MICROORGANISM DETECTION APPARATUS AND METHOD

Abstract: An apparatus for the rapid one-step detection of microorganisms e.g. Salmonella comprising a clear plastic main chamber (1) filled with a liquid non-selective growth medium (7) which communicates with a clear plastic side arm (5). The side arm (5) is plugged by a filtering layer of agar (8) and filled with semi-solid selective growth medium (9). An indicator disc (10) impregnated with antibody is set in the selective medium (9) such that a quantity of antibody diffuses away to form an antibody zone (11). A sample (12) is incubated in the non-selective medium (7) in order to resuscitate dormant microorganisms which multiply and pass into the minor chamber (5) and migrate through the agar (8) into the selective medium (9). The growth of the organism extends visibly around the disc (10) and into the zone (11) where the antibody inhibits growth so as to leave a relatively clear area which thereby confirms the presence of microorganisms in the sample (12).

Description: MICROORGANISM DETECTION APPARATUS AND METHOD. The present invention relates to microorganism detection, more particularly to specific microorganism detection methods in which microorganisms are first multiplied in a non-selective growth medium and then multiplied in a selective growth medium and are then detected by detecting means. The present invention further relates to apparatus for carrying out such methods. Rapid methods for the detection of specific bacteria in a material which may contain other organisms are known wherein a sample of the material is incubated in a non-selective growth medium in order to rapidly increase the concentration of bacteria to levels which are detectable by suitable means. However, this process is not specific and other organisms are likely to outcompete and thus mask the presence of the bacteria of interest. Alternatively it is known to incubate a sample in a selective growth medium which favours the multiplication of the target bacteria. This method has the disadvantage that selective media can lead to slow rates of bacterial growth and a corresponding delay before detectable concentrations are reached. Additionally low levels of the target organism may be. killed by direct selective enrichment. More rapid specific methods are known which entail a two-step test in which firstly a sample is cultured in a non-selective growth medium to resuscitate dormant bacteria and encourage multiplication, and then secondly this medium is used to inoculate a selective growth medium which favours the multiplication of the bacteria of interest. Such methods can generate detectable concentrations of the specific bacteria in relatively isolated form. Such tests have disadvantage in that they may only achieve a realistic presumptive positive/negative result after 48 hours of culture, are either skilled and laborious operations, or involve high capital expenditure. The present invention provides a rapid one-step test method and apparatus for the detection of specific microorganisms, particularly bacteria, addressing some, and preferably all of these problems. According to a first aspect of the present invention there is provided a method of detecting specific microorganisms in a material comprising incubating all or part of the material in a non-selective growth medium such that microorganisms present multiply, incubating microorganisms from the non-selective medium in a selective growth medium which is selective for the specific microorganisms such that any specific microorganims present multiply and are detected by indicating means which indicates the presence of the specific microorganisms in the selective medium, characterised in that the microorganisms can pass freely between the media. The invention embraces the detection of any specific taxonononic grouping of microorganism, eg. species, serotype or strain, for which selective medium and specific detection methods are comprised in the art, or for which such media and methods may later become available. Most suitably the method is used to detect organisms which are motile, as these migrate more rapidly and more reliably within the non -selective and the selective media, and most preferably is used to detect motile bacteria. Preferably, the selective-growth medium employed in the method is in semi-solid or solid form. This mitigates the need to provide further means for limiting diffusion of constituents between the two media and may also facilitate the mounting of and/or the operation of the detecting means in, or in association with, the selective medium. Preferably the microorganisms pass between the media through a filtering means such as a perforated barrier or specific semi-solid media eg. agar. Any means which allow passage or growth of microorganisms between the media while limiting substantial diffusion or efflux of media constituents or other material may be suitable. The use of filtering means such as agar may also promote the expression of flagellar antigens which will in turn increase microorganism motility and may enhance the sensitivity of the detecting means in