WO2017098244A1

WO2017098244A1 - Method of determining the efficacy of a microbial decontamination process

Info

Publication number: WO2017098244A1
Application number: PCT/GB2016/053859
Authority: WO
Inventors: Derek Price
Original assignee: Bactest Limited
Priority date: 2015-12-08
Filing date: 2016-12-08
Publication date: 2017-06-15
Also published as: GB201521629D0

Abstract

A method of and system for determining the efficacy of a microbial decontamination process are discussed. The method provides a determination of efficacy of the decontamination process by monitoring relative changes in the time to detect a threshold event (indicative of microbial growth) in samples taken from the input and output of the decontamination process. If the decontamination process is working, the time taken to detect a threshold event in the output sample will be greater than the time taken to detect a threshold event in the input sample. By comparing the time taken to detect the threshold events when system is calibrated to the input sample (since the composition of the input samples will vary), the level of decontamination of the decontamination process may be estimated, giving the user an estimated measure of the level of logarithmic kills (representing a logarithmic reduction in the number of viable microbes) by the decontamination process. The calibration is performed using the input sample, and one or more dilutions of the input sample.

Description

Method of Determining the Efficacy of a Microbial Decontamination Process

FIELD OF THE INVENTION This invention relates to methods and system for determining the efficacy of a microbial decontamination process.

BACKGROUND TO THE INVENTION Stabilizing large ships to aid steering requires the uptake of large quantities of Ballast Water at one location and subsequent release at another. This is a vehicle by which invasive organisms can move from one ecosystem to another, often thousands of miles apart. Many examples of such invasions by foreign species are now known. To counter this, ballast water purification systems are installed on many ships and regulatory compliance for the level of contamination of foreign organisms at ballast water release will soon be mandatory.

Methods to tests the effectiveness of Ballast Water Management Systems (BWMS's) are required to ensure such systems are operating correctly and able to reduce contamination to levels within the constraints set out by regulatory authorities.

Existing procedures (including EPA-Approved Reference Methods) are not practical on-board ship due to lack of available resource (both scientifically trained personnel and appropriate laboratory equipment). Samples of ballast water that are to be taken, either by on-board crew as part of self-monitoring, or by local port authorities as part of inspections must therefore be transported to an off-site laboratory for assessment.

This process introduces several problems:

Firstly, the microbial content of a sample may change while the sample is in transit meaning that numbers of organisms detected by the laboratory may not reflect the actual numbers present when the sample was taken. This may lead to inaccuracies and confusion as to whether systems really do pass or fail regulatory limitations.

Secondly, the time to transport samples to a laboratory and the time taken to obtain results by existing Approved Reference Methods may mean a delay in excess of 4 days from point of sampling to obtaining a result. This in itself poses two key problems: (i) a vessel may continue to de-ballast for a long time before self-monitoring results are obtained alerting the ship owner that ballast water treatment is ineffective. During this time invasive species may be introduced into new locations, (ii) options for port authorities when inspecting ballast water treatment effectiveness are limited: allow the ship to de-ballast potentially contaminated water whilst awaiting results or hold a ship in port, which becomes a costly exercise for the ship owner.

Other waste treatment management systems also have similar issues. Industrial processes can produce liquids that need to be decontaminated, for example cutting fluids used in milling machines, or process water/machinery in food processing facilities. In many cases, there is a need to monitor rapidly the efficacy of a decontamination process, for example a microbial decontamination process, and often they require samples to be sent away for analysis.

We have therefore identified a need for a more rapid and preferably on-site method of determining the efficacy of a microbial decontamination process. Whilst we will address the needs of assessing a ship's ballast water management system, there is also a need for this type of arrangement in other applications.

SUMMARY OF THE INVENTION

The present invention therefore provides a method of determining the efficacy of a microbial decontamination process, comprising: taking an input sample at an input to a decontamination process, and an output sample at an output of the decontamination process; generating a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, the time to detect a threshold event representing a time to detect a threshold event that is indicative of microbial growth within the sample, determining a time to detect a threshold event in the output sample; comparing the time to detect a threshold event in the input sample and output sample; and determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples. Since the method could be used for a variety of microbial decontamination processes, the variable composition and flora of the samples make exact enumeration of the contents very difficult, even in a lab. However, the method described represents a growth and enumeration method that is largely independent of the flora and matrix composition when combined with a test specific calibration that is created, and is therefore suitable for the described technical applications (Ballast Water Management Systems, industrial processes, machinery in food processing facilities and other such processes). The step of generating a calibration profile response may comprise: generating one or more dilutions of the input sample; determining a time to detect a threshold event in the input sample and the one or more dilutions of the input sample; and defining a relationship between the level of dilution and the time to detect a threshold event for each of the samples and dilutions. Using a number of dilutions in this manner enables a calibration profile of the input sample to be generated, from which an indication of the level of the efficacy of the decontamination process may be made. The one or more dilutions may comprise input samples that are diluted to 50%, 25%, 10% or 1 % of the original concentration of the input sample. Preferably, concentrations of 10% and/or 1 % are used. The input sample is diluted using a sterile and/or filtered solution.

