EA012956B1 - System for rapid analysis of microbiological materials in liquid samples - Google Patents

Abstract

A system for the rapid analysis of microbiological parameters includes a specimen container for containing a liquid sample, a housing having an enclosable chamber shaped for receiving the specimen container, an incubating system mounted within the housing for incubating microbiological materials within the liquid sample, and a spectrophotometer system mounted within the housing for measuring light absorbed, emitted or scattered by the liquid samples as the microbiological materials are incubated by the incubating system over time. The specimen container is filled with a liquid sample to be tested and mixed with a reagent that provides a detectable parameter, and placed inside the apparatus.; The incubation system heats and maintains the temperature of the liquid sample within a preset range while the spectrophotometer system propagates light within the specimen container, and monitors and records changes in the light as the light propagates through the container. A continuous non-intrusive monitoring and recording of the test parameter is achieved as the incubation progresses. Any significant deviation of the signal output is an indication of presence of the detectable parameter, while the time taken to reach the significant deviation provides quantification of the microbiological parameter under investigation.

Description

The present invention relates to methods and apparatus for detecting the presence and quantity of microbiological materials in liquid samples and, in particular, to methods and device for quantitative analysis of pathogenic microorganisms in water samples.

The level of technology

Drinking water and recreational water (water on beaches and other swimming facilities) should be regularly checked, and the results of these checks should be available for a short time to protect the public from dangerous and contagious diseases.

Currently, most of the water sample checks are performed under laboratory conditions away from water facilities or sampling sites. Many of the methods and procedures currently used in conventional microbiological analyzes have been developed more than 100 years ago. These procedures are time consuming and require considerable time both during execution and during data collection. The results of such tests usually remain inaccessible to water operators for approximately 36-72 hours. As a result, operators often cannot take action to clean contaminated water for a long time after the start of consumption or use of such polluted water.

In addition, waterborne diseases remain a major problem, despite attempts to solve it all over the world. This problem is not limited to developing and underdeveloped countries, but is global in nature. Some of its main causes are the following:

1) the current frequency of inspections is not sufficient to provide early warning, therefore, corrective actions cannot be taken to prevent outbreaks of diseases; and (2) currently used testing methods are laborious and time consuming and, therefore, do not contribute to the organization of frequent inspections.

In 1988, the authors are Eagegd 8.S. et al. developed a new technology that is based on the method of a chemically defined substrate MTE, known as Li1oaa1u818 Soshekk (AC): JU1a1 sokGogp apy Eksyaysa soy Ggot yppkshd teakeg: sootkopk \ νί11і zkapyagy tShr1e kye kes11tsIe, Arr1. ETZGOP. Mugoyuk, 54 1595 (1988). This made it possible to simultaneously detect and identify both the total number of coliforms and E.Sok in water at 1 SEU per 100 ml for less than 24 hours. CoSegk® is a medium containing a chromogenic / fluorogenic reagent that contains specific nutrient substances and enzyme substrates with chromophores and fluorophores for simultaneous detection of the total number of coliforms and E. coli In 1989, I8 EPA (US Environmental Protection Agency) approved this method as a means of qualitative testing the total number of coliforms in drinking water.

The development of chromogenic / fluorogenic reagents for microbial testing has opened up the possibility to improve the quality and speed up the testing protocols. In addition, these products provide an additional opportunity to use technology such as optical spectroscopy for biological, microbiological and chemical analysis. Spectrophotometric analysis is very sensitive and, therefore, allows the presence of a very low concentration of color-forming components of interest to be detected in liquid samples (in parts per million). Visually, the human eye can detect color only when its components are present at a fairly high concentration, which leads to the need for an incubation period ranging from 18 to 72 hours. The time required to identify or evaluate the presence of microbiological indicators in water, food and environmental samples can be significantly reduced by combining incubation with photometric analysis.

The use and advantage of spectrophotometric approaches to bacteriological analysis in liquid samples is described in the literature. However, these tests were performed outside the incubation chamber by taking an aliquot of the sample from the incubation tank into the photometric tubes at different intervals and taking measurements using standard spectrometers. This approach requires not only a long time, but it also requires the use of separate incubators, spectrometers and technicians for the test and, in some cases, robotic sampling systems. It is also possible the risk of mutual pollution and human error, if special measures are not taken in the analysis.

In line with this, there is an urgent need for improved methods and devices for testing microbiological materials in drinking water, recreational water, and also in wastewater to enable better management of water facilities, to protect public health and the environment.

Summary of Invention

The present invention relates to a system for conducting a quick quantification.

- 1 012956 Varian analysis of bacteria in fluid samples, such as water. In one aspect, the present invention is directed to a system comprising a sample container, which contains a test sample, and a device having a spectrophotometer system containing an appropriate light emitting source and a detector mounted adjacent to the sample container within the body of the device. A reagent that provides a detectable parameter (for example, color, fluorescence, etc.) is added to the sample to be tested in a sample container. While incubation occurs in the sample, the detector tracks light from a source passing through the sample and sample container. The detector is connected to the spectrophotometer processor, which measures, processes, records and stores information. A processor may also be connected to an appropriate measurement and recording device, such as a computer, multimeter, or any other device that allows you to measure and record the output signals of the detector. This provides non-intrusive continuous incubation and measurement of the growth of the signal of the parameter under study.

