AU2005331515A1 - System for rapid analysis of microbiological materials in liquid samples - Google Patents

System for rapid analysis of microbiological materials in liquid samples Download PDF

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AU2005331515A1
AU2005331515A1 AU2005331515A AU2005331515A AU2005331515A1 AU 2005331515 A1 AU2005331515 A1 AU 2005331515A1 AU 2005331515 A AU2005331515 A AU 2005331515A AU 2005331515 A AU2005331515 A AU 2005331515A AU 2005331515 A1 AU2005331515 A1 AU 2005331515A1
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light
spectrophotometer
specimen container
sample
container
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AU2005331515A
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Ron Emburgh
Ravi Kanipayor
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
    • G01N21/532—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke with measurement of scattering and transmission
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Description

WO 2006/116835 PCT/CA2005/000686 -1 Title: System for Rapid Analysis of Microbiological Materials in Liquid Samples Field of the invention 5 [0001] This invention relates to methods and apparatus for detecting the presence and enumeration of microbiological materials in liquid samples, and in particular, to methods and apparatus for the quantitative analysis of pathogenic microbes in water samples. Background of the invention 10 [0002] Drinking water and recreational water (water at beaches and other swimming facilities) should be tested on a regular basis, arnd the test results should be made available within a short period of time, in order to protect the public from harmful and contagious deceases. [0003] Currently, most water sample tests are carried out in a 15 laboratory environment away from water facilities or locations. Many methods and procedures currently used in routine microbiological analysis were developed over 100 years ago. They are labor intensive and time-consuming procedures both in operation and data collection. The results of such tests are typically not made available to the operators of these facilities for about 36 20 to 72 hours. Consequently, it is often not possible for operato rs of water facilities to take action to correct tainted water until long after the tainted water has been consumed or used. [0004] In addition, the transmission of water-borne diseases remains a major concern despite worldwide attempt to curb the problem. This problem 25 is not confined to developing and under developed countries but is global in nature. Some key reasons for this are: (1) The current testing frequencies are not sufficient to provide early warning so that corrective action can be taken to prevent outbreak of diseases; and 30 (2) The current testing methods are laborious and time consuming and hence discourage frequent testing.
WO 2006/116835 PCT/CA2005/000686 -2 [0005] In 1988, Edberg S.C., et al. developed a new technology based on a chemically defined substrate MTF method known as 'Autoanalysis Colilert (AC): "National filed evaluation of a defined substrate method for the simultaneous evaluation of total coliforms and Escherichia coli from drinking 5 water: comparison with standard multiple tube technique', Appl. Environ. Microbiol., 54 1595 (1988)." This allowed the simultaneous detection and identification of both total coliforms and E.coli in water at 1CFU per 100mIs in less than 24 hours. The Colilert@, a chromogenic-fluorogenic reagent medium, provided the specific nutrients, and enzyme substrates with 10 chromophores and fluorophores for the simultaneous detection of total coliform and E.coli. In 1989, the US EPA approved this method as a means of qualitative testing of total coliform in drinking water. [0006] The development of chromogenic/fluorogenic reagents to conduct microbial testing has opened the door to better and faster testing 15 protocols. In addition, these products provide the additional opportunity to use technology such as optical spectroscopy to conduct biological, microbiological and chemical analysis. Spectrophotometric analyses are very sensitive and hence can detect the presence of a very low concentration of color producing components of interest in liquid samples (in parts per million). 20 Visually, the human eye can only detect the color when these components are present in very high concentration, thus the need for incubation periods ranging from 18 to 72 hours. The time required to identify or estimate the presence of microbiological indicators in water, food and environmental samples can be drastically reduced when combining incubation with 25 photometric analysis. [0007] The use and advantage of spectrophotometric application to microbial analysis in liquid samples have been cited in the literature. However, the tests have been done outside the incubation chamber by drawing an aliquot of a sample from the incubation vessel to photometric tubes at various 30 intervals and measuring using standard spectrometers. This is not only time consuming but requires separate incubators, spectrometers and technical WO 2006/116835 PCT/CA2005/000686 -3 personnel to conduct the test and in some cases robotic sampling systems. There is also a potential risk of cross contamination and human error, if proper care is not applied in conducting the analysis. [0008] Accordingly, there is an urgent need for improved methods and 5 apparatus for testing microbiological materials in drinking water, recreational water and wastewater, to provide better management of water facilities and to protect public health and the environment. Summary of the Invention [0009] The present invention relates to a system for the rapid 10 quantitative analysis of bacteria in fluid samples such as water. One aspect of the present invention is a system comprising a specimen container for containing a test sample and an apparatus having a spectrophotometer system comprising an appropriate light emitting source and a detector proximate to the specimen container within the housing of the apparatus. A 15 reagent that provides a detectable parameter (e.g., color, fluorescence etc.) is added to the test sample in the specimen container. While the sample undergoes incubation, the detector monitors the light from the source passing through the sample and the specimen container. The detector is connected to a spectrophotometer processor that measures, processes, records and stores 20 the information. The processor can also be connected to an appropriate measuring and recording device such as a computer, multimeter or any other device, which can measure, and record the output signal from the detector. This provides a non-intrusive continuous incubation and signal growth measurement of the parameter under investigation. 25 [0010] Another aspect of the present invention is a system for the rapid analysis of microbiological parameters in liquid samples. The system comprises a specimen container for containing the liquid sample, the specimen container being made from a material that allows for the propagation of light, a housing defining an enclosable chamber for holding the 30 specimen container, an incubating system mounted within the housing for incubating microbiological materials within the liquid sample, and a WO 2006/116835 PCT/CA2005/000686 -4 spectrophotometer system mounted within the housing for propagating light within the specimen container and measuring light absorbed, emitted or scattered by the liquid sample as the microbiological materials are incubated by the incubating system over time. 5 [0011] A further aspect of the present invention is an apparatus for detection of microbiological materials in a liquid sample. The apparatus comprises a housing having an enclosable chamber shaped for holding a clear plastic container for containing a liquid sample, incubating apparatus mounted within the housing for incubating any microbiological materials within 10 the liquid sample, and spectrophotometer apparatus mounted within the housing for measuring light absorbed, emitted or scattered by the liquid sample as the microbiological materials are incubated by the incubation apparatus over time. [0012] The present invention is also directed to a method for the rapid 15 analysis of microbiological materials in a liquid sample, comprising the steps of: (a) mixing a liquid sample having an unknown initial population of a microbiological material with a reagent in a specimen container, the reagent providing a detectable parameter 20 indicative of the microbiological material, thereby creating a sample/reagent mixture; (b) placing the specimen container in an enclosable housing and enclosing the housing; (c) incubating the sample/reagent mixture in the enclosed 25 housing at a temperature within a pre-selected temperature range over a period of time; and (d) measuring changes in the detectable parameter as the sample/reagent mixture is being incubated during the period of time.
