TESTING METHOD AND APPARATUS

Abstract

A testing method is described for use in undertaking a test upon a sample (10) for the presence of a specific microbiological material, the method comprising the steps of combining the sample (10) with a viability-preserving medium, optionally with an enrichment medium (12) selected to promote growth and/or reproduction of the specific microbiological material within the sample (10) and incubating the sample (10) and enrichment material (12), undertaking a separation process to separate the live specific microbiological material from the sample (10), and testing the separated material for the presence of the specific microbiological material. An apparatus for use in the method is also described.

Claims

1. A testing method for use in undertaking a test upon a sample for the presence of a specific microbiological material, the method comprising the steps of: combining the sample with a viability-preserving medium selected to keep the microbiological material viable; undertaking a selection process to select the specific microbiological material; and testing the selected material for the presence of the specific microbiological material.

2. The method according to claim 1, wherein the step of combining the sample with a viability-preserving medium comprises combining the sample with an enrichment medium selected to promote one of growth and reproduction of the specific microbiological material within the sample; and incubating the sample and enrichment material.

3. The method according to claim 1, wherein the step of combining the sample with a viability-preserving medium comprises transferring the sample into a phosphate-buffered saline solution.

4. The method according to claim 1, wherein the selection process comprises a separation step in which the selected material is separated from the sample.

5. The method according to claim 4, wherein the separation step is undertaken using a separation material that detects the presence of the specific microbiological material, and attaches thereto.

6. The method according to claim 5, wherein the separation material comprises a suitable antibody material.

7. The method according to claim 5, wherein the separation material is provided upon magnetic beads so that a magnet can be used to separate the specific microbiological material from the remainder of the sample.

8. The method according to claim 4, wherein the separation step comprises at least one of a filtration process and a centrifugation process.

9. The method according to claim 1, wherein the testing step is sensitive to the presence of gram-negative material.

10. The method according to claim 9, wherein the testing step is undertaken using a detection method suitable for detecting the presence of lipopolysaccharide (LPS).

11. The method according to claim 10, wherein the detection method is one of LAL based or TAL based.

12. The method according to claim 1, wherein the testing step is sensitive to the presence of gram-positive material.

13. The method according to claim 12, wherein the detection method detects the presence of at least one of lipoteichoic acid or peptidoglycan.

14. The method according to, wherein the testing step is sensitive to the presence of fungal material.

15. The method according to claim 14, wherein the testing method is a beta-glucan based test.

16. The method according to claim 1, wherein the enrichment medium comprises a modified tryptone soy broth supplemented with at least one of antibiotics and selective media, to positively select and enhance at least one of the growth and the reproduction of the specific microbiological material.

17. The method according to claim 1, wherein the incubation step is undertaken for a period in the range of 15 minutes to 24 hours.

18. (canceled)

19. The method according to claim 1, wherein the incubation step is undertaken at a temperature of 5 to 50° C.

20. The method according to claim 1, wherein a washing step is undertaken before undertaking the testing step.

21. An apparatus adapted for use in the method of claim 1.

Description

[0039] The invention will further be described, by way of example, with reference to the accompanying drawing, FIG. 1, which is a diagram illustrating the steps of a testing method in accordance with an embodiment of the invention.

[0040] Referring to FIG. 1, a testing method in accordance with an embodiment of the invention comprises the steps of acquiring a sample 10, for example of liquid form or liquefied form, of a material to be tested for the presence of a specific microbiological material, in this case E. coli O157. The sample 10 is combined and mixed with an enrichment medium 12 which is selected to enhance the growth and/or reproduction of the specific microbiological material within the sample 10. By way of example, the enrichment medium 12 may take the form of a modified tryptone soy broth which has been supplemented with antibiotics or other selective media so that the growth and/or reproduction of the specific microbiological material is enhanced, but the growth and/or reproduction of other microbiological materials is unaffected or is discouraged. Once the sample 10 and enrichment medium 12 have been combined, the sample 10 and enrichment medium 12 are placed within an incubator for an incubation period of, preferably, 4 hours, the incubator being set to incubate the sample 10 and enrichment medium 12 at a temperature of 37° C. Whilst this specific temperature and duration is suitable for use in testing for the presence of E. coli O157, it will be appreciated that where the test is for the presence of other materials, or where other enrichment media are used, then it may be preferred to incubate for a longer or shorter period of time, and/or for different incubation temperatures to be used. By way of example, incubation may be at a temperature in the range of 20-40° C., and for a duration in the range of 2-8 hours.

