Patent Application
20080293091
This invention relates to methods and apparatus for the rapid photometric detection and enumeration of microbiological materials in fluid samples.
The membrane filtration method is the standard for testing microbiological parameters in water samples. One advantage of the standard membrane filtration method is that it separates the organism under investigation from the potential toxic components present in the original sample matrix. However, the standard membrane filtration method has several drawbacks. The method is over 200 years old and no major modifications have been done to improve the methodology. It is very imprecise, and has problems in dealing with samples containing high microbial counts. The standard membrane filtration method requires manual, visual counting of the growth on the filter as well as experience in identifying false growth. The method is highly error prone due to uncertainty in capturing the target organism within the measurable range.
The development of chromogenic/fluorogenic reagents has opened the door to methods for the analysis of microbiological materials in fluid samples, using optical spectroscopy. These photometric methods are very sensitive, and can detect the presence of a very low concentration of color producing components of interest in fluid samples (in parts per million), whereas the human eye can only detect the color when these components are present in very high concentration. However, most spectrophotomeric methods are performed outside the culture chamber by drawing an aliquot of a sample from the incubation vessel to photometric tubes at various intervals and measuring using standard spectrometers. This is not only time consuming but requires separate incubators and spectrometers, and technical personnel to conduct the tests, using robotic sampling systems in some cases. There is also a potential risk of cross contamination and human error, if proper care is not applied in conducting the analysis.
The present inventors have developed a photometric apparatus and method for the rapid analysis of microbiological materials, which is the subject of U.S. patent application Ser. No. 11/122,089, published under publication No. US2005/0266516 on 1 Dec. 2005. This apparatus comprises a specimen container made from an optically transparent material, a housing having a container holder mounted within an incubation chamber shaped for holding the specimen container, incubation components mounted within the container holder for incubating microbiological materials within the sample, and spectrophotometer components for measuring the light absorbed, emitted or scattered by the liquid as the microbiological materials are incubated over time. This apparatus and method are superior to standard membrane filtration methods, in that they provide for a rapid but simple, reliable and accurate on site testing of microbiological materials in various samples. This apparatus and method also provide a very large linear dynamic range (>105) without the need for dilution. However, this apparatus is not suitable for use in testing some samples, such as samples containing components that are toxic to the microbiological organism under investigation, and samples containing high suspended solids that can interfere with optical measurements.
There accordingly remains a need for further apparatus and methods for the rapid photometric detection and enumeration of microbiological materials, having advantages associated with both the standard filtration method and photometric methods.
The present invention encompasses an apparatus and methods for non-intrusive, automated culturing, and simultaneous rapid photometric detection and quantitation of microbiological, biological, chemical and toxic materials in fluid samples through a continuous, bi-directional diffusion filtration.
The apparatus of the present invention includes a sealed non-intrusive sample cuvette, a housing comprising a base unit with a removable lid, and systems for incubation and photometric detection mounted within the housing. The sample cuvette consists of a clear graduated optically transparent container with two chambers—a culture chamber and a detection chamber—separated by a permeable membrane wall. The cuvette has an upper part and a lower part. The upper part is of different size than the lower part. The sealed top of the cuvette has two fluid inlet/outlet ports for the introduction of the sample into one chamber, while when connected to a suitable vacuum device, the second chamber receives the filtered sample through the permeable membrane. The base of the culture chamber of the cuvette may have an upward indentation to accommodate the temperature sensor cavity of the holder.
The housing has a cuvette holder that is shaped to provide a very snug fit for the cuvette. When placed inside the cuvette holder, the bottom of the upper part of the cuvette rests on top of the holder while the bottom part snugly fits inside the holder. The housing with the cover provides a thermal chamber during simultaneous incubation and photometric enumeration of microbiological parameters. The cuvette holder accommodates a heating element and a temperature sensor. The base of the holder may have an upwardly extending finger cavity to accommodate a temperature sensor. Photometric detection system components, comprising LEDs and detectors, are placed strategically within the holder.
The sealed specimen cuvette acts as a non-intrusive filter apparatus, a specimen culturing vessel as well as an optical sample cuvette. By connecting the outlet from the detection chamber to a vacuum pump, fluid samples can be conveniently drawn into the culture chamber and through the permeable membrane into the detection chamber until the fluid level reaches the volume mark level in the cuvette. The fluid portion in the detection chamber is filtered according to the pore size of the membrane. The pore size can be suitably selected to retain the microbes under investigation in the culture chamber. The membrane is permeable and thus allows movement of fluid sample along with dissolved components in both directions. By connecting both outlets to a bidirectional pump the direction and rate of the flow can be controlled. As an alternative, a closed circulation loop can be created by connecting both outlets through a peristaltic pump or other suitable devices. This way fluid samples from detection chamber can be drawn back into the culture chamber thus providing homogenization of the dissolved components in both chambers at any given time.
