Title of Invention	A KIT AND METHOD FOR RAPID DETECTION AND ENUMERATION OF E.COLI
Abstract	A tool for rapid detection of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development. A method comprising combining thoroughly in a test container, water, where water does not constitute a test sample, and a test sample and observing fluorescence detection time (FDT) and enumerating E. coli using standard graph. Further tool is prepared by (i) preparing test composition (MUG broth), (ii) contacting the said composition with the inert matrix in a conventional manner such as here in described to get predispensed test composition matrix, and dispensing the said matrix in test container, sterilizing in a known manner to obtain tool and storing at ambient temperature.

Title of Invention

A KIT AND METHOD FOR RAPID DETECTION AND ENUMERATION OF E.COLI

Abstract

A tool for rapid detection of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development. A method comprising combining thoroughly in a test container, water, where water does not constitute a test sample, and a test sample and observing fluorescence detection time (FDT) and enumerating E. coli using standard graph. Further tool is prepared by (i) preparing test composition (MUG broth), (ii) contacting the said composition with the inert matrix in a conventional manner such as here in described to get predispensed test composition matrix, and dispensing the said matrix in test container, sterilizing in a known manner to obtain tool and storing at ambient temperature.

Full Text	The present invention relates to a tool and method for rapid detection and enumeration of E. coli FIELD OF THE INVENTION The present invention particularly relates to a tool for qualitative as well as quantitative detection of coliform as well as E. coli from raw material like meat, poultry, meat or poultry products, food, vegetable, as well as water samples. The invention more particularly provides a tool for rapid detection of E. coli that does not require any sophisticated infrastructure and / or skilled trained personnel to operate, is user friendly can be used in field conditions as well as under laboratory conditions, sensitive, and cost effective. The tool makes use of pre-dispensed media matrix, which is stable over one year. The invention also provides a method for rapid detection of coliforms in general and E. coli in particular. The method is rapid and takes up to maximum 13 hours only. The method is sensitive, easy to operate even at field conditions, cost effective, time saving, and single step and does not require special laboratory facilities such as sterile/aseptic atmosphere, sophisticated infrastructure for enumeration or counting of colonies, and skilled personnel. The method employs chemiluminescent agent to detect change in colour in presence of coliforms and appearance of fluorescence in presence of E. coli. Further, the method is adaptable to eliminate false positive as well as negative effects. The invention also provides a process for preparation of tool and a sterile MUG based medium for detecting/enumerating E.coli. More particularly, the process employs easily available ingredients. BACKGROUND OF THE INVENTION: Besides, several serotypes are enteropathogenic, entero-invasive and entero-toxigenic; E. coli groups are themselves pathogenic. E. coli is used as an indicator organism for faecal contamination originating through lack of hygiene and sanitation and/or inadequate exercise of processing controls in food preparation, handling and distribution. Its presence not only signifies cause of the deterioration of food products but also the possible health hazard from potentially pathogenic intestinal organisms if consumed by human or animal. Additionally, this results in to economic loss due to lost productivity and increased expenses on medication. In order to prevent the transmission of food borne pathogens, it is necessary to test raw ingredients as meat, vegetables, fruits well as food samples before putting them in stream of commerce. This is generally expected to be done by the manufacturers or Government Agencies, or users such as hotels or caterers, buying on large scales, to meet the regulatory requirements considering the safety of the consumers. Conventionally, presence of either fecal coliforms or E. coli is ascertained using Membrane technology, or Most Probable Number technology. Though, these methods for detection and enumeration of fecal coliforms or E. coli are well tested and accepted, all these conventional tests are tedious, laborious, require skilled personnel & well equipped laboratories and take more than 3-4 days to produce confirm results and hence are not advisable for regular monitoring of the raw material used for preparing food products and food products so prepared including drinking water for its safety wherein the results are required in a much shorter time. This has more serious consequences with regard to raw or processed meat including meat products, as they are more susceptible to get contaminated. Additionally, the amino acids present in raw meat accelerate the microbial