WO 2006/116835 PCT/CA2005/000686 -5 [0013] The present invention is further directed to a method for the rapid quantitative analysis of microbiological materials in a liquid sample. The method comprises the steps of: (a) placing a liquid sample having an unknown initial population 5 of a microbiological material in a specimen container made from a material that allows for the propagation of light; (b) creating a sample/reagent mixture by mixing the liquid sample with a reagent that provides a detectable parameter indicative of the microbiological rnaterial upon exposure to 10 light; (c) incubating the sample/reagent mixture in an enclosed housing at a temperature within a pre-selected temperature range over a period of time; (d) measuring changes in the detectable parameters as the 15 sample/reagent mixture is being incubated by propagating light of a known intensity within the sample/reagent mixture and measuring changes in the intensity of the light over time; (e) recording the changes in the intensity of the light as a 20 function of time; (f) recording a time of significant deviation at which there occurs an exponential change in the detectable parameter; and (g) determining the initial population by correlating the time of 25 significant deviation with known times of exponential growth for the detectable parameter for known initial populations of the microbiological material.
WO 2006/116835 PCT/CA2005/000686 -6 Brief description of the drawings [0014] The invention will now be described, by way c>f example only, with reference to the following drawings, in which: [0015] Figure 1 is a schematic diagram of the systenri of the present 5 invention; [0016] Figure 2 is a perspective view of apparatus made in accordance with a preferred embodiment of the subject invention; [0017] Figure 3 is an exploded perspective view of the subject apparatus, showing the cap off and the specimen container removed from the 10 base unit; [0018] Figure 4 is a sectional view of the subject apparatus taken along line 4-4 of Figure 2; [0019] Figure 5 is a sectional view of the subject apparatus taken along line 5-5 of Figure 4; 15 [0020] Figure 6 is a sectional top view of the subject apparatus taken along line 6-6 of Figure 4; [0021] Figure 7 is a sectional front view of an apparatus made in accordance with an alternative embodiment of the present invention; [0022] Figure 8 is a flow chart illustrating the method of the subject 20 invention; [0023] Figure 9 is a graph illustrating a typical time of growth curve; [0024] Figure 10 is a data graph displaying exemplary growth curves for two different microbiological parameters; [0025] Figure 11 is a data table showing the results generated by the 25 method of the present invention; [0026] Figure 12 is an exemplary linear correlation curve used in the method of the subject apparatus; [0027] Figure 13 is a test report generated by the subject method; WO 2006/116835 PCT/CA2005/000686 -7 [0028] Figure 14a is a flow chart illustrating the heating control algorithm of the present invention; and [0029] Figure 14b is a flow chart illustrating the ter-nperature control and data collection algorithm of the present invention. 5 Detailed description of the invention [0030] Referring to Figure 1, illustrated therein is a system for the rapid analysis of microbial parameters using non-intrusive in-vessel incubation and detection, made in accordance with the subject invention. The system 10 comprises an incubator-detector apparatus 12, a specimen container 14 for 10 containing a liquid sample 11 mixed with reagent 20, and external data recorder 80. Incubator-detector apparatus 12 comprises a housing 15 having a detection chamber 65 shaped for receiving specirnen container 14, an incubation system 60 mounted in housing 15 for incubating microbiological materials within liquid sample 11, and a spectrophotometer system 62 15 mounted in housing 15. Spectrophotometer system 62 measures the amount of light absorbed, emitted or scattered by the liquid sample 11 in specimen container 14 as microbiological materials are incubated by incubation system 60. [0031] Incubation system 60 includes heating controller 92, and 20 spectrophotometer system 62 includes spectrophotometer controller 94. Power source 90 provides power to incubation system 60 and spectrophotometer system 62. External data recorder 80 preferably comprises a computer 85 having a microprocessor 86 and memory device 88, and an output device such as printer 82 that is connected to computer 85. 25 [0032] Referring now to Figures 2-6, illustrated therein is a preferred embodiment of incubator-detector apparatus 12. Housing 15 of apparatus 12 is a generally cylindrical enclosure comprising a base 16, container holder 18 shaped to hold sample container 14, and a removable cap 50. Base 16 includes an upwardly extending cylindrical lip 55 shaped to receive cap 50. 30 Base 16 houses power source 90, heating controller 92 and spectrophotometer controller 94. Power source 90 can be any suitable power WO 2006/116835 PCT/CA2005/000686 -8 source known in the art, such as a rechargeable battery located vvithin base 16 having power outlet 99 for connection to an external 120 or 220 volt AC power source or a DC power source. Mounted on the exterior of toase 16 are power switch 13, status LEDs 97, and data port 98. 