In another aspect, the present invention is directed to a system for the rapid analysis of microbiological parameters in fluid samples. This system contains a sample container for holding a sample of a liquid, this sample container is made of a material that allows light to propagate, a housing forming a closed chamber for containing a sample container, an incubation system installed inside the housing for incubation microbiological materials in the fluid sample and the spectrophotometer system installed in the housing to pass light through the sample container and measure the amount of absorbed, and ray or the scattered light liquid sample when an incubation of microbiological materials in the incubation system over time.

In an additional aspect, the present invention is directed to a device for detecting microbiological materials in a fluid sample. The device comprises a housing having a closable chamber of such a shape that it contains a transparent plastic container that is designed to hold a sample of liquid, an incubation device installed inside the case, intended to incubate any microbiological materials in a liquid sample, and a spectrophotometer device installed inside the case, intended for measuring the light absorbed, emitted or scattered by a liquid sample as the microbiological materials of the devices are incubated ohm incubation over time.

The present invention is also directed to a method for the rapid analysis of microbiological materials in a fluid sample, comprising the steps of:

(a) mix a sample of liquid having an unknown initial population of microbiological material with the reagent in the sample container, the reagent providing a detectable parameter indicating the microbiological material, thereby creating a sample / reagent mixture;

(b) place the sample container inside the enclosure to be closed, and close the case;

(c) incubating the sample / reagent mixture inside the closed case at a temperature within a predetermined temperature range for a certain period of time; and (b) measuring the change in the detected parameter as the sample / reagent mixture is incubated for a certain period of time.

The present invention is further directed to a method for rapid quantitative analysis of microbiological materials in a fluid sample. The method comprises the steps of:

(a) placing a sample of a fluid having an unknown initial population of microbiological material in a sample container made of a material that allows light to pass through it;

(b) create a sample / reagent mixture by mixing the liquid sample with the reagent, resulting in a detectable parameter showing the microbiological material after exposure to light;

(c) incubating the sample / reagent mixture in a closed case at a temperature within a predetermined temperature range for a period of time;

(b) measure changes in the detected parameter as the sample / reagent mixture is incubated with light passing through a known intensity through the sample / reagent mixture and measuring changes in the light intensity over time;

(e) recording changes in light intensity as a function of time;

(1) record the time of significant deviation at which an exponential change occurs in the detected parameter; and (e) determine the initial population by correlating the time of significant deviation with the known time of the exponential growth of the detected parameter for the known initial populations of microbiological material.

Brief Description of the Drawings

The invention will be described below only as an example with reference to the following drawings, in which:

in fig. 1 is a diagram of a system in accordance with the present invention;

in fig. 2 is a perspective view of a device configured in accordance with the preferred

- 2 012956 embodiment of the subject matter of the invention;

in fig. 3 is a perspective view with an exploded view of the details of the device in accordance with the subject matter of the invention, which shows the removed cap and sample container removed from the main module;

in fig. 4 shows a view of a device in accordance with the subject matter of the invention in section along the line

4-4, indicated in FIG. 2;

in fig. 5 shows a view of a device in accordance with the subject matter of the invention in section along the line

5-5, indicated in FIG. four;

in fig. 6 shows a top view of a device in accordance with the subject matter of the invention in section along line 6-6, indicated in FIG. four;

in fig. 7 shows a front view in section of a device made in accordance with an alternative embodiment of the present invention;

in fig. 8 is a flow chart illustrating a method in accordance with the subject matter of the invention;

in fig. 9 is a graph illustrating a typical growth curve over time;

in fig. 10 shows a graph of data showing approximate growth curves for two different microbiological parameters;

in fig. 11 shows a data table representing the results generated by the method of the present invention;

in fig. 12 shows an exemplary linear correlation curve used in the method of operation of the device that corresponds to the subject matter of the invention;

in fig. 13 shows a test report generated by performing a method that is in accordance with the subject matter of the invention;

in fig. 14a is a flowchart illustrating a heating control algorithm in accordance with the present invention; and in FIG. 14b is a flowchart illustrating an algorithm for temperature control and data acquisition in accordance with the present invention.

The implementation of the invention

FIG. 1 shows a system for express analysis of microbiological parameters using non-intrusive incubation and detection in a reservoir made in accordance with the subject invention. The system 10 contains an incubator detector device 12, a sample container 14 for holding the sample 11 of a liquid mixed with reagent 20, and an external data recorder 80. The incubator-detector device 12 comprises a housing 15 having a detection chamber 65 of such a shape that it can accommodate a sample container 14, an incubation system 60 installed inside the housing 15 for incubating microbiological materials in a liquid sample 11 and a spectrophotometer system 62 installed in housing 15. A spectrophotometer system 62 measures the amount of light absorbed, emitted or scattered by sample 11 of a liquid in a sample container 14 as microbiological materials are incubated in a 60 in system. cubations.

The incubation system 60 includes a heating controller 92, and the spectrophotometer system 62 includes a spectrophotometer controller 94. The power source 90 provides power for the incubation system 60 and the spectrophotometer system 62. The external data recorder 80 preferably comprises a computer 85, having a microprocessor 86 and a memory device 88, as well as an output device, such as a printer 82, which is connected to the computer 85.