In order to promote microbial growth in the samples for determining the efficacy, the sample may be provided with a nutrient. Each of the samples, whether they be input samples, diluted samples or the output sample, may comprise the same fixed final working volume and the same fixed nutrient quantity.

The step of determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples may comprise: determining a relative level of change in the time to detect the threshold event between the input sample and the output sample; comparing the relative level of change in the time to detect a threshold event to a threshold value; and determining the efficacy of the decontamination process when the relative level of change in time is greater than or equal to the threshold value. The difference between the threshold value and the relative level of change in time to detect the threshold events between the input sample and the output sample may be indicative of a level of logarithmic kills, each level of logarithmic kill representing a logarithmic reduction in the number of viable microbes removed by the microbial decontamination process.

The above methods may be applicable in all manner of methods used to detect microbial growth. For example, the step of generating a calibration profile response of the input sample, and the step of determining a time to detect a threshold event in the output sample may be performed using a respiration method, a turbidimetric method or a colorimetric method. When a respiration method is used, each sample is held in a separate culture vessel comprising a sealable chamber for receiving and culturing a liquid sample such that the presence of microbes is detectable by detecting a change in pressure in a headspace of the chamber. In this scenario, the time to detect a threshold event comprises a time to detect a change in pressure in the headspace of the chamber that is greater than or equal to a pressure change threshold value, the change in pressure being indicative of the number of viable microbes in the sample.

For the respirometer methods, generating a calibration profile response of the input sample, and determining a time to detect a threshold event in the output sample comprises: running a test protocol on the respirometer to incubate the samples according to the test protocol, the test protocol defining a temperature to regulate of the liquid sample in the culture vessel; detecting the change in pressure in the headspace of the chamber; monitoring the detected change in pressure; and signalling the time taken to detect the change in pressure when the change in pressure is greater than a pressure change threshold value. The environmental conditions are thus controlled to promote microbial growth, and the time taken to detect a suitable growth event is recorded.

In any of the above methods, the microbial decontamination process may comprise a ballast water management system for a sea vessel, an industrial process, or a food process. The advantage of the above method is that it may be performed at the location of the decontamination process, thus significantly reducing delays in obtaining results when compared to known methods. Furthermore, the above methods allow for the determination of a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, and the determination of a time to detect a threshold event in the output sample to be performed simultaneously. As such, detection times are significantly reduced.

Once the method has determined the efficacy of the decontamination process, this result may be output to a user. This may be in the form of a simple "yes/no" compliance result (that is, the decontamination process is sufficiently efficient or not), or may be more detailed, and may provide a measure of the efficacy. For example, this may be presented as a number of log kills, that is the number of logarithmic kills (by how many logarithmic reductions in the in the number of viable microbes have the samples changes). This may be presented on a display or presented to the user by any other suitable medium.

The present invention also provides a system for determining the efficacy of a microbial decontamination process, comprising: an input sample vessel for receiving an input sample from an input of a decontamination process, and an output sample vessel for receiving an output sample from an output of the decontamination process; and a processor configured to: generate a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, the time to detect a threshold event representing a time to detect a threshold event that is indicative of microbial growth within the sample, determine a time to detect a threshold event in the output sample; compare the time to detect a threshold event in the input sample and output sample; and determine the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples.

For the step of generating a calibration profile response, the processor is configured to: determine a time to detect a threshold event in the input sample and one or more dilutions of the input sample; and define a relationship between the level of dilution and the time to detect a threshold event for each of the samples and dilutions. The one or more dilutions comprise input samples that are diluted to 50%, 25%, 10% or 1 % concentration of the input sample. Preferably, the samples are diluted to 10% and/or 1% of their original concentration. The input sample may be diluted using a sterile and/or filtered solution.