5 [0033] Container holder 18 comprises a base 21 and an cbpen-ended cylindrical wall 19 extending upwardly from base 21. Wall 19 is shaped to surround the lower portion of specimen container 14 when specimen container 14 is placed inside housing 15. Wall 19 includes a pair of inwardly extending, diametrically opposing, generally rectangular indents 23 10 [0034] Removable cap 50 is shaped to fit snugly around wall 19 of container holder 18. Cap 50 preferably comprises a thermally efficient, double wall cylindrical shell having an outer wall 51, inner wall 59, closed top 52 and open-ended bottom flange 53. The outside surface of bottom flange 53 is provided with a bead 66 shaped to thread into groove 67 or, the inside 15 surface of lip 55 of base 16. The inner surface of inner wall 59 includes a protrusion 68 shaped to sealingly engage ring 49 extending arou nd base 21 of container holder 18. Cap 50 may optionally be provided with vacuum or inert gas between the walls 51 and 59. [0035] When cap 50 is placed over container holder 18, cap 50 and 20 container holder 18 define a very efficient thermally insulated incubation detection chamber 65. The inside surface of wall 19 of specimen holder 18 is preferably blackened to make chamber 65 an efficient black box (dark room) for optical detection and measurement. [0036] The incubation system 60 of apparatus 12 comprises heating 25 element 24, temperature sensor 25, and incubation controller 92. Heating element 24 is mounted within heating finger 57 extending upwardly through an aperture in base 21 of container holder 18. Temperature sensor 25 is mounted inside a temperature finger 56 extending upwardly fro" base 21 of container holder 18. The temperature sensor 25 may comprise a thermistor 30 26 placed near the top of finger 56.
WO 2006/116835 PCT/CA2005/000686 -9 [0037] Heating controller 92 controls the heat to the heating element 24 and monitors the temperature of liquid sample 11 through temperature sensor 25. Once the temperature reaches the optimum, heati ng controller 92 maintains the temperature of liquid sample 11 within. Heating controller 92 5 preferably comprises a timer (not shown) for measuring the incubation time from the start and to deactivate the heating at the end of a preset time. [0038] Specimen container 14 comprises a specimen cap 42 and a specimen bottle 44. Specimen bottle 44 is generally cylindrical with bottom cavity 45 to accommodate heating finger 57 and bottom cavity 46 to 10 accommodate temperature sensor finger 56. Specimen bottle 44 also has a pair of diametrically opposed, generally rectangular side recesses 48 shaped to register with indents 23 of wall 19 when specimen container 14 is positioned within container holder 18. Specimen bottle 44 is made from a material that allows for the propagation of light signals, and is preferably made 15 of a clear plastic or other material that is optically transparent. [0039] Spectrophotometer system 62 is a system for measuring light absorbed, emitted or scattered by the liquid sample as the microbiological materials are incubated over time. As best shovvn in Figure 4, spectrophotometer system 62 preferably co mprises three 20 spectrophotometers, a first spectrophotometer comprising light emitting source 30a and detector 35a, a second spectrophotometer comprising light emitting source 30b and detector 35b, and a third spectrophotometer comprising light emitting source 30c and detector 35c. Light emitting sources 30a,b,c propagate beams of light of a given intensity alorg selected optical 25 paths within specimen container 14. Light detectors 35a,b, c are positioned to detect changes in the intensity of the beams of light within a selected field of view related to the optical paths, resulting from light that is absorbed, emitted or scattered by the liquid sample 11 as the microbiological materials are incubated by the incubator system over time. 30 [0040] Each of light sources 30a,b,c preferably comprises a light emitting diode (LED) of a specific wavelength maxirnum, and each of WO 2006/116835 PCT/CA2005/000686 -10 detectors 35a,b,c preferably comprises a phototransistor detector. Light sources 30a,b,c, and detectors 35a,b,c are mounted on printed circuit boards 33a,b,c that are electrically connected to spectrophotometer controller 94. [0041] Light emitting sources 30a, 30b extend through apertures 70a 5 and 70b in base of container holder 18. Light emitting sources 30a, 30b placed in such a way that the light emitting from the sources travels through apertures 70a and 70b, respectively, and upwardly along a selected optical path within specimen container 14. In the case of light emitting source 30a, 30b, the optical paths are shown by arrow 1 and arrow 4, respectively. 10 Container holder 18 also has apertures 75a, 75b for signal detectors 35a, 35b. Detectors 35a, 35b are placed at a 90 degree angle with respect to light emitting sources 30a, 30b, in such a way that the detector window face towards specimen bottle 44 to receive any signal propagating towards detectors 35a, 35b , having fields of view shown by arrow 2 and arrow 5, 15 respectively. [0042] Container holder 18 also includes suitable apertures 70c and 75c for signal-emitting source 30c and signal detector 35c, respectively. Light emitting from light source 30c passes through aperture 70c along a horizontal optical path, with the direction of the propagation shown by arrow 3 through 20 the specimen container 14 and aperture 75c to detector 35c. Detector 35c is positioned so that its field of view is at a 1800 angle to the optical path of light source 30c. [0043] Spectrophotometer controller 94 controls the operation of the spectrophotometer system. Spectrophotometer, controller 94 activates and 25 deactivates and may pulse signal emitting sources 30a,b,c. Spectrophotometer controller 94 also measures and p rocesses the output signals generated by detectors 35a, 35b and 35c. Spectrophotometer controller 94 includes a microprocessor 95 having a built in time clock that functions as a data logger and stores the measured detector signal values 30 along with the corresponding temperatures of the liquid sample 11 and time of the measurements in a specific memory location within microprocessor 95.