growth, which in turn result in spoiling these products faster. Mean counts of E. coli were recently proposed both as the base of microbiological quality grade of meat products as one of the test in hazard analysis critical control points. Hence, it is imperative to develop a rapid method of detection and quantification of E. coli without compromising the precision. Since then number of rapid determination techniques have been developed. PCR, DNA probes etc., can be quoted as some of the available new rapid technologies for detecting target organisms. However, they require sophisticated laboratory facilities and well-trained staff to perform the test. This makes these processes cost extensive and beyond reach of majority people and unfit for field testing particularly for large number of samples. Among the more recently developed rapid methods for detecting coliforms in general and E. coli in particular, is the test based on the enzyme glucuronidase (GUR), a fluorimetric method using 4-methyl umbelliferyl P-D glucuronidase (MUG). The technique was modified by incorporating MUG into liquid and solid media respectively as an improvement over conventionally used Most Probable Number (MPN) and direct plating method. The relevant prior art known to the applicant include two commercially available liquid MPN media called Colilert and ColiSure. It is stated that both total coliforms and E. coli are detected by these tests simultaneously within 24 to 28 hours. Colilert utilizes 2-nitrophenyl-.beta.-D-galactopyranoside (ONPG) and ColiSure uses chlorophenol red-.beta.-D-galactoside as substrates to test for .beta.-galactoside. Both utilize 4-methyl-umbelliferyl-.beta.-D-glucuronide to test for .beta.-glucuronidase. These media are expensive. The tests may be used to detect presence or absence of target organisms and/or may result in an estimate of numbers of organisms rather than in an enumeration of target organisms. Both tests have been approved by the USEPA to test for total coliforms and for E. coli detection in drinking water. Concern about the high false negative rate of Colilert with disinfected drinking water has been raised by Clark, et. Al EC broth with MUG media and Durham tubes of Hardy Diagnostics are available in the market as Cat. No. K 18 and Cat. No. S 18. However the product has poor shelf life. S 18 has a shelf life of 30 days from the date of manufacture and K 18 has a shelf life of 90 days from the date of manufacture. Moreover, the products are light and temperature sensitive and require storing at 2 to 8°C and protect from light excessive moisture, heat, and freezing. Additionally, the products have other limitations of giving false positive results and require standard microbiological supplies and equipments like loops sterile atmosphere etc. The prior art known to the applicant further includes patent application number WO 03/052126. The patent teaches rapid method for detecting spoilage of fish sample by detecting sulfide producing bacteria employing fluorescent agent. The process has to be performed at 300°C for 4 to 17 hours. Maintaining high temperatures at the field site poses problems. Canadian Patent No. 2176895 and US Patent 5605812 by Zomer Elizer described a test kit and method for the quantitative determination of total coliform bacteria and E. coli using chromogenic and fluorogenic methods respectively. The invention intends to improve the then existing prior art and provide a method applicable particularly to large volume liquid test samples, such as water. The improvement resides in providing medium that includes a gelling agent, which in situ with water and test sample provides a gel like semi-solid broth or media thereby restricting the mobility of the bacteria. Thus the medium is in dry form and is reconstituted with water to provide semisolid substrate to facilitate bacterial growth. While reconstituting medium occasionally fingers are to be used to break the possible lumps developed due to presence of gelling agent. This necessitates using the product that is flexible. Every precaution has to be taken to ensure thorough mixing and dissolution of the medium while avoid any leakages. The process requires at least one-generation time to develop colonies. Further, it requires skilled personnel and colony counter to count the fluorescent colonies. Additionally, counting of fluorescent colonies becomes difficult in a crowded plate. The additional drawback of this kit is that the fluorescent agent tends to diffuse throughout the medium when incubated for over 24 hours and pose problems in quantifying the bacteria. US Patent No. 5,843,699 teaches rapid microorganism detection method which is a general guidance of screening methods for detection and identification of target organisms in a sample. It involves different steps like pre-enrichment, selective enrichment, selective isolation and identification by biochemical assays. The drawback here is long procedure of 3-4 days or more, requires laboratory facilities and skilled personnel. US Patent No. 5,738,995 (Wu, et. al) provides methods and apparatus for detecting and discriminating multiple analytes, an inosine containing probe for detecting a target nucleic acid sequence from E. coli O157:H7 microorganisms, E. coli 0157: H7 said probe comprising an oligonucleotide sequence only within a test sample which are simple, user-friendly, cost-effective and fast. In particular, it is preferred that the overall time for sample preparation, nucleic acid sequence amplification, and nucleic acid sequence differentiation be about 5 hours or less, which requires sophisticated equipment and specially trained skilled personnel hence costly. U.S. Pat. No. 4,923,804 to Ley, et al., teaches use of .beta.