[0041] After incubation, it will be appreciated that if the sample initially contained a quantity of the specific microbiological material, the enrichment and incubation steps will have magnified or increased the concentration of the specific microbiological material in the sample, making detection thereof simpler.

[0042] As set out before, in alternative application scenarios, depending on the type of sample, an enrichment and incubation step may not be necessary or beneficial. Without wishing to be bound by theory, it is believed that particularly biofilm material, such as surface colonies of microorganisms, are often already present in relatively high density, and/or are shielded from external chemicals by a polysaccharide matrix. Such microorganisms can be relatively robust, such that it may suffice to merely ensure that the sample collected for subsequent testing remains viable. The sample may be held in a medium that preserves the sample in a viable condition, such as for example phosphate-buffered saline that provides a suitable osmotic environment for a microorganism but is otherwise not designed to promote growth. Any other suitable sample-preserving medium may be used.

[0043] Tests using biofilm material were carried out using test metal discs provided with reference biofilm cultures. Material from such metal discs was harvested using sterile swabs, and transferred into a sterile medium (phosphate-buffered saline), constituting a viability-preserving medium, and vortexed. The microbiological material thus harvested may be present in sufficiently large density (cell count) that no enrichment step is required. Alternatively, an enrichment step in a suitable design growth medium may be carried out to increase the cell count and/or as a further confirmation that the microbiological material in the sample was capable of growing (reproducing).

[0044] After the collection of the material, and/or after incubation, a selection step in the form of a separation step is undertaken in order to select and separate live microbiological material of the specific form from the sample 10. Whilst a range of separation techniques could be used, in this embodiment of the invention the separation is undertaken by adding an antibody material 14 labelled for interaction with the specific microbiological material, the antibody material being attached to magnetic beads, to the sample 10 and enrichment medium 12. The antibody material 14 conveniently takes the form of a suitable immunomagnetic bead material. Once the antibody material 14 has been added, a further short incubation step 15a is undertaken to allow the antibody material 14 to interact or bind with the specific microbiological material contained within sample 10. By way of example, the further short incubation step may be of duration in the range of 2-20 minutes, preferably in the range of 5-15 minutes, and is conveniently in the region of 10 minutes. It will be understood that the either the antibody material 14 is added to the microbiological material or alternatively microbiological material is added to the antibody material.

[0045] After the further short incubation period, in a step 15b a magnet is used to separate the antibody material 14, and the specific microbiological material that has become bound thereto or associated therewith, from the sample 10, and a washing step is undertaken, for example using a small amount of a suitable wash buffer such as a phosphate buffer, and the washed material may be suspended in a further quantity of the wash buffer material.

[0046] When using a magnet to separate antibody material 14, the magnetic separation may be carried out by a magnetic field provided temporarily on a transfer structure of a carrier. In one embodiment, the carrier is provided by a test tube and the temporary magnetic field is provided by a magnetic rod insertable into and removable from the test tube, to temporarily provide a magnetic field at the outer surface of the end of the test tube. The test tube is understood to be of glass or polymer material that is not magnetised by the magnetic rod. Other suitable materials may be used.

[0047] It will be appreciated that the separated material, once washed, consists substantially exclusively of the specific microbiological material (if present in the original sample) and the antibody material bound thereto as part of the separation step. If the specific microbiological material were present in the original sample, then it will be present in relatively high concentration within the washed, separated material and so should be relatively easy to detect. Conversely, if target microbiological material was not present in relevant quantities, then the separation medium will not contain microbiological material. For example, if magnetic beads are used as the separation medium, the magnetic beads will be transferred, using the above-described magnetic transfer, without the antibody-bound microbiological material.