WO 2006/116835 PCT/CA2005/000686 -11 Spectrophotometer controller 94 may indicate the end of the test by activating one or more of status LEDs 97 or an audio signaling device (not shown). Spectrophotometer controller 94 also communicates through data port 98 with external data recording device 80, such as computer 85, a rnLiltimeter or other 5 external signal manipulator. [0044] Microprocessor 95 of spectrophotometer controller 94 includes a memory for storing software that implements the test method of the subject invention. The test method provides the specifications and conditions required to conduct the testing process. Through custom software the method can be 10 programmed and downloaded into the memory of microprocessor 95 through data port 98. The software is also capable of erasing all rrtemory locations within the controller 94. [0045] To utilize apparatus 12 of the present invention to test liquid samples, liquid sample 11 is placed in sample container 14. Liquid sample 11 15 is not limited to water, and may comprise other liquids or other liquid medium containing suspensions such as food particles, filter papers and other solids. An appropriate reagent 20 is then added to liquid sample 11 inside specimen container 14. Reagent 20 may be chemical or biological in nature and provide a detectable parameter such as color, fluorescence, turbidity etc. that 20 indicates the presence or absence of the microbiological material under investigation. [0046] The detection of color, fluorescent or turbidity signal is time dependent and the time of detection is related to the quantity of the bacteria present at the start of the test. Thus quantification of the d etected microbial 25 parameters such as total coliform and e.coli in water sample can be achieved by measuring the signal due to color change or fluorescen ce signal and the time at which they were detected in appreciable amount. [0047] The detection of the color or the detection of the fluorescence signal is measured using spectrophotometer system 62 of apparatus 12. In 30 the preferred embodiment, spectrophotometer system 62 comprises three spectrophotometers that provide colorimetric detection for total coliform, WO 2006/116835 PCT/CA2005/000686 -12 fluorescence detection for e.coli and microbial growth turbidity by nephelometry, respectively. [0048] The built-in time clock of microprocessor 95 of spectrophotometer controller 94 provides the time of growth while the 5 constant temperature of incubation system 60 provides both microbial growth and optical reproducibility. [0049] In a preferred embodiment of system 10 of the present invention, spectrophotometer system 62 comprises three "time-of-growth spectrophotometers" within a single constant temperature incubator, i.e. 10 spectrophotometers that record the growth of specified microbiological parameters as a function of time. The type of spectrophotometric analysis done by each spectrophotometer depends upon the configuration and specification of the source and detector of the spectrophotor-neter. The 180 degree configuration of source detector pair 30c, 35c provides a colorimetric 15 or turbidimetric analysis, while the 90-degree configuration of source-detector pairs 30a, 35a and 30b, 35b provides for either fluorometric or nephelometric analysis. [0050] Source 30c of colorimeter spectrophotorrieter preferably comprises an LED with wavelength maximum at 620 nm, and detector 35c is 20 preferably a phototransistor detector having a signal response ranging across the visible region including the 620 nm. Source 30a of fluorometer spectrophotometer is preferably an UVLED with maximum vvavelength at 380 nm, and detector 35a, placed strategically at 90 degrees to source 30b, is preferably a phototransistor detector having a signal response in the visible 25 region including the 400-500 nm range. The nephelometer configuration is similar to that of the fluorometer except that source 30b is preferably an LED with a maximum wavelength at 400 nm. [0051] After reagent 20 is aseptically added to the sample, specimen cap 42 is fastened to specimen bottle 44, and specimen container 14 is gently 30 shaken to dissolve the reagent, and form a sample/reagent mixture. For simultaneous testing of total coliform and e.coli in water samples, typical WO 2006/116835 PCT/CA2005/000686 -13 reagents provides not only an optimum growth nutrient, but also a color change for total coliform and fluorescence signal for e.coli, if they are present in any quantity in the sample. Examples of typical chromogenic/fluorogenic reagents are: 5 Merck KGaA - Readycult@ IDEXX- Colilert@ [0052] The specimen container 14 is then placed inside the container holder 18 as best shown in Figure 2. Once the removable cap 50 is placed on base 16, incubation-detection chamber 65 provides a black box (dark room) 10 condition ideal for microbial growth and spectrophotometric detection. [0053] Pressing the start button 13 on base 16 of apparatus 12 activates the incubation cycle and the detection process. Optionally, a separated activation button can be used to activate the detection process at a pre-determined time after the start of the incubation. 