The specimen cuvette is made of material that allows the propagation of light. The cuvette may be completely cylindrical or square, with planar vertical surfaces, on opposite sides. Flat, planar surfaces reduces light scattering when using photometric detection system.
In one aspect, the present invention provides a method whereby a fluid sample is introduced into the sterile sealed cuvette through the inlet of the culture chamber using a suction filtration apparatus such as a vacuum pump connected to the outlet of the detection chamber. The culture chamber can be pre-loaded with the appropriate amount of the chromogenic/flurogenic reagent specific to the microbe under investigation or can be introduced as a reagent fluid through the inlet. The fluid sample first enters the culture chamber where the soluble reagent is mixed with it. The fluid sample with the dissolved reagent is then passed through the permeable membrane into the detection chamber. The pores of the membrane is such that it prevents the migration of the microbe under investigation into the detection chamber. The detection chamber receives only the fluid samples and dissolved components which are smaller than the pore size. The fluid is drawn until the level of the fluid reaches the test volume mark in both chambers. A soluble optically active ingredient within the reagent provides the detectable parameter such as color, fluorescence, turbidity, chemiluminescence etc. The detectable parameter provides the detectable signal. The incubation and the detection process are initiated by placing the cuvette directly inside a cuvette holder of the apparatus, placing the removable cap on the apparatus base and initiating the incubation/detection cycle. The detectable signal can be automatically monitored and recorded with time using a computer system and custom software.
In a second aspect, the present invention provides a method whereby a fluid sample is introduced into the sterile sealed cuvette through the inlet of the culture chamber using a suction filtration apparatus such as a vacuum pump connected to the outlet of the detection chamber. The samples are drawn through the inlet of the culture chamber and discarded through the outlet of the detection chamber until the desired volume of the sample is filtered. The permeable diffusion membrane pore size is such that it retains the microbe on the culture chamber and discarding the original sample matrix. Once the desired volume of sample is filtered, the inlet of the culture chamber is connected to a reservoir containing pre-prepared sterile water containing the appropriate chromogenic/flurogenic reagent. By activating the vacuum pump, this reagent solution is then introduced into both chambers until the level of the solution reaches the mark in both chambers. A soluble optically active ingredient within the reagent provides the detectable parameter such as color, fluorescence, turbidity, chemiluminescence etc. The detectable parameter provides the detectable signal. The incubation and the detection process are initiated by placing the cuvette directly inside a cuvette holder of the apparatus, placing the removable cap on the apparatus base and initiating the incubation/detection cycle. The detectable signal can be automatically monitored and recorded with time using a computer system and the custom software.
The detectable time is measured and is correlated to the initial population of the microbe under investigation. Alternately, the apparatus can be left alone to complete the analysis and the data can then be downloaded into the computer using custom software and hardware.
The invention will now be described, by way of example only, with reference to the following drawings, in which:
Referring now to
As best shown in
The hollow sidewall block 53 defines the heating element chamber 65. An indentation at the bottom of the cuvette holder 35 defines the temperature controller cavity 67. Optionally, the opposed side wall block 52 may define the temperature controller chamber 68 to accommodate a second sensor 69 for the optical chamber. The heating element chamber 65 accommodates a heating element 70 while temperature controller cavity 67 accommodates a temperature sensor 72. The heating element 70 and the temperature sensors 69, 72 are controlled by a temperature controller located in base unit 15.
The generally solid sidewall block 85 acts as an LED block while the opposite sidewall block 82 acts as a detector block. The LED block 85 accommodates a light emitting source such as LED 90 placed strategically facing the cavity 40. The detector block 82 accommodates a suitable detector 92, to receive the light emitted from the LED 90 and propagated through the sample cuvette 100. Another suitable LED 95 is placed strategically at the bottom of the cuvette holder 35 near the detector block 82 such that the light emitted from this LED 95 propagates upwardly into the sample cuvette 100. The detector 92 also receives the light emitted by the sample in the cuvette 100 when the sample is exposed to the light scattered from LED 95. The LEDs 90, 95 and the detector 92 are controlled by a microprocessor controller located in the base unit 15. The microprocessor controller processes the signals from the detector 92 and generates a record of the signals as a function of time.
The specimen cuvette 100 comprises a generally cylindrical upper portion 110 having a round cross-section, and a bottom portion 115 having a generally square cross-section. As shown in
As best shown in
The bottom of the culture chamber 140 has an upwardly projecting indentation 75 to accommodate the sensor cavity 67 when the cuvette 100 is placed inside the sample cavity 40.