those methods where such antigens form the basis of the detection system. Preferably the interface between the non-selective medium and either the filtering means if present or the selective medium is not parallel to the surface of the non-selective medium in which the material is incubated (is a 'non parallel interface'). By using an interface which is eg. substantially perpendicular to, as opposed to one which is parallel to, the surface, any particulate matter from the test material which rises to the surface of the non-selective medium will not be forced into the selective phase. By contrast, in embodiments where the interface is parallel to the surface of the non-selective medium (for instance when the selective medium is held in a carrier which dips or projects into the surface of the non-selective medium) the test may be spoiled because non-specific microorganisms from the test material may be forced into, and multiply within, the selective phase thereby masking the test results. A 'non parallel interface' is particularly desirable when testing fermentable materials in which gas production could cause material to rise to the surface of the non-selective phase. Such an interface also helps prevent efflux of the selective medium into the non-selective phase under the force of gravity or otherwise. Preferably the microorganisms pass between the bulk of the non-selective and selective media through a partially air filled conduit eg. a partially air filled horizontal tube connnecting the media. By bulk of the media is meant the main bodies of the media. The presence of air may function as a further filter between the selective and non-selective phases and thereby assist in preventing entry of particulate matter from the non-selective phase into the selective phase or efflux of the selective phase into the the non-selective phase. The presence of air in the arm may also serve as an attractant to certain specific microorganisms to be detected. The detecting means preferably comprises inhibitory material specific to the microorganism to be detected but most suitably this material is immunologically derived, such as antibodies raised against specific bacterial antigens. The presence of antibodies in a defined area of semi-solid or solid selective medium would inhibit growth of the microorganism matrix or lawn in that area, thus indicating the presence of the target microorganism in the sample. The use of more than one antibody area at different points in the selective medium could increase the certainty of any presumption reached or could be used to detect more than one type of microorganism. In a second aspect of the invention, there is provided an apparatus for carrying out the methods of the present invention, said apparatus comprising a non-selective growth medium in connection with a selective growth medium such that the microorganisms can pass freely between them. Preferably the media are held in separate volumes which are separated by filtering means comprising a perforated physical barrier (eg. a perforated plastic plate or other divider) such as to limit diffusion between them. In a further embodiment, the filtering means may comprise a specific semi solid media eg. agar. Preferably the interface between the non-selective medium and the filtering means if present or the selective medium is a 'non parallel interface' and preferably it is substantially perpendicular to the surface of the non-selective medium. Preferably the selective medium is held in a side arm which is connected to the side of a main chamber containing the selective medium. Most preferably the side arm is substantially of L-form with the selective medium being present ostensibly in the vertical portion, and being laterally spaced from the main chamber by the horizontal portion. Preferably the filtering means is positioned such as to form a barrier between the horizontal portion of the side arm and the vertical portion. Preferably the apparatus is such that non-selective medium partly fills the horizontal portion of the side arm. Most preferably air is also present within the horizontal portion. Preferably detecting means for indicating the presence of the microorganisms is provided in the selective medium but such may involve separate apparatus. An apparatus in which there are several separate side arms, each containing a different selective medium and indicating means combination, and all connected to a single chamber containing non -selective medium, is also disclosed. This allows identification of several different types of specific microorganism from one sample in a single operation. Alternatively, each side arm could contain the same selective medium, but have a different type of indicating means. This would allow tests for the detection of a single type of microorganism to run in parallel thus increasing the certainty of the result. In a third aspect of the invention there is provided a container for use in the methods described above whereby the container contains selective growth medium and is in the form of a side arm adapted to be connected to the side of a chamber comprising a