FIG. 2-6 show a preferred embodiment of the device 12 of the incubator detector. The housing 15 of the device 12 is made, in General, in the form of a cylindrical casing containing the base 16, the container holder 18, having such a shape that it holds the sample container 14, and a removable cap 50. The base 16 includes a continuing vertical cylindrical bead 55 , having the form in which the cap 50 is installed. In the base 16, a power supply 90, a heating controller 92 and a spectrophotometer controller 94 are installed. The power source 90 may be any suitable power source known in the art, such as a rechargeable battery installed inside the base 16, having an output connector 99 for connecting to an external 120 or 220 V alternating power source or to a constant power source voltage. On the outer surface of the base 16 has a power switch 13, indicator LEDs 97 and data port 98.

The container holder 18 comprises a base 21 and a cylindrical wall 19 with an open end extending upwardly from the base 21. Wall 19 is shaped in such a way that it surrounds the bottom portion of sample container 14 when sample container 14 is placed inside housing 15. Wall 19 includes a pair of inward, diametrically opposed, generally rectangular recesses 23.

Removable cap 50 has such a shape that ensures its tight fit around the wall 19 of the holder 18 of the container. The cap 50 preferably contains a heat-efficient, cylindrical shell with double walls, having an outer wall 51, an inner wall 59, a closed top

- 3 012956 end 52 and an open bottom flange 53. An edge 66 is provided on the outer surface of the lower flange 53, which is shaped so that it is screwed into the groove 67 on the inner surface of the flange 55 of the base 16. The inner surface of the inner wall 59 includes a protrusion 68 having such a shape that it is sealed to the ring 49 extending around the base 21 of the holder 18 of the container. If necessary, a vacuum may be formed in the space between the walls 51 and 59 of the cap 50 or it may be filled with an inert gas.

When the cap 50 is mounted on the container holder 18, the cap 50 and the holder 18 of the container form a very efficient heat-insulated incubation detection chamber 65. The inner surface of the wall 19 of the sample holder 18 is preferably blackened so that the camera 65 forms an effective black box (dark room) for optical detection and measurement.

The incubation system 60 of the device 12 comprises a heating element 24, a temperature sensor 25 and an incubator controller 92. The heating element 24 is installed inside the heating pin 57, continuing vertically upward through the hole in the base 21 of the holder 18 of the container. The temperature sensor 25 is installed inside the pin 56 for measuring temperature, which extends upward from the base 21 of the holder 18 of the container. The temperature sensor 25 may include a thermistor 26 located near the top of the pin 56.

The heating controller 92 controls the heating of the heating element 24 and monitors the temperature of the liquid sample 11 using the temperature sensor 25. After the temperature reaches the optimum value, the heating controller 92 maintains the temperature of the sample 11 of the liquid inside. The heating controller 92 preferably includes a timer (not shown) designed to measure the incubation time from its start and to turn off the heating at the end of a predetermined time.

The sample container 14 contains a sample cap 42 and a sample flask 44. Sample flask 44 is generally cylindrical with a lower cavity 45 for installing a heating pin 57 into it and a lower cavity 46 for installing a temperature sensor pin 56 into it. The sample flask 44 also has a pair of diametrically opposed, generally rectangular side cutouts 48, which are shaped so that the grooves 23 of the wall 19 are installed when the sample container 14 is installed inside the container holder 18. Sample flask 44 is made of a material that allows light signals to propagate, and is preferably made of transparent plastic or other material that is optically transparent.

A spectrophotometer system 62 is a system for measuring the light absorbed, emitted, or scattered by a sample fluid, as microbiological materials are incubated over time. As best shown in FIG. 4, the spectrophotometer system 62 preferably comprises three spectrophotometers, a first spectrophotometer containing a light emitting source 30a and a detector 35a, a second spectrophotometer containing a light emitting source 30B and a detector 35b, and a third spectrophotometer containing a light emitting source 30c and the detector 35c. From light emitting sources 30a-c, the rays of light propagate with a given intensity along selected optical paths inside the sample container 14. The light detectors 35a-c are positioned so that they detect a change in the intensity of light rays within a selected field of view associated with optical paths obtained from light that is absorbed, emitted or scattered by fluid sample 11 as microbiological materials are incubated over time. .

Each of the light sources 30a-c preferably contains a light-emitting diode (LED) with a specific maximum wavelength, and each of the detectors 35a-c preferably contains a phototransistor detector. Light sources 30a-c and detectors 35a-c are mounted on printed circuit boards 33a-c, which are electrically connected to the spectrophotometer controller 94.

The light emitting sources 30a, 30b continue through the openings 70a and 70b at the base of the container holder 18.

The light emitting sources 30a, 30b are installed in such a way that the light emitted by the sources propagates through the openings 70a and 70b, respectively, and up along the selected optical path inside the sample container 14. In the case of a light emitting source 30a, 30b, the optical paths are shown by arrow 1 and arrow 4, respectively. The container holder 18 also has openings 75a, 75b for the signal detectors 35a, 35b. The detectors 35a, 35b are located at an angle of 90 ° relative to the light emitting sources 30a, 30b in such a way that the detector window is facing the flask 44 for a sample to receive any signal propagating in the direction of the detectors 35a, 35b, and have fields of view shown by arrow 2 and arrow 5, respectively.

The container holder 18 also includes corresponding apertures 70c and 75c for the signal emitting source 30c and the signal detector 35c, respectively. The light emitted by the light source 30c passes through the opening 70c along the horizontal optical path with the direction of propagation shown by arrow 3 through the sample container 14 and the opening 75c to the detector 35c. The detector 35c is set so that its field of view is at an angle of 180 ° relative to the optical path of the source 30c of light.