15 [0054] Activation of the detection process may include turning the power to the signal emitting sources 30a,b,c and detectors 35a,b,c, pulsing the signal emission and monitoring the signal output of detectors 35a, b, c. [0055] Heating controller 92 brings and maintains the temperature of liquid sample 11 at a constant temperature within a preset temperature range. 20 For coliform and E.coli testing in water a temperature of 36±10 C is preferred. However, the temperature depends upon the reagent used and may vary from one reagent to another. The temperature also depends upon the test method specification. For example, E.coli can be tested at either 360C or 410C using the same reagent. 25 [0056] Spectrophotometer controller 94 continuously monitors, records and stores the output signals from the detectors 35a,b,c. In the method of the preferred embodiment, spectrophotometer controller 94 also records and stores the time and temperature of each output signal. Controller 94 may be connected to external data recorder 80 that is programmed to record the 30 signal either continuously or at a pre determined intervals. External data WO 2006/116835 PCT/CA2005/000686 -14 recorder 80 may also record the time of each signal measured and the corresponding temperature of the sample, and generate a "time dependent growth signal pattern" (TDGSP) of the microbial parameters under investigation. 5 [0057] The colorimetric TDGSP of total coliform and fluorometric TDGSP of e.coli are preferably recorded simultaneously along with a nephelometric TDGSP of increasing turbidity due to bacterial growth in water. [0058] A significant deviation of the output signal from the initial base line is an indication of the presence of the parameter under investigation while 10 the time needed to reach the significant deviation from the start provides an indication of the original amount of the test parameter. [0059] The time clock of microprocessor 95 provides the date and time test started, the time of each measured signal and the corresponding temperature, the time at which the test completed or terminated. The end of 15 the test can be indicated through status LEDs 97 and/or audio signals or can be controlled through a software program. [0060] Referring now to Figure 7, illustrated therein is a schematic view of apparatus 112 made in accordance with an alternative embodiment of the present invention. Apparatus 112 is generally similar to apparatus 12 of the 20 preferred embodiment as shown in Figures 2-6 except for a few modifications. [0061] Apparatus 112 comprises a specimen container 114, and a housing 115 comprising a base 116, a cylindrical container holder 118, and a removable cap 150. Container holder 118 has a cylindrical base 155 and an open-ended cylindrical wall 160. Container holder base 155 has a 25 temperature controller finger 156 extending upwards to accommodate temperature controller 180. Temperature controller 180 may be a bimetal switch or any other suitable device, which can activate and deactivate the heating element. [0062] A heating element 124 is mounted within the open ended 30 cylindrical wall 160 of the sample holder 150. As shown, heating element 124 WO 2006/116835 PCT/CA2005/000686 -15 comprises a resistor wire 125. Alternatively, heating element 124 may be a resistor coil, resistor foil etc. The length of the resistor wire is dictated by the resistor temperature and the ohm per foot rating of wire 125. [0063] Specimen container 114 comprises a specimen cap 142 and a 5 specimen bottle 144. The specimen bottle is generally cylindrical with a bottom cavity 145 shaped to accommodate temperature controller finger 156. [0064] Heating controller 192 maintains a constant preset temperature range within the sample. Optionally it may comprise a timer (not shown) for measuring the incubation time from the start and to deactivate the heating at 10 the end of a preset time. [0065] The spectrophotometer system of apparatus 112 is similar to that of apparatus 12 of the preferred embodiment. Light emitting source 130a emits light in the direction of arrow 1 and detector 135a detects emitted or scattered light traveling in the direction of arrow 2. Light emitting source 130b 15 emits light in the direction of arrow 4 and detector 135b detects emitted or scattered light traveling in the direction of arrow 5. Light emitting source 130c emits light in the direction of arrow 3 and detector 135 detects light traveling in the direction of arrow 3. The spectrophotometer system also includes spectrophotometer controller 194 for controlling the operation of light sources 20 130a,b,c and detectors 35a,b,c, and power source 190. [0066] Referring now to Figure 8-13, illustrated therein is a preferred embodiment of the quantitative analysis method of the present invention. [0067] The quantitative analysis method of the present invention is based on the recognition that there is a relationship between initial population 25 and growth population with time. The time interval between the start of the test (starting population) and a fixed growth population is a function of the initial population, the incubation temperature and the growth media. Thus keeping the incubation temperature and growth media as constants, the time required to reach the fixed growth population is a direct function of the initial 30 population.