The sample may be provided with a nutrient in order to promote microbial growth. When the system is arranged as such, each of the samples comprises the same fixed final working volume and the same fixed nutrient quantity in order to ensure the results are consistent across the variety of samples.

For the step of determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples, the processor may be configured to: determine a relative level of change in the time to detect the threshold event between the input sample and the output sample; compare the relative level of change in the time to detect a threshold event to a threshold value; and determine the efficacy of the decontamination process when the relative level of change in time is greater than or equal to the threshold value. The difference between the threshold value and the relative level of change in time to detect the threshold events between the input sample and the output sample may be indicative of a level of logarithmic kills, each level of logarithmic kill representing a logarithmic reduction in the number of viable microbes removed by the microbial decontamination process.

In any of the above systems, a respirometer, a turbidimetric process or a colourmetric process may be used.

When a respirometer is used, each sample is held in a separate culture vessel comprising a sealable chamber for receiving and culturing a liquid sample such that the presence of microbes is detectable by detecting a change in pressure in a headspace of the chamber. The time to detect a threshold event may comprise a time to detect a change in pressure in the headspace of the chamber that is greater than or equal to a pressure change threshold value, the change in pressure being indicative of the number of viable microbes in the sample.

In the system using a respirometer, the respirometer comprises a temperature controller coupled to the processor to control a temperature in the chamber, and wherein when generating a calibration profile response of the input sample, and when determining a time to detect a threshold event in the output sample, the processor is configured to: run a test protocol on the respirometer to incubate the samples according to the test protocol, the test protocol defining a temperature to regulate of the liquid sample in the culture vessel; detect the change in pressure in the headspace of the chamber; monitor the detected change in pressure; and signal the time taken to detect the change in pressure when the change in pressure is greater than a pressure change threshold value.

In any of the above systems, the microbial decontamination process may comprise a ballast water management system for a sea vessel, an industrial process, or a food process. Preferably, the system is located at the location of the decontamination process. As such, the results of the efficacy of the decontamination process may be determined much faster than current known systems and methods.

For the system, the determination of a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, and the determination of a time to detect a threshold event in the output sample may be performed simultaneously. As such, the determination of the efficacy may be known sooner than compared to known systems. Once the method has determined the efficacy of the decontamination process, this result may be output to a user by the process and some suitable indicator. This may be in the form of a simple "yes/no" compliance result (that is, the decontamination process is sufficiently efficient or not), or may be more detailed, and may provide a measure of the efficacy. For example, this may be presented as a number of log kills, that is the number of logarithmic kills (by how many logarithmic reductions in the in the number of viable microbes have the samples changes). This may be presented on a display or presented to the user by any other suitable medium.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will now be further described, by way of example only, with reference to the accompanying Figures in which:

Figure 1 shows a method according to the present invention; Figure 2 shows a calibration method according to the present invention;

Figures 3a and 3b show a culture vessel for use in embodiments of the invention under, respectively, normal atmospheric pressure and reduced pressure;

Figure 4 shows an example of a test unit configured to accept a culture vessel of the type shown in Figure 3;

Figure 5 shows a block diagram of a test unit of the type shown in Figure 4 configured to implement a test method according to an embodiment of the invention;

Figure 6 shows the sampling arrangement for an example Ballast Water Management System; Figure 7 shows calibration profile data;

Figure 8 shows time to detection data for a number of samples;

Figure 9 shows the sampling arrangement for a second example Ballast Water Management System;

Figure 10 shows calibration profile data for the arrangement of figure 9; and

Figure 1 1 shows time to detection data for a number of samples taken from the arrangement of figure 9.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In brief, the invention provides a method in which the efficacy of a decontamination process, for example a microbial decontamination process, without the need to determine accurate numbers of active microbes within a number of samples. Instead, the method of the invention provides a determination of efficacy of the decontamination process by monitoring relative changes in the time to detect a threshold event (indicative of microbial growth) in samples taken from the input and output of the decontamination process. If the decontamination process is working, the time taken to detect a threshold event in the output sample will be greater than the time taken to detect a threshold event in the input sample. By comparing the time taken to detect the threshold events when system is calibrated to the input sample (since the input samples will vary), the level of decontamination of the decontamination process may be estimated, giving the user an estimated measure of the level of logarithmic kills (representing a logarithmic reduction in the number of viable microbes) by the decontamination process. The calibration is performed using the input sample, and one or more dilutions of the input sample. In the method according to the invention, there is no requirement to make an accurate determination of the number of viable microbes within a sample to determine the efficacy of a microbial decontamination process. Instead, the method looks for changes or events that indicate the presence or growth of viable microbes over a period in time. Figure 1 outlines the broad principle of the method according to the invention.