- 4 012956

The spectrophotometer controller 94 controls the operation of the spectrophotometer system. The spectrophotometer control controller 94 activates and deactivates the signal emitting sources 30a-c and can ensure their pulse operation. Spectrophotometer controller 94 also measures and processes the output signals generated by the detectors 35a, 35b and 35c. The spectrophotometer controller 94 includes a microprocessor 95, which has a built-in clock, which performs the function of a data recorder and saves the signal values measured by the detector along with the corresponding temperature of the sample 11 liquid and the measurement time at a specific location in the memory of the microprocessor 95. The spectrophotometer controller 94 can indicate the end test, including one or more LEDs 97 or an audible alarm device (not shown). The spectrophotometer controller 94 also communicates via a data transfer port 98 with an external data recorder 80, such as a computer 85, a multimeter, or another external signal handler.

The microprocessor 95 of the spectrophotometer controller 94 includes a memory device for storing software that embodies the method of testing the subject matter of the invention. The testing method determines the specifications and conditions required for the testing process. Through the use of specialized software, this method can be programmed and loaded into the memory of microprocessor 95 via data port 98. The software also allows erasing all areas of the storage device of the controller 94.

To use the device 12 in accordance with the present invention for testing liquid samples, a liquid sample 11 is placed in a sample container 14. The liquid sample 11 is not limited to water and may contain other liquids or other liquid media containing suspensions, such as food particles, filter paper and other solids. The corresponding reagent 20 is then added to the liquid sample 11 inside the sample container 14. Reagent 20 may be chemical or biological in nature and may provide a detectable parameter, such as color, fluorescence, turbidity, etc., which indicates the presence or absence of the microbiological material under investigation.

The detection of a color, a fluorescent signal or a signal representing the degree of turbidity depends on time, and the detection time is linked to the number of bacteria present at the beginning of the test. Thus, it is possible to obtain quantitative indicators of the detected microbiological parameters, such as the total number of coliforms and Ε.οοίί in the water sample, by measuring the signal obtained as a result of a color change, or a fluorescent signal and the time during which they are detected in appreciable quantities.

The detected color or the detected fluorescent signal is measured using a device 12 spectrophotometer system 62. In a preferred embodiment, the spectrophotometer system 62 contains three spectrophotometers that provide colorimetric detection to determine the total coliform, fluorescence detection for the Ε.εοίί and cloudiness associated with microbiological growth , for nephelometry, respectively.

The microprocessor 95 built-in clock of the spectrophotometer controller 94 provides growth time, while the constant temperature of the incubation system 60 provides both the reproducibility of microbial growth and optical reproducibility.

In a preferred embodiment of the system 10 in accordance with the present invention, the spectrophotometer system 62 comprises three spectrophotometers measuring growth time with a single incubator with a constant temperature, i.e. spectrophotometers that record the growth of certain microbiological parameters as a function of time. The type of spectrophotometric analysis performed by each spectrophotometer depends on the configuration and specification of the source and detector of the spectrophotometer. The 180 ° configuration of the source and detector pair 30c, 35c and colorimetric analysis of the degree of turbidity, while the configuration of the source detector’s 90 ° pair 30a, 35a and 30b, 35b provides either fluorometric or nephelometric analysis.

The source 30c of the colorimetric spectrophotometer preferably comprises an LED with a maximum wavelength of 620 nm, and the detector 35c is preferably a phototransistor detector having a signal response located in the visible light region including 620 nm. The source 30a of the fluorometric spectrophotometer is preferably an ultraviolet LED with a maximum wavelength of 380 nm, and the detector 35a, located generally at an angle of 90 ° to the source 30b, is preferably a phototransistor detector having a signal response in the visible range that includes range of 400-500 nm. The configuration of the nephelometer is similar to that of the fluorometer, except that the source 30b is preferably an LED with a maximum wavelength of 400 nm.

After aseptic reagent 20 is added to the sample, sample cap 42 is fixed to sample flask 44, and sample container 14 is gently shaken to dissolve the reagent and form the sample / reagent mixture. To simultaneously test the total number of coliforms and .οοίί in samples

- 5 012956 water, the usual reagents provide not only the optimal growth medium, but also a color change in accordance with the total number of coliforms and the fluorescence signal for E. coli, if they are present in any quantity in the sample. Examples of typical chromogenic / fluorogenic reagents are

Megsk KSaA - Weiussi®

HOEHH-SUSHEY®

The sample container 14 is then placed inside the container holder 18, as best shown in FIG. 2. After the removable cap 50 is placed on the base 16, the detection incubation chamber 65 provides black box conditions (dark room), ideal for microbial growth and spectrophotometric detection.

After pressing the start button 13 on the base 16 of the device 12, the incubation cycle and the detection process are activated. If necessary, you can use a separate activation button to activate the detection process at a specified time after the start of incubation.

Activating the detection process may include turning on the power of the signal emitting sources 30a-c and the detectors 35a-c, turning on the pulsed emission signal and tracking the output signal of the detectors 35a-c.

The controller 92 heating brings and maintains the temperature of the sample 11 of the liquid at a constant value of temperature within a predetermined temperature range. To test coliforms and E. coli in water, it is preferable to maintain a temperature of 36 ± 1 ° C. However, the temperature depends on the reagent used and may vary from one reagent to another. The temperature also depends on the specification of the test method. For example, E. coli can be tested either at 36 ° C or at 41 ° C using the same reagent.