volume such that the selective growth medium and the volume are in communication such as to allow microorganisms to pass freely between them. Preferably the side arm is of L-form with the horizontal portion of the 'L' being connectable to the side of the chamber. Preferably the L-form side arm further comprises filtering means such as agar and/or a plastic divider within, or at one extremity of, the horizontal portion of the side arm. Preferably the filtering means is arranged such that in use, when the side arm is connected to the chamber and when the volume comprised within the main chamber is filled with non-selective medium to a level above the horizontal portion of the container, the horizontal portion can be all or partly filled with non-selective medium. Most preferably the filtering means is so arranged such that when the main chamber is so filled, air may also be present within the horizontal portion. In a further embodiment, the invention provides a container for use in the methods described above whereby the container contains selective growth medium and comprises filtering means such as agar and/or a plastic divider and is adapted to be connected to a chamber comprising a volume such that the selective growth medium and the volume are in communication such as to allow microorganisms to pass freely between them. Preferably the container further includes indicating means for indicating the presence of the microorganisms in the selective medium. A number of such containers may be connected to a main chamber containing non-selective growth medium. This may allow the identification of several different types of specific microorganism, or increase the certainty of the detection of a single type of specific microorganism, from a single sample in a single operation. In a fourth aspect the invention provides a kit for use in any of the above methods comprising one or more growth medium containers as described above and further comprising one or more of: a chamber adapted to be connected to the medium container, non-selective medium, filtering means or detecting means. Thus the invention makes available methods and apparatus for the detection of specific microorganisms which address many, and in preferred forms all, of the problems of the prior art. The use of the one-step test method provides a mechanism for delivering an accurate positive/negative result within 24 hours of the start of culture. The relative simplicity of carrying out the method will enable small laboratories employing staff who are not experienced bacteriologists to obtain rapid results with the minimum of equipment. The use of a side arm, 'non parallel interface' or partially air-filled conduit reduces the problems of entry of particulate matter from the non-selective phase into the selective phase or efflux of the selective phase into the the non-selective phase. The methods are particularly suited to the detection of clinical and carrier infection in animals and man, and contamination of foodstuffs (animal and human, including fermentable foodstuffs), or environmental samples with, for example. Salmonella. The facility for obtaining a Salmonella test result within 2k hours opens up the possibility of bonding of livestock, foodstuffs, premises or equipment pending approval, withdrawal or further testing in cases of test failure. The method and apparatus of the present invention will now be described by way of illustration only by reference to the following examples and figures. Other embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these. FIGURES Fig ' l (a) and (b) show longitudinal sections of the apparatus and medium container described in Example 1 Fig 2 shows a longitudinal section of the apparatus described in Example 2 Fig 3 shows a perspective view of the apparatus described in Example 3 Fig 4 shows a perspective view of the main chamber of the apparatus described in Example 4 Fig 5 shows a perspective view of a minor chamber of the apparatus described in Example 4 EXAMPLES Example 1. An apparatus for the detection of Salmonella in a sample was provided as shown in Fig 1 (a) comprising a clear plastic chamber (1) having a short threaded arm (2) and a cap (3) incorporating a breathing hole (4). A clear plastic container (5) in the form of an approximately 'L' shaped side arm is screwed into the arm (2). The vertical and horizontal portions of the side arm are separated by a plastic partition (16) having a 1 mm diameter hole (6) in it. The vertical portion of the side arm (5) is plugged at the base by a filtering layer of agar (8) and filled with semi-solid selective growth medium (9). An indicator disc (10) impregnated with anti-Salmonella flagellar antibody is set in the selective medium (9) such that a quantity of antibody diffuses away from the disc (10) into the surrounding selective medium (9) to form an antibody zone (11). The side arm is sealed with an air-tight cap (15) • The main chamber is filled with a liquid non-selective growth medium (7) to a level which is above the arm (2) such that non-selective medium partly fills the horizontal portion of the side arm, leaving