At step 10, an input sample is taken at an input to a decontamination process, and an output sample at an output of the decontamination process. In step 12, a calibration profile response of the input sample is generated. Since the type of microbes are likely to be unknown, the method performs a calibration step in order to calibrate the system to the response of the input sample, so that the system has a baseline or calibration factor from which a determination of the performance of the decontamination process can be made. In the generation of the calibration profile of the input sample, the system is controlling the environmental conditions of the input sample in order to promote growth of any viable microbes within the sample, that is controlling one or more of a temperature or pressure or other environmental factors. A nutrient may also be present in order to promote microbial growth. The condition of the input sample is monitored over a period of time and when the activity of the viable microbes reaches a threshold level (triggering a threshold event condition), the time taken to reach this point is recorded and stored for later use. The calibration steps will be discussed in more detail later.

The same process is used in step 14 to determine a time to detect a threshold event in the output sample. That is, the system controls the environmental conditions of the output sample in order to promote growth of any viable microbes within the sample. The condition of the output sample is monitored over a period of time and when the activity of the viable microbes reaches a threshold level (triggering a threshold event condition), the time taken to reach this point is recorded and stored for later use.

In step 16, a comparison of the time to detect the threshold events in the input and output samples is made. Since the input sample is used to generate the calibration profile, this comparison of the times to detect the threshold events in the two samples becomes a relative comparison of the system's response to the growth of the microbes within the samples. There is no need to make an absolute determination of the number of viable microbes, only the change times to detect events between the two samples.

Thus, in step 18, a determination of the efficacy of the microbial decontamination can be made. If the decontamination process is working, the time taken to detect the threshold event in the output sample should be longer than the time taken to detect the threshold event in the input sample. And the difference between the two samples may give an estimate of how effective the decontamination process is, that is by how much the contaminant has been reduced between the input and output samples. The different values of time taken to trigger the threshold events in the input and output samples may give an indication of a level of logarithmic kills ("log kills"), where each level of logarithmic kill represents a logarithmic reduction in the number of viable microbes removed by the microbial decontamination process. In decontamination processes, it is desirable to have 1 , 2, 3, 4 or 5 log kills.

Figure 2 shows the calibration process in a little more detail. In this process, an input sample is taken in step 20, and then one or more dilutions of the input sample are made in step 22. The input sample may be diluted using a sterile and/or filtered solution (ideally mimicking the sample but without contaminating organisms), and it is diluted to provide samples having, for example, 50%, 25%, 10% or 1 % of its original concentration. Preferably, the method uses the (neat) input sample, and two diluted samples, one at 10% of its original concentration, and a second at 1 % of its original concentration. However, the method is not limited to this number, or selection of diluted input samples. In order to perform the calibration step, the input sample and at least one dilution is required. Once the dilutions have been provided, the time taken to detect a threshold event is determined in step 24. This is the same process as in step 14 of Figure 1 . That is, for each of the samples, the system controls the environmental conditions of the sample in order to promote growth of any viable microbes within the sample, that is controlling one or more of a temperature or pressure or other environmental factors. A nutrient may also be present in order to promote microbial growth. The condition of the input sample is monitored over a period of time and when the activity of the viable microbes reaches a threshold level (triggering a threshold event condition), the time taken to reach this point is recorded and stored for later use.

After the time to detect the threshold events have been determined for each of the samples, a relationship between the dilution level and the time to detect the threshold event may be defined. This relationship serves as a calibration profile or baseline profile for the system based on the surrounding environment, that is, the microbes present in the input sample at the site of the decontamination process. This calibration profile may then be used in the method described with reference to Figure 1 to determine the efficacy of the decontamination process. In order to obtain a determination of the efficacy of the decontamination process and to obtain an estimate of the effectiveness of that decontamination process, it is preferable that the samples are provided and processed in a consistent manner. In any of the samples provided to the method, whether they be the input sample, a diluted input sample or the output sample, each of the samples comprises preferably the same fixed final working volume and the same fixed nutrient quantity.