-glucuronides to test for E. coli and that the indoxyl-.beta.-D-glucuronide is a preferred agent. (See Ex. 2 of the reference cited) However, he teaches, at column 1, 1. 50-68 that the use of MUG compounds to test for E. coli on a membrane filter test is not appropriate since the fluorescent light can be subject to interference in a membrane filter test. Hence, the teaching of Ley would discourage one from use of an agent having a 4-methylumbelliferyl fluorescent moiety in a membrane filter test. The medium differs from the substrate of the invention in several respects, 1) The medium of Ley can only detect E. coli and does not provide for detection of total coliforms. Because of this, a second medium would be required to identify total coliforms, thereby increasing the time, labor, material, and cost to the laboratory performing the analysis. 2) The base medium of Ley contains glycerol as a nutrient and lacks an inducer and an inhibitor of gram negative bacteria that can give a false positive response. Glycerol in media also causes spreading of colonies making enumeration and discrimination difficult. 3) The medium of Ley is incubated at an elevated temperature (44.5.degree. C.) that would be detrimental to the recovery of injured microorganisms. U.S. Pat. No. 4,591,554 to Koumura, et al., discloses use of fluorescence analysis using umbelliferone derivatives, including phosphates and galactosides. That reference also teaches use of lactose as an inducer. The organisms are first inoculated into broth for growth. There is no inhibitor in the media, and the reference indicates, at column 3, lines 40-45 that the test also picks up Erwinia, Proteus, and Salmonella—gram negative organisms not usually classified among the coliform bacteria. The media recommended by Koumura can also promote the growth of many other types of organisms such as gram positive bacteria, yeasts and fungi that may also be present in the samples. (See Table 2 of that reference.) Some of the non-coliform organisms are able to inhibit the growth of coliforms. Hence, the tests of Koumura are not appropriate for use wherein there is a desire to find the total coliform populations. Babelona, et al., (J. Micro. Meth. 12: 235-245) discloses use of MU-glucuronide complexes in testing for E. coli as does much of the prior art. There is no test using a non-fluorescing chromogen-glucuronide to test for E. coli, nor is there any MU-galactoside test for total coliforms. US Patent No. 6,670, 145, (Brenner et al) discloses a method for the detection of coliforms and E. coli using a chromogenic and glucurogenic substates. an ingredient that will encourage growth and repair of injured coliforms, buffers to maintain a pH in the range of 6.5-8, at least one agent that suppresses growth of gram positive cocci and spore-forming organisms, at least one active agent that will suppress growth of non-coliform gram negative bacteria, and at least one chromogen or fluorogen has been used effectively and is cost effective. In the preferred embodiment, both a fluorogen and chromogen were used. Preferred methods include use of filter and/or plates containing the growth-promoting ingredients and the indicators. Results are obtained in 16-24 hrs. Antibiotics are also used in the medium to prevent the growth of other bacteria. Since it is membrane filter technology, it can be performed by skilled personnel in a laboratory only. The medium is particularly useful for routine testing of drinking water. US Patent No. 5,861270, discloses a two stage enzymatic method for the detection of coliform bacteria or E. coli, in a liquid or liquefied sample in a shorter time, for TC for example within a detection time of 7 hours wherein bacteria are concentrated on a membrane filter. This filter is placed on a growth medium containing nutrients, including preferably minerals, a protein hydrolysate and a sugar, preferably maltose or a polyalcohol, preferably mannitol, an inducer of a marker enzyme, in particular .beta.-galactosidase or .beta.-glucuronidase and inhibitors of the growth of competing bacteria. After a pre incubation step, the filter is placed on an assay medium containing a fluorogenic or chemiluminogenic enzyme substrate and a membrane permeabilizer. The membrane filter and the assay medium are incubated to allow cleavage of the enzyme substrate producing fluorescent or chemiluminescent microcolonies on the membrane filter after triggering of light emission. Bacteriological Analytical Manual (BAM) ONLINE January 2001 and September 2002 respectively described various media and methods for determining /enumerating coliforms and E.coli by LST-MUG method in shellfish and different microorganism which requires laboratory facilities. The existing technologies, as can be seen from the disclosure herein above, suffer from one or the other disadvantages given below: 1. Require laboratory facility. 