an air bubble (27). The horizontal portion of the side arm thus forms a partially air filled conduit between the non-selective and selective media. The interface provided by the the perforated partition (16) between the non-selective medium (7) and agar (8) is vertical i.e. perpendicular to the surface of the non-selective medium (7). In use, the the non-selective medium (7) is inoculated with a sample (12) taken from a source suspected to be contaminated with Salmonella. The sample (12) is incubated at a suitable temperature in the non -selective medium in order to resuscitate dormant bacteria which multiply and pass into the vertical portion of the side arm (5) through the hole (6) in the partition (16). The bacteria migrate through the filtering layer of agar (8) into the selective medium (9) which favours Salmonella multiplication so that growth of the organism extends visibly towards and around the disc (10) . The antibody in the zone (11) inhibits the growth of Salmonella so as to leave a relatively clear area. Thus the presence of this visually clearer area is presumptive positive identification of the presence of Salmonella contamination in the sample. If required, Salmonella colonies can be isolated from the apparatus by streaking a sample of the furthest point of bacterial growth extension from the side arm (5) onto selective plating agar. Following this full biochemical or serological confirmation and identification can be carried out in using conventional methods. One operation of the apparatus in Example 1 can be further understood from the following experiment. The main chamber (1) was filled with 225 ml of buffered peptone water plus beef heart infusion, the latter enhancing the growth of Salmnnel1a relative to other competing organisms so as to give a clearer result. The side arm (5) was plugged at the base by a filtering layer of Cragie agar which also served to promote the expression of flagellar antigens and enhance Salmonella motility. The minor chamber was filled with semi-solid Rappaports medium plus Novobiocin. The Novobiocin reduces the growth of Proteus spp. thus selecting against hyper-motile organisms which could out-compete Salmonella. An indicator disc impregnated with poly 'H' Salmonella antiserum was set in the Rappaports such that poly 'H' , which inactivates flagellar motility, could diffuse away from the disc to form an antibody zone. 25g of animal feed contaminated with Salmonella was placed in the buffered peptone water. The apparatus was then incubated at room temperature for 24 hours. After this period a well defined clear zone within the bacterial lawn was visible around the poly 'H' disc. The apparatus was further validated with using 219 simulated Salmonella contaminated samples and 819 naturally contaminated samples. For 855 animal feed and other environmental samples the detection rate after 24 hours was 39 % * as compared with 3^ % after 96 hours using a standard Rappaports test. Similarly using 183 poultry post-mortem samples the detection rate after 24 hours was 7^ % , as compared with 71 % after 72 hours using a standard Sellenite test. All positive samples were confirmed by further analysis. Fig 1(b) shows a selective growth medium container for use in the methods of the present invention. The container (5) is in the form of an approximately 'L' shaped side arm adapted to be connected to the side of a non-selective growth medium chamber. The vertical and horizontal portions of the side arm are separated by a plastic wall (16) having a 1 mm diameter hole (6) in it. The vertical portion of the side arm (5) is plugged at the base by a filtering layer of agar (8) and filled with semi-solid selective growth medium (9). The container is sealed at the top with a cap (15). Example 2. An apparatus for the detection of Salmonella in a sample is provided as shown in Fig 2 comprising a clear plastic tube (13) having a cap at each end (14,15) and a partition (16) defining two volumes within the tube (17,18) and incorporating a 1 mm diameter hole (19). In order to prepare the apparatus for use the second volume (18) is partly filled with agar (8) and then filled with semi-solid selective growth medium (9) in which an antibody impregnated disc (10) is set. The second volume (18) is then sealed with a cap (15). The tube is inverted and the first volume (17) pertly filled with liquid non-selective medium (7) and closed with a cap (16). In use, the the non-selective medium (7) is inoculated with a sample taken from a source suspected to be contaminated with Salmonella and the sample is incubated at a suitable temperature in the non-selective medium in order to resuscitate dormant bacteria which multiply and pass from the first volume (17) into the second volume (18) through the hole (19). The bacteria migrate through the filtering layer of agar (8) into the selective medium (9). The presence of Salmonella in the sample can then be deduced as in Example 1. Example 3. An apparatus corresponding to that of Example 1 but capable of detecting four different types of bacteria in a sample is provided as shown in Figs 3a and 3b comprising