Turning to the system used to detect the triggering event, that is to the measure the time to detect the threshold event indicative of active microbial growth, a number of systems are suitable for this. Some such systems include, but are not limited to, turbidimetric systems, which identify viable microbial growth by measuring the turbidity of the sample, colorimetric systems, which measure the colour change response in a gel within the sample chamber (since viable microbes produce acids or alkalis that can react with specific gels), or a respirometer. The remainder of this invention will be discussed in relation to the use of a respirometer, however other methods may be used to detect the threshold event and determine the time taken to do so. We have previously described a system for monitoring the metabolism/growth of microorganisms, the system comprising a sealed chamber with a flexible diaphragm to provide sensitive measurements of variations of gas pressure in the headspace above a culture liquid. For background, reference may be made, for example, to US8,389,274. The system is known as Speedy Breedy (RTM). We have also previously described methods of enumerating the number of organisms in a sample using this system. Background reference of this method can be found in WO2015/136254.

Figures 3a and 3b show, schematically, an embodiment of a similar device 100 under, respectively, normal atmospheric pressure and negative pressure (in operation either negative pressure or positive pressure may be produced). Thus a culture 102 of biological material undergoes metabolism and growth during which it exchanges gases with the aqueous liquid (water) carrying cells depending upon various factors gas may be used and/or produced, for example the cells may produce carbon dioxide during respiration. A gaseous headspace 104 of the sealed culture chamber 106 thus experiences changes in pressure due to exchange of gas with the culture medium, and these are monitored by a diaphragm 108 and converted to an electronic pressure signal 1 10 which may, for example, be digitised and processed electronically by hardware, software or a combination of the two. Preferably the system also includes an agitator 1 12 and temperature control (not shown), as well as a sealable inlet/outlet port 1 14. Figure 4 shows a physical embodiment of a test device 200 with two chambers 202, 204, each configured to accept a culture vessel 100 of the general type shown in Figure 3. Each chamber provides a magnetic drive for agitator 1 12 and a temperature control system to allow the temperature in each culture vessel to be regulated, in embodiments in the range 14 to 44 degrees or 10 to 60 degrees. The device also has a user interface 206 comprising a display and user controls, and communications such as a USB connection and/or wired or wireless network connection. In preferred embodiments the system can be used independently or connected to a separate computer; the external connection facilitates, but is not necessary for, control of the system. For example a quality assurance manager may design and download protocols for the system and upload test results for analysis and/or audit. Connection to an external computer system facilitates downloading a custom test protocol to the device. In embodiments the system also has a removable Flash™ memory card for storing experimental results and/or data defining test protocols. Preferred embodiments of the device are relatively lightweight and portable and are powered by a low voltage dc source.

In use samples are introduced into the two culture vessels and the system controls the growth conditions and logs changes in pressure. As previously described and shown in Figure 1 the pressure sensing in embodiments uses a non-invasive process which isolates the sensors and, importantly, provides a barrier to protect both the biological culture and the operator.

The system operates as a sensitive microbial respirometer, detecting metabolic activity by measuring pressure transients relating to gaseous exchanges within the closed culture vessel (which in embodiments has a volume in the range 10ml-100ml, preferably 50ml) as a result of microbial respiration. The mixing helps to homogenise the culture and also facilitates gaseous exchange which is important to convert the effects of metabolic processes into detectable pressure transients (which may be positive or negative).

The above described systems are very effective at detecting the growth of bacteria and other living biological entities.

Referring now to Figure 5, this shows a block diagram of a test unit 300 of the general type shown in Figure 4. The block diagram is simplified to facilitate understanding omitting, for example, safety features. Thus a processor 302 is coupled to one or more communications interfaces 304, such as a USB port and/or network connection, and to an operator interface 306, for example similar to interface 206 at Figure 4. The processor is also coupled to a temperature control system 308, for each culture vessel. This comprises a temperature sensor and heater and/or cooler, operating under control of processor 302 according to control software stored in programmed memory 312. A pressure sensor 310 senses the pressure of the or each culture vessel, again for use by the control software. Optionally other sensors may be included, for example a colour sensor to sense a colour or colour change of a culture medium. In a simple embodiment this may comprise a wavelength-selective optical illumination source and/or a wavelength-selective detector, for example an optical detector with a colour filter. The system includes working memory 314 and non-volatile (in embodiments Flash™) memory 316, optionally on a removable card or the like. The non-volatile memory 316 stores data defining a set of test protocols and stores logged experimental data, in particular pressure, temperature, and time data.

Speedy Breedy uses a culture technique to amplify the number of microbes (if present) and determines their presence by measuring sensitive pressure changes within a closed culture vessel due to microbial respiration. An internal algorithm then defines a significant pressure event and assigns a Time to Detection which is indicative of the contamination level in the original sample.