2. Require skilled personnel. 3. Not suitable for field conditions. 4. Involve more than one step. 5. Time consuming. Requires minimum 4 -6 hrs and maximum of 48 hrs. 6. Cost extensive. 7. Require additional ingredients or confirmatory test. 8. Poor shelf life and require special storing infrastructure. 9. Recommend high MUG concentration (50 to lOOmg/L) It may be worthwhile to mention here that none of these methods are suitable for field conditions and shelf life over 1 year at ambient temperature. Thus there is an imperative need to develop a tool for rapid detection and enumeration of E.coli, which is simple, suitable for field conditions, highly stable, can obviate the disadvantages associated with the existing methods, and can help ascertaining quality of product for consumption or further processing before entering in to Commerce. After continuous and painstaking research for prolonged period the applicant is successful in developing a tool and method for rapid detection of E. coli that can be used in turn to asses the quality of goods as well as preparing products thereof for consumption. Further the tool has diverse applicability. SUMMARY OF THE INVENTION The main object of the present invention is to provide a tool and method for rapid detection of E. coli obviating the disadvantages associated with the existing processes. The other object is to provide a tool for qualitative as well as semi-quantitative detection of coliform and E. coli from raw materials such as meat vegetable, food products, as well as water samples. Yet other object is to provide a tool having stability over one year at ambient temperature. Another object is to provide a tool for rapid detection of E. coli, that is user friendly can be used in field conditions as well as under laboratory conditions. Yet other object is to provide a tool that does not require any sophisticated infrastructure and /or skilled trained personnel to operate while maintaining precision. Yet another object is to provide a tool that is cost effective. The tool makes use of pre-dispensed media matrix. Further, the invention intends to provide a method for rapid detection of coliforms in general and E. coli in particular using the said tool. Still other object is to provide a single step method, which is rapid takes up to 13 hours only. Still another object is to provide a method that is sensitive, easy to operate even at field conditions, does not require special laboratory facilities such as sterile/aseptic atmosphere, sophisticated infrastructure for enumeration or counting of colonies, and skilled personnel, cost effective, time saving. Yet other object is to provide a method that employs chemiluminescent agent to detect change in colour for presence of coliforms and appearance of fluorescence for presence of E. coll. Yet another object is to provide a method capable of eliminating false positive as well as negative effects. The invention also aims to provide a process for preparation of the tool as herein above described and a sterile MUG based medium for detecting/enumerating E.coli, using easily available ingredients. STATEMENT OF INVENTION Accordingly the present invention provides a tool for rapid detection of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development. According to one of the embodiments of this invention the test container may be glass or plastic bottle or tube wherein the adaptability of opening and resealing is imparted by providing stopper/ cap. According to other embodiment the test container may be transparent or semitransparent having ability to allow the light transmission and airtight. According to another embodiment the biologically inert matrix may be sand/ plastic and or glass beads of 10 to 30 mesh. According to yet other embodiment the source of nitrogen and other conventional nutrients may be proteose peptone and yeast extract; buffering agent may be phosphate buffer; inhibitor may be tergitol 7; chemiluminescent agent may be 4-methylumbelliferyl p-D glucuronoid (MUG). According to yet another embodiment the test composition may be MUG broth consisting of proteose peptone in the range of — 3 to 5 g, yeast extract ~ 3g, buffer to adjust pH to 6.8 to 7.2, tergitol 7 O.lml MUG in the range of 20 to 100mg/l, and water 200ml. In accordance with other aspect pf this invention there is provide a method for rapid detection and enumeration of E. coli comprising combining thoroughly in a test container, water, where water does not constitute a test sample, and a test sample and observing fluorescence detection time (FDT) and enumerating E. coli using standard graph. The method as disclosed herein above may employ the matrix ranging from 4 to 5 times of MUG broth and pre-dispensation may be conducted by coating the matrix