WO 2006/116835 PCT/CA2005/000686 -16 [0068] A chromogenic/fluorogenic reagent such as Readycult@ (Merck KgaA) or Colilert@ (IDEXX) provides the mechanism by which the microbial growth population (total coliform and e.coli) can be monitored and measured in this invention through photometric detection process. The specific enzymes 5 produced by these organisms (for example _-galactosidase (total coliform) and _-glucuronidase (specific to e.coli)) will metabolize the nutrient-indicator and releases the chromophor or fluorophor into the liquid medium. The concentration of the chromophors or flurophors, at a given time, in the detection medium is proportional to the growth population at that time and 10 hence the change in the signal intensity due to the increase in concentration of the colour components is a measure of the time based growth population. [0069] The population detection time (tpop), which is defined as the time taken to reach a detectable population size, has been used to estimate bacterial growth parameters. The detection time has been shown to be 15 inversely proportional to the logarithm of the inoculum (initial population of the microbe) level. tpop - 1/log Xo {1} where Xo = initial bacterial population. 20 [0070] The time of significant deviation (TSD) is the time at which the measured photometric signal quantity above the baseline signal is statistically significant. TSD also depends on the initial concentration of the bacteria in 25 the sample and higher the initial bacterial count shorter the TSD. Since the increase in population size can be measured using increase in signal output, the time required to obtain a significant deviation (TSD) of the signal output from the baseline should corresponds to tpop if measured in the growth phase. The method is 30 tpop = TSD {2} And therefore, TSD - 1/log X 0 {3} WO 2006/116835 PCT/CA2005/000686 -17 [0071] This linear correlation curve equation- LCCE between TSD and initial population of the microbe under investigation (Xo) provides, in addition to detecting the presence, quantitative information of the bacterial population. 5 [0072] Figure 8 is a flow chart describing the steps of the subject method. At block 200, reagent 20 is mixed with liquid sample 11 having an unknown initial population of a microbiological material in specimen container 14, thereby creating a sample/reagent mixture. Reagent 20 provides a detectable parameter, such as colour or fluorescence, indicative of the 10 microbiological material. At block 202, specimen container 14 is placed inside housing 15 and cap 50 is placed on housing 15. At block 204, the incubation process is initiated, and the sample/reagent mixture is incubated in the enclosed housing at a constant temperature over a period of time. [0073] At block 206, data collection is initiated, and the changes in the 15 detectable parameter are measured as the sample/reagent mixture is incubated over time. These changes are measured by propagating light of a known intensity within the sample/reagent mixture in specimen container 14, and detecting changes in the intensity of the light over the period of incubation. 20 [0074] During the incubation period, data relating to the time, temperature and photometric signal indicative of the changes in light intensity are collected by spectrophotometer controller 94. An increase in microbial population with time is accompanied by the increase in photometric signal. This generates a real-time microbial growth curve, such as that shown in 25 Figure 9. At block 208, the testing is terminated, and the collected data is stored in the memory of microprocessor 95 of spectrophotometer controller 94. [0075] At block 210, the data is downloaded to external data recorder 80, preferably computer 85 installed with custom software. At block 212, 30 computer 80 processes the data and displays the result both in graphical WO 2006/116835 PCT/CA2005/000686 -18 format and tabular format. Figure 10 illustrates the growth curves of all selected parameters of a typical sample, as recorded on a real time basis or downloaded from apparatus 12, as well as the incubation temperature profile. The left hand side vertical values indicate signal intensity (arbitrary numbers). 5 The right hand side is the temperatures in OC (Celsius). The bottom horizontal scale represents the time in hours. Figure 11 illustrates a data table that contains the values of the parameter signals along with the time and temperature for a typical sample. [0076] At block 214, the software of computer 85 automatically 10 calculates the TSD based upon a pre-set value of the photometric signal above the baseline signal value defined by the analyst. At block 216, a built in pre-defined linear correlation curve equation is used to calculate the initial population (expressed in Colony Forming Unit (CFU) in a given sample volume) of the microbial parameter under investigation. To obtain this linear 15 correlation curve equation, a series of split samples of varying initial microbial population (for example E.coli) are run both using the method of the present invention and a standard method (Membrane Filtration). The pre-defined linear correlation curve equation (LCCE) is generated by plotting TSD values obtained from the present invention and the corresponding initial population 20 values (Xo) from the membrane filtration method. A sample linear correlation curve is shown in Figure 12. [0077] At block 218, the initial population values are displayed on a computer screen such as that shown in Figure 13. [0078] The method of the present invention accordingly provides a 25 continuous, non-intrusive monitoring and recording of one or more detectable parameters as the incubation process progresses. A significant deviation of the output signal is an indication of the presence of the detectable parameter, while the time taken to reach the significant deviation provides a quantitative analysis of the parameter. 30 [0079] Referring now to Figures 14a and 14b, illustrated therein are the heating and temperature control and data collection algorithms of controller 92 WO 2006/116835 PCT/CA2005/000686 -19 and 94. Pressing the power switch 13 at the command block 300 initiates the controller 92 program. [0080] At command block 305 the controllers 92 and 94 perform a quality check to verify the incubation and detection systems and components 5 are functioning properly. [0081] At command block 310 if the logic is "Yes" the controllers 92 and 94 will proceed to block 320. If the logic is "No" the controllers will initiate the appropriate LEDs to signal "Failed Unit". [0082] At command block 320 the controller 92, through heating 10 element 24, heats the sample 11 in container 14 and at a pre-determined interval monitors the temperature of the sample 11 through temperature sensor 25. [0083] If the logic at command block 330 is "No" then the controller 92 continues heating and monitoring the temperature of sample 11 in container 15 14. [0084] If the logic at command block 330 is "Yes" then the temperature of the sample 11 has reached a pre-determined temperature value as measured by the