more in the balloon than the fixed quantity of water necessary for the analysis. In addition, the use of an internal monitoring enables later evaluation of the filtration, transport and analysis in the laboratory, this significantly increasing the quality of the result. All this contributes toward a standardization of the whole legionella monitoring process. Because water draw-off, monitoring and analysis are standardized, the margin of error in the whole process will decrease and the quality of the whole monitoring system will thus be increased.

The device can be reused frequently, although a new balloon and filter holder with a filter therein will have to be placed for each sample. Gas cartridges, ready-to-use filter holders and balloons must always be replaced as non-durable items and preferably in the form of disposables. In the case of reuse as also described in the French patent application FR

2857606 contamination can never be precluded. This in contrast to the device according to the invention, wherein contamination can be 100% precluded due to the use of disposables and collection of the water in a disposable balloon.

As already stated above, the detection of bacteria preferably takes place on the basis of DNA testing. This method is quicker and more sensitive than the culture method generally used and, since it is based on the detection of DNA, it does not matter whether the bacteria survive transport or not. Another detection method, such as the culture method followed by counting, can of course also be applied and this will generally take place in a laboratory. Whichever detection method is used, the result will answer the question of whether the legionella bacteria are present in the filtered water and in what quantity.

The present invention is further elucidated on the basis of the following drawings of two embodiments of the invention for collecting micro-organisms from a fluid in a filter according to the invention and the balloon holder applied in this device.

Figure 1 shows an oblique front view of the device according to the invention comprising a bottle (1) with balloon holder (3) and an O-ring (2) therebetween, a balloon (4); a filter holder comprising a filter (8) with filter plate (6), on the underside a lower filter shell part (5), an O-ring (7) and on the upper side an upper filter shell part (9); a cover (19) comprising a pressure-relief valve (14) with venting spring (11) and venting closure (12) and venting O-ring (13), an O-ring (10) between upper filter shell part (9) and cover (19), a part for opening the gas cartridge comprising a punch (18), insert part (17) and a press-through pin (16) with O-ring (15), and outlet for the liquid (21) and a connection point (20) for a gas cartridge.

Figure 2 shows balloon holder (3) comprising a ring with recesses (3') and provided with a first edge (3") lying substantially perpendicularly of ring (3') and wherein the inner ring of the ring is reinforced with a second edge (3'").

Figure 3 is an oblique front view of the device according to the invention as shown schematically in Figure 1 but with the addition of hose (22) and a gas cartridge provided with a valve (23).

The figures are predominantly schematic and not drawn to scale. Corresponding parts are designated in the figures with the same reference numerals.

Figure 1 shows in detail an oblique front view of the device according to the invention. For the purpose of collecting micro-organisms, in particular legionella bacteria, from water it is applied as follows. The device consists of a bottle (1) in which, in a balloon holder (3), a balloon (4), preferably of latex, can be placed. The bottle is preferably of plastic, but can also be of any other suitable material such as tin, aluminium or glass. The balloon is preferably of latex, but can also be of any other suitable material, such as polyester. The water sample is received in the balloon, and added to the water as control is for instance an internal standard with which the quality of the filtration, transport and laboratory analysis can be demonstrated and which therefore significantly increases the reliability of the result. Subsequently placed on the opening of the balloon is a filter holder with a filter (8) therein, for instance of polyester, with a pore size such that micro-organisms, in particular bacteria such as for instance legionella bacteria, are stopped, and a filter plate (6). A cover (19) is then screwed onto the bottle (1) so that the filter holder clamps onto the balloon. Situated in the cover are: centrally an outlet (21) for the liquid, generally water, a connection point (20) for a gas bottle such as a gas cartridge, and a valve (14) for preventing overpressure. Once cover (19) has been closed and the gas bottle screwed tight, the gas cartridge is opened by pushing upward punch (18), and thereby press-through pin (16). Pressure is developed between the wall of bottle (1) and balloon (4) by the outflowing gas, whereby the water is pressed through the filter. Pressure- relief valve (14) ensures that the pressure in the bottle does not become too high. When all the water has been filtered, cover (19) is removed and the filter holder with filter (8) therein is sent to the laboratory for analysis.

Figure 2 shows balloon holder (3) for a balloon (4) as shown in Figure 1 , which is an advantageous element in the device according to the invention. The recesses serve to create a fixed system in which nothing can be changed, whereby the draw-off of water is standardized. When balloon holder (3) is arranged on bottle (1), first edge (3") then drops into balloon (3). The lower filter shell part (5) shown in Figure 1 fits into the second edge (3"').

Figure 3 shows the device according to Figure 1 , but now with an additional hose (22) for the purpose of allowing more chance of success in suctioning in the solutions with microorganisms for analysis, emptying the whole balloon and allowing the micro-organisms to be collected wholly on the filter. In addition or furthermore, the gas supply is preferably also controllable, for instance by applying a valve (23) during use of a gas cartridge.

Although the invention has been elucidated above with reference to an embodiment and application, it will be apparent that the invention is by no means limited thereto. On the contrary, many other variations are possible within the scope of the invention for the person with ordinary skill in the art.