with the MUG broth. Further, the water used for combining may ranges from 4 to 80 ml and test sample ranges from 1 to 20g. The FDT may be performed under long wavelength UV (366mm) at hourly intervals against water as blank. The enumeration may be carried out comparing the FDT with corresponding colony count on the graph. In accordance with the other aspect of this invention there is provided a method for preparing tool consisting the following steps: (i) preparing test composition (MUG broth), (ii) contacting the said composition with the inert matrix in a conventional manner such as here in described to get predispensed test composition matrix, (iii) dispensing the said matrix in test container, sterilizing in a known manner to obtain tool and storing at ambient temperature. DETAILED DESCRIPTION OF THE INVENTION As required detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention. The following examples are included solely to aid in a more complete understanding of the invention described and claimed herein. The examples do not limit the scope of the claimed invention in any fashion. However, one of the ordinary skilled in the art appreciates the modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or a solution to occur or become more pronounced are not to be construed as critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the dependency of this application and all equivalents of those claims as issued. EXAMPLES Working example I Bacto grade microbiological media ingredients- proteose peptone, tergitol 7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade) and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable, transparent, screw capped glass bottles/tubes were procured from reputed manufacturers. MUG broth was prepared using (i) proteose peptone No. 3 — 5g, (ii) yeast extract — 3g, (iii) dipotassium hydrogen orthophosphate — 0.3g, (iv) tergitol 7 — 0.1 ml, MUG — 0.03g, water — 200ml. pH was adjusted to 7.2  MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating.  Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per standard procedures.  Then MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.  Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be dispensed in clean individual glass tubes/bottles and mixed thoroughly.  Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap.  After the tubes/bottles were cooled the caps were tightened and stored at room temperature.  To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.  l-20g of the test sample was added to the bottle and shaken well.  To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.  Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.  Tubes/bottles were observed for blue fluorescence under long wave length (366mm) UV at hourly intervals till the detection of fluorescence or for 13 h which ever is early and FDT was recorded.  Control tubes with no fluorescence serve as negative control.  Absence of visible fluorescence after 13 h of incubation indicated the absence of E.coli in the sample.  Concentration of E.coli was determined by comparing the FDT with corresponding cell count on the standard graph. Working example II  Bacto grade microbiological media ingredients- proteose peptone, tergitol 7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade) and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable, transparent, screw capped glass bottles/tubes were procured. »«« MUG broth was prepared using proteose peptone No. 3-5g, yeast extract- 3g, dipotassium hydrogen orthophosphate-0.3g, tergitol 7-0.1 ml, MUG- 0.04g, water 200ml. pH was adjusted to 7.2.  MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating.  Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per standard procedures.  The MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.  Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be dispensed in clean individual glass tubes/bottles and mixed thoroughly.  Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap.  After the tubes/bottles were cooled the caps were tightened and stored at room temperature. To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.  l-20g of the test sample was added to the bottle and shaken well.  To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.  Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.  Tubes/bottles were observed for blue fluorescence under long wave length (366mm) UV at hourly intervals till the detection of fluorescence or for 13 h which ever is early and FDT was recorded.  Control tubes with no fluorescence serve as negative control. Absence of visible fluorescence after 13 h of incubation indicated the absence of E.coli in the sample.  Concentration of E.coli was determined by comparing the FDT with corresponding cell count on the standard graph. Working example III  Bacto grade microbiological media ingredients- proteose peptone, tergitol 7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade) and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable, transparent, screw capped glass bottles/tubes were procured from reputed manufacturers.  