Speedy Breedy is a rapid, portable, safe and easy-to-use microbial detection tool which offers a number of key advantages:

· Like for like, existing data shows that the time it takes to obtaining results in

Speedy Breedy is significantly, often days, quicker than existing methods that are in use

• Speedy Breedy allows testing to commence at the point of sampling, therefore eliminating the need to send samples to a laboratory and providing a further reduction in time to results

• On-site testing and the reduction in time to results allows for effective local testing of water treatment systems, helping operators and enforcement officers ensure the effectiveness of these processes. The outcome is a reduced risk of transferring invasive species without the slow and costly off-site testing.

In the embodiments shown of the Speedy Breedy, two chambers are provided. In order to perform the method as described above, two Speedy Breedy devices are required in order to provide the data on the input sample, one dilution sample, and the output sample when processing the samples simultaneously, as is the preferred method. Since two Speedy Breedy devices actually provides four vessels, this enables a further dilution sample to be tested, which improves the accuracy of the calibration profile.

Results of a real test will now be described. In the real-life test, the method was used to determine the efficacy of a Ballast Water Management System. The aim of the study was to demonstrate the method (using Speedy Breedy devices) as a rapid on-ship test to assess microbial contamination levels before and after processing ballast water through a Ballast Water management System (BWMS). Further, to assess the ease of use on a ship by non-technical operatives.

Sampling ballast water:

Testing was conducted while passing ballast water through the ship's Optimarin BWMS. Ballast water was loaded into three separate tanks (or port and starboard pairs of tanks, depending on requirements for ship stability).

Referring to Figure 6, during uptake of sea water 602 into the ballast water tank 606, samples were collected at sample port A (before treatment by the ballast water management system 604) and sample port B (after treatment by the ballast water management system 604), using the operational set-up shown, and with the valves 608 in operational position as shown. Similarly, on discharge of the ballast water in the ballast water tank 606 into the sea 602, samples were collected at sample ports A and B, using the operational set-up described in the Figure 9 after 5 days holding time in the ballast tank 606.

Experiment 1 : Uptake of sea water to ballast tank:

Sterile Speedy Breedy vessels containing powdered Tryptone Soya Broth medium (Oxoid CM0129) equivalent to 50 ml of final working volume were filled with varying sample volumes of marine ballast water either neat or diluted in 0.2 um filtered sea water to bring the final working volume to 50 ml. The final nutrient quantity was therefore constant across the test range.

The cultures were run in Speedy Breedy at 35°C and mixed continually at 60 rpm according to a Speedy Breedy "TVC 35 Deg" protocol within the instrument. Results:

From the above data, the calibration curve shown in Figure 7 was generated using the methods described above. Figure 7 shows the input sample concentration level against the mean time to detect the threshold event. Figure 8 shows the neat (100% sea water) input sample before the BWMS for replicates 1 and 2. The graph shows the pressure response of the culture vessel over time for the shown temperature. The red line indicated the determined time to detect the event.

Experiment 2: Discharge of ballast tank to sea:

Sterile Speedy Breedy vessels containing powdered Trytone Soya Broth medium (Oxoid CM0129) equivalent to 50 ml of final working volume were filled with varying sample volumes of marine ballast water either neat or diluted in 0.2 um filtered sea water to bring the final working volume to 50 ml. The final nutrient quantity was therefore constant across the test range.

The cultures were run in Speedy Breedy at 35°C and mixed continually at 60 rpm according to a Speedy Breedy "TVC 35 Deg" protocol within the instrument.

Results:

% TTD TTD Mean sample (H:S) (Mins) (Mins)

Sample DD/S1/26 Replicate 1 100% 5.38 338

Before BWMS Replicate 2 100% 5.38 338 337

Replicate 3 100% 5.49 334

Sample DD/S1/26 Replicate 1 10% 7.18 438

Before BWMS Replicate 2 10% 9.34 574 514

Replicate 3 10% 8.49 529

Sample DD/S1/26 Replicate 1 1 % 9.03 543

Before BWMS Replicate 2 1 % 8.42 522 533

Vessel

Replicate 3 1 % 4.29 Fail

Sample DJ/S2/26 Replicate 1 100% 9.23 563

After BWMS Replicate 2 100% no event 565

Replicate 3 100% 9.26 566 From the above data, the calibration curve shown in Figure 10 was generated using the methods described above. Figure 10 shows the input sample concentration level against the mean time to detect the threshold event. Figure 10 shows the response for the sample after the BWMS for replicates 1 and 2. The graph shows the pressure response of the culture vessel over time for the shown temperature. The red line indicated the determined time to detect the event. Replicates 2 and 3 show no detection in the left chamber (green line) meaning that this sample had no viable bacteria in the sample and a long TTD for the right chamber (blue line) meaning that there were very few organisms still viable, possibly a single organism. The third replicate (not shown) similarly gave a very long TTD.