The spectrophotometer controller 94 continuously monitors, records and stores the output signals of the 35a-c detectors. In a method in accordance with a preferred embodiment, the spectrophotometer controller 94 also records and maintains time and temperature for each output signal. Controller 94 can be connected to an external data recorder 80, which is programmed to record the signal either continuously or at specified intervals. The external data recorder 80 can also record the measurement time of each measured signal and the corresponding sample temperature and can generate a graph of growth signal versus time (GSTD, ΤΏΟ8Ρ) for the microbiological parameters under study.

Colorimetric ΤΏΟ8Ρ total number of coliforms and fluorometric ф8Ρ E.coH are preferably simultaneously recorded together with nephelometric ометри8Ρ degree of increased turbidity due to the growth of bacteria in water.

A significant deviation of the output signal from the original baseline is an indicator of the presence of the parameter under study, while the time required to achieve a significant deviation from the initial position provides an indicator of the initial amount of the parameter being tested.

The microprocessor clock 95 represents the date and time of the test, the time of each measurement signal and the corresponding temperature, the time at which the test was completed or terminated. The end of the test can be indicated by the state of the LEDs 97 and / or by audible signals or it can be monitored using a software application.

FIG. 7 is a schematic representation of a device 112 manufactured in accordance with an alternative embodiment of the present invention. The device 112 is generally similar to the device 12 in accordance with the preferred embodiment, as shown in FIG. 2-6, with the exception of a few modifications.

The device 112 comprises a sample container 114 and a housing 115 comprising a base 116, a cylindrical container holder 118 and a removable cap 150. The container holder 118 has a cylindrical base 155 and a cylindrical wall 160 with an open end. The cylindrical base 155 of the container holder has a temperature controller pin 156, extending upward, for mounting the temperature controller 180 therein. The temperature controller 180 may be a bimetallic switch or any other suitable device that can activate and deactivate the heating element.

The heating element 124 is installed inside the cylindrical wall 160 with an open end of the sample holder 150. As shown in the drawing, the heating element 124 contains a resistive wire 125. Alternatively, the heating element 124 may be a resistive coil, resistive foil, etc. The length of the resistive wire is determined by the temperature of the resistor and the resistance in ohms per feet of wire 125

The sample container 114 contains a sample cap 142 and a sample flask 144. The sample flask is made generally cylindrical with a lower cavity 145, the shape of which allows the temperature controller pin 156 to be inserted into it.

The heating controller 192 maintains a constant predetermined temperature range in the sample. AT

- 6 012956 if necessary, it may contain a timer (not shown) for measuring the incubation time from its start to the shutdown of heating at the end of a specified time.

The spectrophotometer system of device 112 is similar to that of device 12, in accordance with the preferred embodiment. The light emitting source 130a emits light in the direction of arrow 1, and the detector 135a detects emitted or diffused light propagating in the direction of arrow 2. The emitting light source 130b emits light in the direction of arrow 4, and the detector 135b detects emitted or diffused light propagated in the direction of arrow

5. The light emitting source 130c emits light in the direction of arrow 3, and the detector 135 detects light propagating in the direction of arrow 3. The spectrophotometer system also includes a spectrophotometer controller 194 designed to control the operation of the light sources 130a-c and the detectors 135a-c, as well as sources of 190 power.

FIG. 8-13 illustrate a preferred embodiment of a quantitative analysis method in accordance with the present invention.

The method of quantitative analysis in accordance with the present invention is based on the understanding that there is an interdependence between the original population and the population obtained over time as a result of growth. The time interval between the start of the test (the initial population) and the population with fixed growth is a function of the initial population, incubation temperature and growth medium. Thus, if the incubation temperature and the growth medium are kept constant, the time required to reach a population with a fixed height is a direct function of the original population.

A chromogenic / fluorogenic reagent, such as Keabusi® (Megsk KdAL) or SyoChey® (ΙΌΕΧΧ) provides a mechanism by which the present invention can monitor and check and measure the population resulting from the growth of microbes (total number of coliforms and E. soy ) using the photometric detection process. The specific amount of enzymes produced by these organisms (for example, galactosidase (total number of coliforms) and glucuronidase (specific for E. soy)) will metabolize the nutrient indicator and release the chromophore or fluorophore into a liquid medium. The concentration of chromophores or fluorophores at a given time in the detection environment is proportional to the population growth over time, and therefore the change in signal intensity resulting from an increase in the concentration of color components is a measure of population growth as a function of time.

The time (1 _pp ) of the detection of a population, which is defined as the time required to reach a detectable population size, is used to estimate the growth parameters of the bacteria. It was determined that the detection time is inversely proportional to the logarithm of the inoculum level (the initial microbial population).

TSRR “1 / 1ODHO {1} where X ₀ = initial population of bacteria.

The time of significant deviation (GUS, Τ8Ό) is the time at which the value of the measured photometric signal above the baseline signal becomes statistically significant. Τ8Ό also depends on the initial concentration of bacteria in the sample and the higher the initial count of bacteria, the shorter the Τ8Ό. Since an increase in population size can be measured using an increase in the output signal, the time required to obtain a significant deviation (Τ8Ό) of the output signal from the baseline should correspond to 1 _ppm if it is measured during the growth phase.

The method is ⁼ Τ5ϋ {2} and therefore

Τ3ϋ wasp 1 / 1od ho 13}

Such an equation of the linear correlation curve (CCLC, HCSE) between Τ8 Ό and the initial population of the microbes studied (X0) provides, in addition to the presence detection, quantitative information about the bacterial population.