a clear plastic main chamber (1) having four identical threaded arms (2a,2b,2c,2d) and a cap (3) incorporating a breathing hole (4). Four clear plastic side arms (5a,5bι5c,5d) are screwed onto each of the arms (2a-d) . Each side arm is divided into horizontal and vertical portions by partitions (I6a,l6b,l6c,l6d) incorporating 1 mm diameter holes (6a,6b,6c,6d) . Each side arm (5a-d) is prepared as in Example 1 but contains a different semi-solid selective growth medium, which is selective for a different type of target bacteria, and different indicator discs each impregnated with antibodies raised against one of the different types of target bacteria. In use, the main chamber is filled with a liquid non-selective growth medium (7) to a level which covers the arms (2a-2d) and is inoculated with a sample (12) taken from a source suspected to be contaminated with one or more of the target bacteria. The sample (12) is incubated in the non-selective medium at a temperature chosen to resuscitate dormant bacteria which multiply and pass into the vertical portions of the side arms (5a-d) through the holes (6a-6d). As with Example 1, the bacteria migrate through the filtering layers of agar into the different selective media wherein the target bacteria multiply preferentially such that bacterial growth enriched with the target organism extends visibly towards and around the corresponding indicator disc. The antibody in the zone inhibits the growth of the target bacteria so as to leave a relatively clear area. Thus the presence of this visually clearer area is presumptive positive identification of the presence of the target bacteria in the sample. Example 4. A further apparatus for the detection of more than one type of bacteria in a sample was provided as shown in Figs 4 and 5 comprising a clear plastic main chamber (20) having a screw cap (21) incorporating a breathing hole (22) . The upper surface of the chamber (20) incorporates four openings (23a,23b,23c,23d) each of which is adapted to receive a clear plastic container (24) which forms a sealable fit with the opening. The container (24) incorporates a plastic supporting flange (25) for engaging the upper surface of the main chamber (20) and can communicate with the main chamber (20) via a 1 mm diameter hole (26) . The container is plugged at the base by a filtering layer of agar (8) and filled with a semi-solid selective growth medium (9) which is selective for one type of target bacteria. An indicator disc (10) impregnated with antibodies raised against the target bacteria is set in the selective medium (9) such that a quantity of antibody diffuses away from the disc (10) to form an antibody zone (11). In use, a container (24) is inserted into one or more of the openings (23a-d) , the remaining openings (if any) being sealed with suitable means. The main chamber is then filled with a liquid non-selective growth medium (7) to a level above the base of the container or containers (24). The non-selective medium (7) is then inoculated and incubated as with Example 3 *

Claims: 1. A method of detecting specific microorganisms in a material comprising incubating all or part of the material in non-selective growth medium such that microorganisms present multiply, incubating microorganisms from the non-selective growth medium in a growth medium which is selective for the specific microorganisms, and using a detecting means to indicate the presence of the specific microorganisms in the selective medium characterised in that the microorganims can pass freely between the non-selective medium and the selective medium. 2. A method as claimed in claim 1 further characterised in that the microorganisms pass between the media through filtering means 3. A method as claimed in claim 2 wherein the filtering means comprise a perforated barrier. 4. A method as claimed in claim 2 wherein the filtering means comprise agar. 5. A method as claimed in any previous claim further characterised in that the interface between the non-selective medium and either the filtering means if present or the selective medium is not substantially parallel to the surface of the non-selective medium in which the material is incubated. 6. A method as claimed in claim 5 wherein the interface is substantially perpendicular to the surface of the non-selective medium in which the material is incubated. 7. A method as claimed in any previous claim further characterised in that the microorganisms pass between the bulk of the media through a partially air filled conduit 8. An apparatus for the detection of specific microorganisms characterised in that the apparatus comprises a non-selective growth medium in communication with a selective growth medium such that the microorganisms can pass freely between the two. 9. An apparatus as claimed in claim 8 further characterised in that the selective medium is held in a first volume which is in communication with a second volume containing the non-selective growth medium phase. 10. An apparatus as claimed in claim 8 or claim 9 further characterised in that there is filtering means between the non-selective medium and the selective medium. 