Conclusions:

The method demonstrates a high degree of correlation between the Times to Detection in replicates, indicating that single tests rather than the mean of replicates may hold sufficient validity for an indicative test for degree of contamination.

The method, in a real life scenario, indicated an approximate 70% reduction in viable numbers during the first pass through the BWMS (from the sea to the ballast tanks).

The method indicated an approximate 3 log reduction in viable numbers during the second pass through the BWMS (from the ballast tanks to the sea).

The method therefore provides a simple, sensitive, rapid method for monitoring efficacy of microbial decontamination systems.

As discussed above, whilst we have explained the method in conjunction with a respirometer, this method also lends itself to turbidimetric methods and colorimetric methods as mentioned above. Instead of monitoring pressure changes in a vessel, a turbidimetric would monitor changes in the turbidity (cloudiness) of the sample, and a colorimetric method would monitor changes in the colour of a reactant gel in the sample chamber. Each system could be configured to monitor changes in these conditions as a measure of time to detect a time to detect an event indicative of microbial growth in the sample, and record the data for determination of the efficacy of the decontamination process. No doubt many other effective alternatives will occur to the skilled person. It will be understood that the invention is not limited to the described embodiments and encompasses modifications apparent to those skilled in the art lying within the scope of the claims appended hereto.

Claims

CLAIMS:

1. A method of determining the efficacy of a microbial decontamination process, comprising:

taking an input sample at an input to a decontamination process, and an output sample at an output of the decontamination process;

generating a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, the time to detect a threshold event representing a time to detect a threshold event that is indicative of microbial growth within the sample,

determining a time to detect a threshold event in the output sample;

comparing the time to detect a threshold event in the input sample and output sample; and

determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples.

2. A method according to claim 1 , wherein the step of generating a calibration profile response comprises:

generating one or more dilutions of the input sample;

determining a time to detect a threshold event in the input sample and the one or more dilutions of the input sample; and

defining a relationship between the level of dilution and the time to detect a threshold event for each of the samples and dilutions.

3. A method according to claim 2, wherein the one or more dilutions comprise input samples that are diluted to 50%, 25%, 10% or 1 % concentration of the input sample.

4. A method according to claim 3, wherein the input sample is diluted using a sterile and/or filtered solution.

5. A method according to any preceding claim, wherein the sample is provided with a nutrient to promote microbial growth.

6. A method according to claim 5, wherein each of the samples comprises the same fixed final working volume and the same fixed nutrient quantity.

7. A method according to any preceding claim, wherein the step of determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples comprises:

determining a relative level of change in the time to detect the threshold event between the input sample and the output sample;

comparing the relative level of change in the time to detect a threshold event to a threshold value; and

determining the efficacy of the decontamination process when the relative level of change in time is greater than or equal to the threshold value.

8. A method according to claim 7, wherein the difference between the threshold value and the relative level of change in time to detect the threshold events between the input sample and the output sample is indicative of a level of logarithmic kills, each level of logarithmic kill representing a logarithmic reduction in the number of viable microbes removed by the microbial decontamination process.

9. A method according to any preceding claim, wherein the step of generating a calibration profile response of the input sample, and the step of determining a time to detect a threshold event in the output sample is performed using a respiration method, a turbidimetric method or a colorimetric method.

10. A method according to claim 9, wherein, when a respiration method is used, each sample is held in a separate culture vessel comprising a sealable chamber for receiving and culturing a liquid sample such that the presence of microbes is detectable by detecting a change in pressure in a headspace of the chamber.

1 1. A method according to claim 10, wherein the time to detect a threshold event comprises a time to detect a change in pressure in the headspace of the chamber that is greater than or equal to a pressure change threshold value, the change in pressure being indicative of the number of viable microbes in the sample.