MUG broth was prepared using proteose peptone No. 3-5g, yeast extract-3g, dipotassium hydrogen orthophosphate-0.3g, tergitol 7-0.1 ml, MUG-0.05g, water 200ml. pH was adjusted to 7.2.  MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating. Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per standard procedures. Then MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.  Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be dispensed in clean individual glass tubes/bottles and mixed thoroughly.  Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap. After the tubes/bottles were cooled the caps were tightened and stored at room temperature.  To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.  l-20g of the test sample was added to the bottle and shaken well. To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.  Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.  Tubes/bottles were observed for blue fluorescence under long wave length (366mm) UV at hourly intervals till the detection of fluorescence or for 13 h which ever is early and FDT was recorded.  Control tubes with no fluorescence serve as negative control. Absence of visible fluorescence after 13 h of incubation indicated the absence of E.coli in the sample.  Concentration of E.coli was determined by comparing the FDT with corresponding cell count on the standard graph. MUG concentration A minimum of 20mg/L of MUG in the medium is required to get just detectable fluorescence and 30mg/L is effective contrary to lOOmg/L as described in prior art. Table-1 (Table Removed) ADVANTAGES (i) The process and the test kit developed are simple and cost effective. (ii) It is rapid and easy to perform and no skill/training required (iii) It does not require laboratory facilities (iv) Suitable for field conditions (v) The test kit is stable for more than 1 year in room temperature (vi) Concentration of E.coli in the sample can be obtained from the graph by the determination of FDT. (vii) MUG concentration could be reduced to 30 mg/L contrary to lOOmg/L as in conventional methods. WE CLAIM: 1. A tool and method for rapid detection and enumeration of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development. 2. A tool as claimed in claim 1, wherein the test container is transparent or semitransparent glass or plastic airtight bottle or tube wherein the adaptability of opening and resealing is imparted by providing stopper/ cap. 3. A tool as claimed in claims 1 to 3 wherein the biologically inert matrix is sand/ plastic and or glass beads of 10 to 30 mesh. 4. A tool as claimed in claims 1 to 4 wherein the source of nitrogen and other conventional nutrients is proteose peptone and yeast extract; buffering agent is phosphate buffer; inhibitor is tergitol 7; chemiluminescent agent is 4- methylumbelliferyl p-D glucuronoid (MUG). 5. A tool as claimed in claim 1 wherein test composition is MUG broth consisting of proteose peptone in the range of -- 3 to 5 g, yeast extract — 3g, buffer to adjust pH to 6.8 to 7.2, tergitol 7 O.lml MUG in the range of 20 to 1 OOmg/1, and water 200ml. 6. A method for rapid detection and enumeration of E. coli comprising combining thoroughly in a test container, water, where water does not constitute a test sample, and a test sample and observing fluorescence detection time (FDT) and enumerating E. coli using standard graph. 7. A method as claimed in claim 6 wherein the matrix used is 4 to 5 times of MUG broth and pre-dispensation is conducted by coating the matrix with MUG broth. 8. A method as claimed in claims 7 & 8 wherein the water used for combining ranges from 4 to 80 ml and test sample ranges from 1 to 20g. 9. A method as claimed in claims 7 to 9 wherein the FDT is performed under long wavelength UV (366mm) at hourly intervals against water as blank and enumeration is carried out comparing the FDT with corresponding colony count on the graph. 10. A method for preparing tool consisting the following steps: (i) preparing test composition (MUG broth), (ii) contacting the said composition with the inert matrix in a conventional manner such as here in described to get predispensed test composition matrix, (iii) dispensing the said matrix in test container, sterilizing in a known manner to obtain tool and storing at ambient temperature. 11. A tool and method for rapid detection and enumeration of E. coli and a method therefor substantially as herein described.

Full Text

The present invention relates to a tool and method for rapid detection and enumeration of E. coli
FIELD OF THE INVENTION
The present invention particularly relates to a tool for qualitative as well as quantitative detection of coliform as well as E. coli from raw material like meat, poultry, meat or poultry products, food, vegetable, as well as water samples. The invention more particularly provides a tool for rapid detection of E. coli that does not require any sophisticated infrastructure and / or skilled trained personnel to operate, is user friendly can be used in field conditions as well as under laboratory conditions, sensitive, and cost effective. The tool makes use of pre-dispensed media matrix, which is stable over one year.