FIG. 8 is a flow chart describing the steps of the method of the invention. At step 200, reagent 20 is mixed with liquid sample 11, which has an unknown initial population of microbiological material in the sample container 14, thereby creating a sample / reagent mixture. Reagent 20 provides a detectable parameter, such as color or fluorescence, which is an indicator of microbiological material. At step 202, the sample container 14 is placed inside the housing 15, and the cap 50 is placed on the housing 15. In step 204, the incubation process is initiated, and the sample / reagent mixture is incubated in a closed housing at a constant temperature for a certain period of time.

At step 206, data collection begins, and the change in the detected parameter is measured as the sample / reagent mixture incubates over time. These changes are measured by transmitting light of known intensity through a sample / reagent mixture in a container 14 for

- 7 012956 samples and detection of changes in light intensity during the incubation period.

During the incubation period, data relating to time, temperature, and a photometric signal indicating the change in light intensity is collected using a spectrophotometer controller 94. The increase in the microbial population over time is accompanied by an increase in the photometric signal. Thus, a microbial growth curve is formed in real time, such as shown in FIG. 9. At step 208, testing is stopped, and the collected data is stored in the memory of the microprocessor 95 of the spectrophotometer controller 94.

At step 210, the data is loaded into an external data recorder 80, preferably a computer 85, in which specialized software is installed. In step 212, computer 80 processes the data and displays the result both in graphical format and in table format. FIG. 10 illustrates the growth curves of all selected parameters of a typical sample, recorded in real time or downloaded from device 12, and the incubation temperature profile. Vertical values located on the left side indicate the signal intensity (random numbers). On the right side is the temperature in ° C (degrees Celsius). The lower horizontal scale represents the time in hours. FIG. Figure 11 illustrates a data table that contains the values of the parameter signals, along with time and temperature for a typical sample.

At step 214, the software of computer 85 automatically calculates "8" based on a predetermined value of the photometric signal greater than the value of the baseline signal determined by the technician. At step 216, the embedded, predetermined equation of the linear correlation curve is used to calculate the initial population (expressed in colony-forming units (CFU, SEI) in a given sample volume) of the microbiological parameter under study. To obtain this equation of the linear correlation curve, tests are performed with a sequence of separated samples with different initial microbial populations (for example, E. coli), simultaneously using the method in accordance with the present invention and the standard method (membrane filtration). The given equation of the linear correlation curve (LCCE) is formed by plotting the values of Τ8Ό obtained in accordance with the present invention and the corresponding values (X ₀ ) of the initial population obtained using the membrane filtration method. The linear correlation curve of the sample is shown in FIG. 12.

At step 218, the original values of the population are displayed on the computer screen, as shown in FIG. 13.

The method in accordance with the present invention appropriately provides continuous, non-intrusive monitoring and recording of one or more of the detected parameters as the incubation process proceeds. A significant deviation of the output signal is an indicator of the presence of a detectable parameter, while the time required to achieve a significant deviation provides a quantitative analysis of this parameter.

FIG. 14a and 14b illustrate the heating and temperature control and data acquisition algorithms of the controllers 92 and 94. When the power switch 13 is pressed at the command stage 300, the controller program 92 is initiated.

At the command stage 305, controllers 92 and 94 perform a quality check to verify the correct functioning of the incubation and detection systems and components.

In command step 310, if the logical Yes is present, the controllers 92 and 94 go to step 320. If the logical No is present, the controllers turn on the corresponding LEDs to signal the Faulty module.

At the command stage 320, the controller 92 heats the sample 11 in the container 14 through the heating element 24 and monitors the temperature of the sample 11 through a temperature sensor 25 for a predetermined interval.

If logical No is present at the command stage 330, the controller 92 continues heating and monitoring the temperature of the sample 11 in the container 14.

If logical Yes is present at the command stage 330, then the temperature of sample 11 has reached a predetermined temperature value measured by the temperature sensor 25. The controller 92 then sets the test time to zero and continues to monitor the temperature of sample 11 in the container 14. The controller 92 also begins to track the time.

If at the command stage 350 a logical No is obtained, the controller 95 continues heating and monitoring the temperature of the sample 11.

If logical command Yes is received at the command stage 350, the controller 92 transfers the processing to step 355 and stops heating the sample 11 in the container 14, and transfers the processing to the command step 365.

At command stage 365, controller 92 begins collecting temperature values and time data, while controller 94 begins collecting signal data. The controller 92 also initiates a temperature control loop 1 to maintain the incubation temperature in a predetermined range. If at step 400 inside cycle 1 there is a logical Yes, then the controller returns the processing back to step 355 and continues the operation of the cycle.

If there is a logical No at step 400, then the controller moves the processing to command step 410 and initiates heating of sample 11 in container 14. Loop 2 continues until

- 8 012956 until the logical Yes appears at step 400 and the processing returns to loop 1.

Regardless of logical cycles 1 and 2, both controllers 92 and 94 continue to collect their respective data at specified intervals and store this data in the processor's memory.

At step 370, the indications of the indicator LEDs 97 are updated to indicate the progress of the test.

If logical command No is received at command stage 375, controllers 92 and 94 return processing to command stage 365 and continue data collection, thus initiating cycle 3 of the data collection cycle. If at step 375 logical Yes is received, then the test is considered complete and controllers 92 and 94 stop monitoring and data collection and turn off both the incubation system and the testing system, and go to command stage 385 to standby mode (standby mode), and await further instructions from the user or technician.