temperature sensor 25. The controller 92 will then set the test time zero and continue monitoring the temperature of the sample 11 in 20 container 14. The controller 92 also starts monitoring the time. [0085] If the logic in command block 350 is "No", the controller 95 will continue heating and monitoring the temperature of sample 11. [0086] If the logic in command block 350 is "Yes", the controller 92 moves to block 355 and stops heating sample 11 in container 14 and moves 25 to command block 365. [0087] At command block 365 controller 92 starts collecting temperature and time data while controller 94 starts collecting the signal data. Controller 92 also initiates the temperature control Loop 1 to maintain the incubation temperature at the pre-set range. If the logic at block 400 within WO 2006/116835 PCT/CA2005/000686 -20 Loop 1 is "Yes" then the controller falls back to block 355 and continues the loop. [0088] If the logic at block 400 is "No" then the controller will go to command block 410 and initiate heating of sample 11 in container 14. Loop 2 5 will continue until the logic at block 400 becomes "Yes" and falls back to Loop 1. [0089] Irrespective of the logic loops 1 and 2, both controllers 92 and 94 will collect their respective data at a pre-set time interval and store the data in the processor memory. 10 [0090] At block 370 the status LEDs 97 are updated to indicate the test in progress. [0091] If the logic at command block 375 is "No" the controllers 92 and 94 will go back to command block 365 and continue collecting data, thus initiating a data collection loop - Loop 3. If the logic at block 375 is "Yes" then 15 the test is deemed completed and the controllers 92 and 94 stop monitoring and collecting data and shut down both incubation and detection systems and go into idle (stand by) mode at command block 385 and await further instruction from the user or analyst. [0092] The methods and apparatus of the subject invention provide a 20 number of advantages over standard membrane filtration methods. The subject methods and apparatus provide for a rapid but simple, reliable and accurate onsite testing of microbiological material in various types of liquid samples, including drinking water and recreational water. Other advantages include less interference from turbidity, no need for dilution because of large 25 dynamic analytical range, simplified operation through total automation, and build in quality control (QC) providing auto QC for every test. [0093] It should be appreciated that various modifications can be made to the embodiments of the methods and apparatus described herein. While the spectrophotometer system of the preferred embodiment comprises three 30 spectrophotometers, it should be understood that the apparatus could WO 2006/116835 PCT/CA2005/000686 -21 comprise a different number of spectrophotometers. As well, the spatial configuration of the source-detectors could be altered significantly without departing from the present invention. Also, each spectrophotometer can be configured for detecting different test parameters and can be operated 5 independently or simultaneously. [0094] It should also be appreciated that the light emitting sources are not limited to LEDs (as they could be lasers, or laser diodes), and the detectors are not limited to phototransistors, (as they could be photodiodes, photoresistors, CCDs, etc.) 10 [0095] Furthermore, the method of the present invention is not limited to the detectable parameters of the preferred embodiment, as the present method could be used to detect light emission resulting from bioluminescence or chemiluminescence processes resulting from a biological or chemical component in reagent 20 within the sample container 14. This would allow 15 the method and apparatus of the present invention to be used for toxicity studies using bioluminescence bacteria. [0096] Also, the light emitting source and the detector of any or all of the spectrophotometers could be placed outside of the incubation-detection chamber 65 but within apparatus 10 and used to monitor the signal growth 20 through fiber optics placed strategically within the chamber 65. [0097] Accordingly, various modifications can be made to the embodiments of the invention described and illustrated herein without departing from the present invention, the scope of which is defined in the appended claims.

Claims (28)

1. A system for rapid analysis of microbiological materials in a liquid sample, comprising of: (a) a specimen container for containing a liquid sample, the 5 specimen container being made from a material that allows for propagation of light; (b) a housing having an enclosable chamber shaped for holding the specimen container; (c) an incubating system mounted within the housing for 10 incubating any microbiological materials within the liquid sample; and (d) a spectrophotometer system mounted within the housing for propagating light within the specimen container and measuring the light absorbed, emitted or scattered by the 15 liquid sample as the microbiological materials are incubated by the incubating system over time.
2. The system defined in claim 1, wherein the spectrophotometer system comprises at least one spectrophotometer, the spectrophotometer comprising a light emitting source positioned to propagate light within 20 the specimen container, a detector positioned for detecting changes in amount of the light as the light propagates through the microbiological materials and for producing a detector signal indicative of the changes, and a spectrophotometer controller for controlling the light emitting source and the detector. 25
3. The system defined in claim 2, wherein the spectrophotometer controller comprises a microprocessor for processing the detector signal and for generating a record of the changes in the light as a function of time. WO 2006/116835 PCT/CA2005/000686 -23
4. The system defined in claim 2, wherein the light emitting source propagates light having a known intensity along a first optical path within the specimen container, and the detector detects the changes in the intensity of the light within a field of view related to the first optical 5 path.
5. The system defined in claim 3, wherein the field of view is positioned at a 1800 angle to the first optical path.
6. The system defined in claim 3, wherein the field of view is positioned at a 900 angle to the first optical path. 10
7. The apparatus defined in claim 2, wherein the light emitting source comprises a light emitting diode, and the detector comprises a phototransistor.
8. The system defined in claim 1, wherein the spectrophotometer system comprises a first spectrophotometer and a second spectrophotometer, 15 wherein the first spectrophotometer comprises a first light emitting source for propagating a first beam of light within the specimen container and a first detector for detecting changes in the first light beam, and the second spectrophotometer comprises a second light emitting source for propagating a second beam of light within the 20 specimen container, and a second detector for detecting changes in the second beam of light.