12. A method according to claim 1 1 , wherein generating a calibration profile response of the input sample, and determining a time to detect a threshold event in the output sample comprises:

running a test protocol on the respirometer to incubate the samples according to the test protocol, the test protocol defining a temperature to regulate of the liquid sample in the culture vessel;

detecting the change in pressure in the headspace of the chamber;

monitoring the detected change in pressure; and

signalling the time taken to detect the change in pressure when the change in pressure is greater than a pressure change threshold value.

13. A method according to any preceding claim, wherein the microbial decontamination process comprises a ballast water management system for a sea vessel, an industrial process, or a food process.

14. A method according to any preceding claim, wherein the method is performed at the location of the decontamination process.

15. A method according to any preceding claim, wherein the determination of a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, and the determination of a time to detect a threshold event in the output sample are performed simultaneously.

16. A method according to any preceding claim, comprising outputting the determined efficacy of the decontamination process to a user.

17. A system for determining the efficacy of a microbial decontamination process, comprising:

an input sample vessel for receiving an input sample from an input of a decontamination process, and an output sample vessel for receiving an output sample from an output of the decontamination process; and

a processor configured to:

generate a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, the time to detect a threshold event representing a time to detect a threshold event that is indicative of microbial growth within the sample,

determine a time to detect a threshold event in the output sample;

compare the time to detect a threshold event in the input sample and output sample; and

determine the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples.

18. A system according to claim 17, wherein for the step of generating a calibration profile response, the processor is configured to:

determine a time to detect a threshold event in the input sample and one or more dilutions of the input sample; and

define a relationship between the level of dilution and the time to detect a threshold event for each of the samples and dilutions.

19. A system according to claim 18, wherein the one or more dilutions comprise input samples that are diluted to 50%, 25%, 10% or 1 % concentration of the input sample.

20. A system according to claim 18 or 19, wherein the input sample is diluted using a sterile and/or filtered solution.

21 . A system according to any one of claims claim 17 to 20, wherein the sample is provided with a nutrient to promote microbial growth.

22. A system according to claim 21 , wherein each of the samples comprises the same fixed final working volume and the same fixed nutrient quantity.

23. A system according to any one of claims 17 to 22, wherein for the step of determining the efficacy of the decontamination process based on the comparison of the time to detect threshold events in the input and output samples, the processor is configured to:

determine a relative level of change in the time to detect the threshold event between the input sample and the output sample; compare the relative level of change in the time to detect a threshold event to a threshold value; and

determine the efficacy of the decontamination process when the relative level of change in time is greater than or equal to the threshold value.

24. A system according to claim 23, wherein the difference between the threshold value and the relative level of change in time to detect the threshold events between the input sample and the output sample is indicative of a level of logarithmic kills, each level of logarithmic kill representing a logarithmic reduction in the number of viable microbes removed by the microbial decontamination process.

25. A system according to any one of claims 17 to 24, comprising using a respirometer, a turbidimetric process or a colourmetric process.

26. A system according to claim 25, wherein, when a respirometer is used, each sample is held in a separate culture vessel comprising a sealable chamber for receiving and culturing a liquid sample such that the presence of microbes is detectable by detecting a change in pressure in a headspace of the chamber.

27. A system according to claim 26, wherein the time to detect a threshold event comprises a time to detect a change in pressure in the headspace of the chamber that is greater than or equal to a pressure change threshold value, the change in pressure being indicative of the number of viable microbes in the sample.

28. A system according to claim 27, wherein the respirometer comprises a temperature controller coupled to the processor to control a temperature in the chamber, and wherein when generating a calibration profile response of the input sample, and when determining a time to detect a threshold event in the output sample, the processor is configured to:

run a test protocol on the respirometer to incubate the samples according to the test protocol, the test protocol defining a temperature to regulate of the liquid sample in the culture vessel;

detect the change in pressure in the headspace of the chamber;

monitor the detected change in pressure; and signal the time taken to detect the change in pressure when the change in pressure is greater than a pressure change threshold value.

29. A system according to any one of claims 17 to 28, wherein the microbial decontamination process comprises a ballast water management system for a sea vessel, an industrial process, or a food process.

30. A system according to any one of claims 17 to 29, wherein the system is located at the location of the decontamination process.

31 . A system according to one of claims 17 to 30, wherein the determination of a calibration profile response of the input sample to determine a calibration time to detect a threshold event in the input sample, and the determination of a time to detect a threshold event in the output sample are performed simultaneously.

32. A system according to any one of claims 17 to 31 , wherein the processer is configured to output the determined efficacy of the decontamination process to a user.