The permeable membrane 170 of the membrane cartridge 130 is made of suitable material with a specific pore size to allow only the fluid and the dissolved components to diffuse back and forth between the two chambers. The microbes under investigation and other non-specific particles are blocked by the membane 170 and retained in the culture chamber 140, while the fluid and the parameters attached to enzymes produced by the microbes when subjected to the reagent pass through the membrane 170 into the detection chamber 145. Examples of suitable permeable membrane materials include mixed cellulose, ester, polycarbonate, Millipore® Durapore PVDF.
The culture chamber 140 has an inlet 150 to allow the introduction of the test sample and the detection chamber 145 has an outlet 155, which when connected to a suitable vacuum pump, such as nano pump 158 shown placed on top of cuvette 100 in
Referring now to
The base unit 215 is generally similar to base unit 15, except base unit 215 is much larger and is shaped to accommodate multiple, independent cuvette holders 235. Each cuvette holder 235 contains a sample cavity 240, incubation components 260 and optical detection components 280. The sample cavity 240 receives the sample cuvette 100 as shown in
TEST METHOD—In a preferred sample cuvette such as that shown in
To provide homogenization of fluid and dissolved components in both chambers at any given time during the incubation and photometric detection, the pump is activated at a pre-set frequency to draw fluid sample from detection chamber into culturing chamber and through the permeable membrane back into detection chamber. As an alternative homogenization of the fluid in the two chambers can be achieved through a diffusion of fluid through the permeable membrane in both directions using a reversible pump.
For simultaneous testing of total coliform and e.coli in water samples, typical reagent solution provides not only nutrient for the growth but also colorimetric or fluorometric detection signals from the growth of total coliform and e.coli respectively. Examples of typical chromogenic/fluorogenic reagents for total coliform and e.coli are Merck KGaA—Readycult Coliform 100®, IDEXX—Colilert®, and CPI-Colitag™.
The reagent is either pre deposited in the incubation chamber 140 of the sealed cuvette 100 or can be added along with the fluid drawn through the inlet 150. After adding portion of the fluid or fluid-reagent solution, gently shake several times the specimen cuvette 100 until the reagent is completely dissolved in the fluid. Draw the remaining fluid until the level of the fluid reached the mark 160 in both chambers.
The sample cuvette 100 can also be used as a sampling vessel whereby samples are drawn in through the inlet 150 and drawn out through the outlet 155 using a suitable vacuum pump attached to the outlet 155. This way large amount of samples (>100 mis) can be analyzed without the need of dilution and eliminate the toxic effect of the matrix on the microbes during culturing. The reagent and sterile water for culturing can be then introduced into both culture chamber 140 and detection chamber 145 through the diffusion filtration process.
Referring now to
It will be apparent that the present system and method combines membrane filtration with optical photometric detection, and that this combination provides several advantages. Removing the microbes from the toxic medium prior to testing provides more consistent results when analyzing various types of matrices. Secondly, accuracy and precision can be improved by filtering volumes of samples greater than 100 mis (standard reporting volume). In the standard membrane filtration method it is not possible to filter more sample because of the limitation of growing microbes on a small filter and the error in reading them, when present in high numbers.
The present system provides for the rapid but simple, reliable and accurate onsite testing of biological, microbiological and chemical parameters in drinking water, recreational water, wastewater, food, medical and environmental samples to provide a better management of production, treatment and other facilities to protect public health and the environment. By combining membrane filtration and optical detection method, higher speed, accuracy and precision can be achieved, and it is believed that apparatus of the present invention should be of great value to those to conduct routine microbiological testing whether in the lab or in remote areas.
The advantages of the apparatus and methods in accordance with the present invention include the following:
It is to be understood that the present invention is not to be limited in scope by the specific embodiments, methods or applications as described herein. The above descriptions are intended as an illustration of one aspect of the invention and any modification or alternative embodiments are within the scope of the invention and will be apparent to those skilled in the art. For example the embodiment described herein can be applied for detection and enumeration of parameters biological, microbiological and chemical in nature and the method can be colorimetric, fluorometric, turbidimetric, chemiluminescence or bioluminescence. It should be also appreciated that the scope of the present invention is not limited from DNA and semiconductor-nano-technology based monitoring methods. Also, the cuvette can be “U” shaped with the filter cartridge sitting at the middle of the bottom curve to provide two separate chambers, or inner and outer containers providing two chambers with the walls of the inner chamber made of diffusion membrane can also be envisaged.
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.
| Number | Date | Country | |
|---|---|---|---|
| 60940214 | May 2007 | US |