[0048] Once washed, a test step 16 is undertaken on the washed material, to identify whether the washed material contains biological material. By way of example, where the testing method is being used to test for E. coli O157 or other Gram-negative biological material then the test step 16 may involve the use of a test sensitive to the presence of LPS. By way of example, the test may be a LAL or TAL based test. However, where the microbiological material that the testing method is being used to detect is of a Gram-positive form, for example staphylococcus, listeriae or fungal materials, then the test step 16 may be of a different form, for example testing for the presence of lipoteichoic acid or peptidoglycan. The test step 16 is preferably undertaken using a test that produces an output not only indicative of the presence of the material under test, but also allowing enumeration of the level of contamination. As the level of contamination within the sample is enhanced through the use of the enrichment medium, it will be appreciated that some adjustment of the level of contamination indicated by the result of the test step 16 may need to be made to provide an accurate indication of the level of contamination of the sample 10 with the microbiological material under test.

[0049] Due to the selective nature of the separation step, a non-result provides a high level of confidence that no target microbiological material was present.

[0050] Conducting TAL or LAL based tests, for example of the gel clotting or colour change form, to identify whether LPS is present in the separated material, and using this as a marker for the presence of the specific microbiological material within the sample, and the approximate level thereof, may be undertaken in a simple and conveniently manner, such tests being well known and simple to use. Conveniently, the washed, separated material is placed within a series of sterile tubes or the like, in which the tests are undertaken. By suitably diluting the samples or using different test methods, an assessment of the concentration of the specific microbiological material can be made.

[0051] It will be appreciated that the testing method of the invention is advantageous in that testing may be undertaken in a relatively quick manner for the presence of specific microbiological materials. Limited specific equipment is required in order to undertake the tests, and so the testing method can potentially be undertaken in environments remote from laboratories, and testing may be undertaken without requiring individuals undertaking the tests to be skilled in laboratory techniques.

[0052] Whilst a specific embodiment of the invention is described herein with reference to the accompanying drawing, it will be appreciated that a wide range of modifications and alterations may be made thereto without departing from the scope of the invention as defined by the appended claims.

[0053] The invention is thought to be useful in scenarios in which a lengthy testing time in the region of several days is not appropriate or convenient. Certain embodiments of the invention are further thought to be of benefit for microorganisms that tend to be difficult to grow in traditional enrichment media, particularly microorganisms present in biofilms.

[0054] The methods disclosed herein may be used to test for the presence or absence of microbiological material, including but not exclusively biofilms, on medical devices, for human and/or veterinary use, on any surfaces thereof and particularly inside and/or luminal regions of medical devices, such as those of catheters, needles, reservoirs, tubing, connector surfaces, surgical instruments, drills, implants, dental corrective devices and dentures etc., as well as wound management, dressings, wound swabs etc.

[0055] The methods disclosed herein may be used to test for the presence or absence of microbiological material, contamination measurements and/or measurement of contamination levels, including but not exclusively biofilms, on food items, for human and animal consumption, feed, dairy products, eggs, fish, crustaceans, molluscs, plants, meat, cosmetics, as well as processing lines, packaging, bottling, and processing areas and equipment for such articles and combinations thereof. Likewise, the methods may be used for testing of farming and reproduction equipment, aquaculture, ballast water and others.

[0056] Likewise, the methods disclosed herein are thought to be useful in the testing of fuel processing, particularly biofuel processing where contamination with microorganisms can remain otherwise undetected.

[0057] The methods may be used in the form of a series and/or array of tests. For instance, test swabs of surfaces with suspected biofilm growth may be taken at spaced-apart intervals to map out the spread of a biofilm. It will be appreciated that such series of tests, repeatedly taken over a period of time, allow dynamics of biofilm development to be investigated. The method may be used to understand microbiological growth under external influences, to observe the attenuation and/or inhibition effect of anti-microbial agents, barriers, radiation etc. and different combinations and concentrations thereof. The method may therefore be used to study resistance and tolerance levels of microbiological organisms.

[0058] The methods disclosed herein may be combined and used in parallel. For instance, a sample from one source may be processed in accordance with embodiments without exposure to an enrichment medium, and another sample from the same source may be processed in accordance with embodiments comprising exposure to an enrichment medium. It will be understood that the non-enriched sample may be processed sooner, potentially instantaneously, and the enriched sample may be processed a few hours later, after an enrichment phase. Any differences between the results of a pre-enriched sample and an enriched sample may be considered in the analysis of the microbiological material.