The methods and apparatus of the invention in accordance with the subject matter of the invention provide many advantages over standard membrane filtration methods. The methods and apparatus in accordance with the subject matter of the invention provide a quick, but simple, reliable and accurate on-site test of microbial material in various types of fluid samples, including drinking water and recreational water. Other advantages include less clutter related to cloudiness, no need for dilution due to a large dynamic analytic range, simplified operation, full automation and integrated quality control (QC, OS), which provides an automatic OS for each test.

It should be understood that various modifications may be made to the embodiments and methods described herein. Although the spectrophotometer system in accordance with the preferred embodiment contains three spectrophotometers, it should be understood that the device may contain a different number of spectrophotometers. In addition, the spatial configuration of the source-detectors can be significantly changed without departing from the scope of the present invention. In addition, each spectrophotometer can be configured to detect different test parameters and can operate independently or simultaneously.

You should also understand that light emitting sources are not limited to LEDs (lasers or laser diodes can also be used instead), and detectors are not limited to phototransistors (photodiodes, photoresistors, SSEs (CCD, charge-coupled device), etc.) can be used instead. .).

Furthermore, the method according to the present invention is not limited to detectable parameters in accordance with the preferred embodiment, since the present method can be used to detect light emission resulting from bioluminescence or chemiluminescence processes as a result of the presence of a biological or chemical component in reagent 20, container 14 for the sample. This can provide the possibility of using the method and device in accordance with the present invention for toxicity studies using bioluminescent bacteria.

In addition, the light emitting source and detector of any or all spectrophotometers can be located outside the incubation-detection chamber 65, but inside the device 10, and can be used to monitor signal growth using fiber-optic means generally installed inside the camera 65.

Accordingly, various modifications can be made in the embodiments and embodiments described and presented herein without departing from the scope of the present invention, the scope of which is defined in the appended claims.

Claims (33)