9. The system defined in claim 8, wherein the spectrophotometer system comprises a third spectrophotometer, the third spectrophotometer comprising a third light emitting source for propagating a third beam of 25 light within the specimen container and a third detector for detecting changes in the third beam of light.
10. The system defined in claim 2, wherein the housing comprises an upwardly extending, generally cylindrical container holder having an WO 2006/116835 PCT/CA2005/000686 -24 open top, and a removable cap for enclosing the housing, the removable cap being shaped to surround the container holder.
11. The system defined in claim 10, wherein the light emitting source and the detector are mounted in the container holder at a location 5 proximate to the specimen container when the specimen container is placed in the housing.
12. The system defined in claim 10, wherein the container holder comprises an inwardly extending indent, and the specimen container comprises a side recess shaped to register with the indent when the 10 specimen container is placed in the container holder.
13. The system defined in claim 10, wherein the container holder comprises a pair of diametrically opposed indents, and the specimen container comprises a pair of diametrically opposed recesses shaped to register with the indents. 15
14. The system defined in claim 1, wherein the incubating system comprises a heating element, a temperature sensor, and a heating controller responsive to the temperature sensor for controlling the heating element.
15. The system defined in claim 14, wherein the temperature sensor 20 extends upwardly into the chamber, and the specimen container has a bottom cavity shaped to accommodate the temperature sensor.
16. The system defined in claim 14, wherein the heating element extends upwardly into the chamber, and the specimen container has a bottom cavity shaped to accommodate the heating element. 25
17. Apparatus for incubating and detecting microbiological materials in a liquid sample, comprising: WO 2006/116835 PCT/CA2005/000686 -25 (a) a housing having an enclosable chamber shaped for holding a clear plastic specimen container for containing a liquid sample; (b) incubating apparatus mounted within the housing for 5 incubating any microbiological materials within the liquid sample, and (c) spectrophotometer apparatus mounted within the housing for propagating light within the specimen container and measuring light absorbed, emitted or scattered by the 10 liquid sample as the microbiological materials are incubated by the incubation apparatus over time.
18. The apparatus defined in claim 17, wherein the spectrophotometer apparatus comprises a light emitting source for propagating a beam of light of a known intensity along a first optical path within the specimen 15 container, and a detector for detecting changes in the intensity of the light along a second optical path related to the first optical path.
19. The apparatus defined in claim 18, wherein the housing comprises a base, a container holder mounted on the base for holding the specimen container, the container holder having an open top, and a removable 20 cap shaped to enclose the top of the sample holder.
20. The apparatus defined in claim 19, wherein the incubating apparatus comprises a heating element, a temperature sensor, and a heating and temperature controller coupled to the temperature sensor for controlling the heating element. 25
21. A method for the rapid analysis of microbiological materials in a liquid sample, comprising the steps of: (a) mixing a liquid sample having an unknown initial population of a microbiological material with a reagent in WO 2006/116835 PCT/CA2005/000686 -26 a specimen container, the reagent providing a detectable parameter indicative of the microbiological material, thereby creating a sample/reagent mixture; (b) placing the specimen container in an enclosable housing 5 and enclosing the housing; (c) incubating the sample/reagent mixture in the enclosed housing at a temperature within a pre-selected temperature range over a period of time; and (d) measuring changes in the detectable parameter as the 10 sample/reagent mixture is being incubated during the period of time.
22. The method of claim 21, further comprising the steps of recording the changes in the detectable parameter as a function of time.
23. The method defined in claim 21, wherein the step of measuring 15 changes in the detectable parameter comprises propagating light within the sample/reagent mixture in the specimen container and detecting changes in the light.
24. The method defined in claim 22, further comprising the steps of: (a) recording a time of significant deviation at which there 20 occurs an exponential change in the detectable parameter; and (b) determining the initial population by correlating the time of significant deviation with known times of exponential growth for known initial concentrations of the 25 microbiological material.
25. The method defined in claim 21, wherein the pre-selected temperature range is ±1 0 C. WO 2006/116835 PCT/CA2005/000686 -27
26. The method defined in claim 21, wherein the detectable parameter is selected from a group comprising colour, fluorescence and turbidity.
27. The method defined in claim 21, wherein the detectable parameter comprises bioluminescence or chemiluminescence resulting from a 5 biological or chemical component in the reagent.
28. A method for the rapid quantitative analysis of microbiological materials in a liquid sample, comprising the steps of: (a) placing a liquid sample having an unknown initial population of a microbiological material in a specimen 10 container made of a material that allows for the propagation of light; (b) creating a sample/reagent mixture by mixing the liquid sample with a reagent that provides a detectable parameter indicative of the microbiological material upon 15 exposure to light; (c) incubating the sample/reagent mixture in an enclosed housing at a temperature within a pre-selected temperature range over a period of time; (d) measuring changes in the detectable parameter as the 20 sample/reagent mixture is being incubated over the period of time by propagating light within the sample/reagent mixture in the specimen container and detecting changes in the intensity of the light; (e) recording the changes in the intensity of the light as a 25 function of time; WO 2006/116835 PCT/CA2005/000686 -28 (f) recording a time of significant deviation at which there occurs an exponential change in the intensity of the light; and (g) determining the initial population by correlating the time of 5 significant deviation with known times of exponential growth for known initial concentrations of microbiological materials.
AU2005331515A 2005-05-05 2005-05-05 System for rapid analysis of microbiological materials in liquid samples Abandoned AU2005331515A1 (en)

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