11. An apparatus as claimed in claim 10 wherein the filtering means comprise a perforated barrier. 12. An apparatus as claimed in claim 10 wherein the filtering means comprise agar. 13- An apparatus as claimed in any one of claims 8 to 12 further characterised in that the interface between the non-selective medium and either the filtering means if present or the selective medium is not substantially parallel to the surface of the non-selective medium. 14. An apparatus as claimed in claim 13 wherein the interface is substantially perpendicular to the surface of the non-selective medium 1 . An apparatus as claimed in one any of claims 9 to 14 wherein the selective growth medium is present in a side arm which communicates with a main chamber containing non-selective growth medium 16. An apparatus as claimed in claim 15 wherein the side arm is substantially of L-form. 17. An apparatus as claimed in claim 16 wherein the selective medium is present ostensibly in the vertical portion of the side arm and is laterally spaced from the main chamber by the horizontal portion of the side arm. 18. An apparatus as claimed in claim 17 wherein the non-selective medium partly fills the horizontal portion of the side arm. 19• An apparatus as claimed in claim 18 wherein air partly fills the horizontal portion of the side arm. 20. An apparatus as claimed in any one of claims 8 to 19 wherein there is more than one selective growth medium in communication with the non-selective medium. 21. An apparatus as claimed in any one of claims 8 to 20 further characterised in that the apparatus includes detecting means. 22. A container for use in a method for detecting specific microorganisms wherein the container contains selective growth medium and is characterised in that the container is in the form of a side arm adapted to be connected to the side of a chamber comprising a volume such that the selective growth medium and the volume are in communication such as to allow microorganisms to pass freely between them. 23- A container as claimed in claim 22 wherein the side arm is of L-form with the horizontal portion of the 'L' being connectable to the side of the chamber. 24. A container as claimed in claim 23 wherein the side arm further comprises filtering means in or at one extremity of the horizontal portion of the side arm. 2 . A container as claimed in claim 24 wherein the filtering means is arranged such that in use, when the side arm is connected to the chamber and when the volume comprised within the main chamber is filled with non-selective medium to a level above the horizontal portion of the container , the horizontal portion can be all or partly filled with non-selective medium. 26. A container for use in a method for detecting specific microorganisms wherein the container contains selective growth medium and is characterised in that the container comprises filtering means and is adapted to be connected to a chamber comprising a volume such that the selective growth medium and the volume are in communication such as to allow microorganisms to pass freely between them. 27. A container as claimed in any one of claims 24 to 26 wherein the filtering means comprise a perforated barrier. 28. A container as claimed in any one of claims 24 to 26 wherein the filtering means comprise agar. 29. A container as claimed in any one of claims 22 to 28 wherein the container is further characterised in that it comprises detecting means for indicating the presence of the microorganisms in the selective medium. 30. A kit for use in a method for detecting specific microorganisms comprising one or more containers as claimed in any one of claims 22 to 29 and further comprising one or more of a chamber, non-selective medium, filtering means or detecting means. 31. A method, apparatus, container or kit as claimed in any one of claims 1 to 7ι 21, 29 or 30 wherein the detecting means is associated with the selective growth medium. 32. A method, apparatus, container or kit as claimed in claim 19 wherein the detecting means comprises inhibitory material specific for the microorganisms. 33- A method, apparatus, container or kit as claimed in claim 3 wherein inhibitory material is immunological material. 34. A method, apparatus, container or kit as claimed in any one of claims 1 to 33 wherein the selective medium is in semi-solid or solid form. 35> A method, apparatus, container or kit as claimed in any one of claims 1 to 34 wherein the microorganisms are motile. 36. A method, apparatus, container or kit as claimed in any one of claims 1 to 35 wherein the microorganisms are bacteria. 37• A method, apparatus, container or kit as claimed in claim 3 wherein the bacteria are Salmonella. 38. An apparatus or container for the detection of a specific microorganism substantially as described hereinbefore with reference to Figures 1 to 5.

Inventors: DAVIES ROBERT HAROLD, DAVIES, ROBERT, HAROLD
Applicants: DAVIES ROBERT HAROLD, MINI AGRICULTURE & FISHERIES, DAVIES, ROBERT, HAROLD, THE MINISTER OF AGRICULTURE FISHERIES AND FOOD IN 
Application year: 1995
Main classification: C12Q1
IPC codes: C12M1, C12Q1