The invention also provides a method for rapid detection of coliforms in general and E. coli in particular. The method is rapid and takes up to maximum 13 hours only. The method is sensitive, easy to operate even at field conditions, cost effective, time saving, and single step and does not require special laboratory facilities such as sterile/aseptic atmosphere, sophisticated infrastructure for enumeration or counting of colonies, and skilled personnel. The method employs chemiluminescent agent to detect change in colour in presence of coliforms and appearance of fluorescence in presence of E. coli. Further, the method is adaptable to eliminate false positive as well as negative effects.
The invention also provides a process for preparation of tool and a sterile MUG based medium for detecting/enumerating E.coli. More particularly, the process employs easily available ingredients.
BACKGROUND OF THE INVENTION:
Besides, several serotypes are enteropathogenic, entero-invasive and entero-toxigenic; E. coli groups are themselves pathogenic. E. coli is used as an indicator organism for faecal contamination originating through lack of hygiene and sanitation and/or inadequate exercise of processing controls in food preparation, handling and distribution. Its presence not only signifies cause of the deterioration
of food products but also the possible health hazard from potentially pathogenic intestinal organisms if consumed by human or animal. Additionally, this results in to economic loss due to lost productivity and increased expenses on medication. In order to prevent the transmission of food borne pathogens, it is necessary to test raw ingredients as meat, vegetables, fruits well as food samples before putting them in stream of commerce. This is generally expected to be done by the manufacturers or Government Agencies, or users such as hotels or caterers, buying on large scales, to meet the regulatory requirements considering the safety of the consumers.
Conventionally, presence of either fecal coliforms or E. coli is ascertained using Membrane technology, or Most Probable Number technology. Though, these methods for detection and enumeration of fecal coliforms or E. coli are well tested and accepted, all these conventional tests are tedious, laborious, require skilled personnel & well equipped laboratories and take more than 3-4 days to produce confirm results and hence are not advisable for regular monitoring of the raw material used for preparing food products and food products so prepared including drinking water for its safety wherein the results are required in a much shorter time. This has more serious consequences with regard to raw or processed meat including meat products, as they are more susceptible to get contaminated. Additionally, the amino acids present in raw meat accelerate the microbial growth, which in turn result in spoiling these products faster. Mean counts of E. coli were recently proposed both as the base of microbiological quality grade of meat products as one of the test in hazard analysis critical control points. Hence, it is imperative to develop a rapid method of detection and quantification of E. coli without compromising the precision.
Since then number of rapid determination techniques have been developed. PCR, DNA probes etc., can be quoted as some of the available new rapid technologies for detecting target organisms. However, they require sophisticated laboratory facilities and well-trained staff to perform the test. This makes these processes cost extensive and beyond reach of majority people and unfit for field testing particularly for large number of samples. Among the more recently developed rapid methods for detecting coliforms in general and E. coli in particular, is the test
based on the enzyme glucuronidase (GUR), a fluorimetric method using 4-methyl umbelliferyl P-D glucuronidase (MUG). The technique was modified by incorporating MUG into liquid and solid media respectively as an improvement over conventionally used Most Probable Number (MPN) and direct plating method.
The relevant prior art known to the applicant include two commercially available liquid MPN media called Colilert and ColiSure. It is stated that both total coliforms and E. coli are detected by these tests simultaneously within 24 to 28 hours. Colilert utilizes 2-nitrophenyl-.beta.-D-galactopyranoside (ONPG) and ColiSure uses chlorophenol red-.beta.-D-galactoside as substrates to test for .beta.-galactoside. Both utilize 4-methyl-umbelliferyl-.beta.-D-glucuronide to test for .beta.-glucuronidase.