CLAIM 1. A device for the rapid analysis of microbiological materials in a liquid sample containing: (a) a sample container for holding a liquid sample, wherein the sample container is made of a material that allows light to propagate; (b) a casing having an incubation chamber, the shape of which allows the container for the sample to be contained therein, the casing comprising a container holder mounted in the incubation chamber, wherein the container holder is configured to hold the sample container; (c) incubation means installed inside the container holder, designed to incubate any microbiological material in a liquid sample; and (b) spectrophotometer components installed in the container holder to distribute light in the sample container and to measure the light absorbed, emitted, or scattered by the liquid sample as the microbiological materials incubate inside the incubation chamber over time. - 9 012956 2. The device according to claim 1, in which the components of the spectrophotometer contain a light-emitting source located so that the light propagates inside the sample container, and a detector installed to detect changes in the amount of light when light propagates through microbiological materials and to obtain a detector signal that is an indicator of these changes. 3. The device according to claim 2, also containing a spectrophotometer controller for controlling the light emitting source and the detector, the spectrophotometer controller comprising a microprocessor for processing the detector signal and for generating a record of light changes as a function of time. 4. The device according to claim 2, in which the light emitting source distributes light having a known intensity along the first optical path in the sample container, and the detector detects changes in light intensity within the field of view with respect to the first optical path. 5. The device according to claim 3, in which the field of view is located at an angle of 180 ° relative to the first optical path. 6. The device according to claim 3, in which the field of view is located at an angle of 90 ° relative to the first optical path. 7. The device according to claim 2, in which the light emitting source contains an LED, and the detector contains a phototransistor. 8. The device according to claim 1, additionally containing a first spectrophotometer and a second spectrophotometer, the first spectrophotometer containing a first light emitting source installed inside the container holder and designed to propagate the first light beam in the sample container, and a first detector installed inside the container holder and designed to detect changes in the first light beam, and the second spectrophotometer contains a second light emitting source mounted inside the container holder and The values for the propagation of second ray of light in the specimen container, and a second detector mounted within the container holder and adapted for detecting change in the second beam of light. 9. The device of claim 8, further comprising a third spectrophotometer, the third spectrophotometer comprising a third light emitting source mounted inside the container holder and designed to propagate a third light beam in the sample container, and a third detector mounted inside the container holder and designed to be detected changes in the third ray of light. 10. The device according to claim 2, in which the housing contains a base, and the container holder comprises an upwardly extending generally cylindrical wall mounted on the base, the wall having an open top end formed to receive the sample container and a removable cap connected in a removable manner with a base, the removable cap having such a shape that it surrounds and closes the container holder. 11. The device according to claim 10, in which the light emitting source and the detector are installed in the container holder in a place located in close proximity to the sample container when the sample container is placed in the housing. 12. The device according to claim 10, in which the lower part of the wall of the container holder contains at least one inwardly extending cutout, and the sample container contains at least one side recess having such a shape that it corresponds to a cutout when the sample container is placed in the container holder. 13. The device of claim 10, in which the lower part of the wall of the container holder contains a pair of diametrically opposite cutouts, and the container for the sample contains a pair of diametrically opposite recesses having such a shape that they correspond to the cutouts. 14. The device according to claim 1, in which the means for incubation contain a heating element and a temperature sensor. 15. The device according to 14, in which the temperature sensor extends up into the container holder, and the sample container has a lower cavity having such a shape that it houses a temperature sensor. 16. The device according to 14, in which the heating element extends upward into the container holder, and the sample container has a lower cavity of such a shape that it contains a heating element. 17. The device of claim 8, further comprising a spectrophotometer controller for controlling the first spectrometer and the second spectrometer so as to simultaneously detect changes in the first light beam and changes in the second light beam. 18. The device according to item 12, in which at least one detector and a light emitting source are installed in at least one recess. 19. The device according to item 13, in which the detector is installed in one of the two recesses, and the light emitting source is installed in the other of the two recesses. 20. A device for incubation and detection of microbiological materials in a sample of liquid, containing: (a) a housing having an incubation chamber of such a shape that it stores one container for - 10 012956 samples designed to contain a sample of liquid, and the container for the sample is made of a material that allows light to propagate; (b) incubation means installed inside the chamber, designed to incubate any microbiological materials in the liquid sample, and (c) spectrophotometer components installed inside the chamber, designed to distribute light in the sample container and measure the light absorbed, emitted or scattered by the sample liquids as incubation of microbiological materials inside the incubation chamber over time. 21. The device according to claim 20, in which the components of the spectrophotometer contain a light emitting source located close to the sample container and designed to propagate a light beam of known intensity along the first optical path in the sample container, and a detector located close to the sample container and designed to detect changes in light intensity along the second optical path relative to the first optical path. 22. The device according to item 21, in which the housing contains a base, a container holder mounted on the base for holding the sample container, the container holder having an open upper end formed to receive the sample container, and a removable cap connected in a removable manner to the base, and the removable cap has such a shape that it surrounds and closes the container holder and thereby forms a chamber. 23. The device according to item 22, in which the means for incubation contain a heating element mounted inside the container holder, and a temperature sensor installed inside the container holder. 24. A device for incubation and analysis of microbiological materials in a sample of liquid, containing: (a) a sample container for holding a sample of liquid, wherein the sample container is made of a material that allows light to propagate; (b) a housing comprising a base, one container holder mounted on the base, the container holder being formed to hold the sample container, and a removable cap removably connected to the base, the removable cap having such a shape that it surrounds and closes the container holder in order to create an isolated and opaque incubation detection chamber for the sample container; (c) a heating device for heating and maintaining the fluid sample at a constant temperature, the heating device comprising a heating element mounted inside the container holder and a temperature sensor mounted inside the container holder; and (6) at least one spectrophotometer designed to propagate light in a sample container and measure light absorbed, emitted or scattered by microbiological materials while maintaining a liquid sample at a constant temperature inside the incubation detection chamber over time, the spectrometer comprising a light emitting source mounted inside the container holder for distributing light in the sample container, and a detector installed inside the container holder for children ktirovaniya changes in the quantity of light when the light propagates through microbiological materials and for generating a detection signal indicative of changes. 25. The device according to paragraph 24, in which the holder of the container contains extending upward, in General, a cylindrical wall mounted on the base, and the lower part of the wall contains a pair of diametrically opposite cutouts, the detector is installed in one of two recesses, and the light emitting source is installed in the other of the two recesses, and the sample container contains a pair of diametrically opposed recesses having such a shape that they correspond to the notches. 26. A method for the rapid analysis of microbiological materials in a liquid sample, comprising stages in which: (a) mixing a liquid sample having an unknown initial population of microbiological material with a reagent in a sample container contained in a device according to any one of claims 1 to 25, wherein the reagent provides a detectable parameter showing microbiological material, thereby creating a sample / reagent mixture ; (b) place the sample container in a case contained in a device according to any one of claims 1 to 25, and close the case; (c) incubating the sample / reagent mixture inside the closed case at a temperature within a predetermined temperature range for a certain period of time; and (6) measuring the change in the detected parameter as the sample / reagent mixture is incubated for a certain period of time. 27. The method according to p, optionally containing stages, which record the changes in the detected parameter as a function of time. 28. The method of claim 26, wherein the step of measuring the changes in the detected parameter comprises the step of passing light through the sample / reagent mixture in the sample container and the children - 11 012956 imitate changes in light. 29. The method according to item 27, further comprising stages in which: (e) record the time of a significant deviation at which an exponential change in the detected parameter occurs; and (1) determine the initial population by correlating the time of a significant deviation with the known time of exponential growth for known initial concentrations of microbiological material. 30. The method according to p, in which the specified temperature range is ± 1 ° C. 31. The method according to p, in which the detected parameter is selected from the group consisting of color, fluorescence and degree of turbidity. 32. The method according to p, in which the detected parameter contains bioluminescence or chemiluminescence as a result of the presence of a biological or chemical component in the reagent. 33. A method for the quantitative express analysis of microbiological materials in a fluid sample, comprising the steps of: (a) place a liquid sample having an unknown initial population of microbiological material in a sample container made of a material that allows light to pass through it and contained in a device according to any one of claims 1 to 25; (b) creating a sample / reagent mixture by mixing the liquid sample with the reagent, resulting in a detectable parameter showing microbiological material after exposure to light; (c) performing the incubation of the sample / reagent mixture in the housing contained in the device according to any one of claims 1 to 25, at a temperature within a predetermined temperature range for a period of time; (b) measuring changes in the detected parameter as the sample / reagent mixture is incubated for a certain period of time by passing light through the sample / reagent mixture in the sample container and detecting changes in light intensity; (e) record changes in light intensity as a function of time; (1) record the time of a significant deviation at which an exponential change in light intensity occurs; and (e) determine the initial population by correlating the time of a significant deviation with the known time of exponential growth for known initial concentrations of microbiological materials.