These media are expensive. The tests may be used to detect presence or absence of target organisms and/or may result in an estimate of numbers of organisms rather than in an enumeration of target organisms. Both tests have been approved by the USEPA to test for total coliforms and for E. coli detection in drinking water. Concern about the high false negative rate of Colilert with disinfected drinking water has been raised by Clark, et. Al
EC broth with MUG media and Durham tubes of Hardy Diagnostics are available in the market as Cat. No. K 18 and Cat. No. S 18. However the product has poor shelf life. S 18 has a shelf life of 30 days from the date of manufacture and K 18 has a shelf life of 90 days from the date of manufacture. Moreover, the products are light and temperature sensitive and require storing at 2 to 8°C and protect from light excessive moisture, heat, and freezing. Additionally, the products have other limitations of giving false positive results and require standard microbiological supplies and equipments like loops sterile atmosphere etc.
The prior art known to the applicant further includes patent application number WO 03/052126. The patent teaches rapid method for detecting spoilage of fish sample by detecting sulfide producing bacteria employing fluorescent agent. The
process has to be performed at 300°C for 4 to 17 hours. Maintaining high temperatures at the field site poses problems.
Canadian Patent No. 2176895 and US Patent 5605812 by Zomer Elizer described a test kit and method for the quantitative determination of total coliform bacteria and E. coli using chromogenic and fluorogenic methods respectively. The invention intends to improve the then existing prior art and provide a method applicable particularly to large volume liquid test samples, such as water. The improvement resides in providing medium that includes a gelling agent, which in situ with water and test sample provides a gel like semi-solid broth or media thereby restricting the mobility of the bacteria. Thus the medium is in dry form and is reconstituted with water to provide semisolid substrate to facilitate bacterial growth. While reconstituting medium occasionally fingers are to be used to break the possible lumps developed due to presence of gelling agent. This necessitates using the product that is flexible. Every precaution has to be taken to ensure thorough mixing and dissolution of the medium while avoid any leakages. The process requires at least one-generation time to develop colonies. Further, it requires skilled personnel and colony counter to count the fluorescent colonies. Additionally, counting of fluorescent colonies becomes difficult in a crowded plate. The additional drawback of this kit is that the fluorescent agent tends to diffuse throughout the medium when incubated for over 24 hours and pose problems in quantifying the bacteria.
US Patent No. 5,843,699 teaches rapid microorganism detection method which is a general guidance of screening methods for detection and identification of target organisms in a sample. It involves different steps like pre-enrichment, selective enrichment, selective isolation and identification by biochemical assays. The drawback here is long procedure of 3-4 days or more, requires laboratory facilities and skilled personnel.
US Patent No. 5,738,995 (Wu, et. al) provides methods and apparatus for detecting and discriminating multiple analytes, an inosine containing probe for detecting a target nucleic acid sequence from E. coli O157:H7 microorganisms, E. coli 0157:
H7 said probe comprising an oligonucleotide sequence only within a test sample which are simple, user-friendly, cost-effective and fast. In particular, it is preferred that the overall time for sample preparation, nucleic acid sequence amplification, and nucleic acid sequence differentiation be about 5 hours or less, which requires sophisticated equipment and specially trained skilled personnel hence costly.
U.S. Pat. No. 4,923,804 to Ley, et al., teaches use of .beta.-glucuronides to test for E. coli and that the indoxyl-.beta.-D-glucuronide is a preferred agent. (See Ex. 2 of the reference cited) However, he teaches, at column 1, 1. 50-68 that the use of MUG compounds to test for E. coli on a membrane filter test is not appropriate since the fluorescent light can be subject to interference in a membrane filter test. Hence, the teaching of Ley would discourage one from use of an agent having a 4-methylumbelliferyl fluorescent moiety in a membrane filter test. The medium differs from the substrate of the invention in several respects, 1) The medium of Ley can only detect E. coli and does not provide for detection of total coliforms. Because of this, a second medium would be required to identify total coliforms, thereby increasing the time, labor, material, and cost to the laboratory performing the analysis. 2) The base medium of Ley contains glycerol as a nutrient and lacks an inducer and an inhibitor of gram negative bacteria that can give a false positive response. Glycerol in media also causes spreading of colonies making enumeration and discrimination difficult. 3) The medium of Ley is incubated at an elevated temperature (44.5.degree. C.) that would be detrimental to the recovery of injured microorganisms.
U.S. Pat. No. 4,591,554 to Koumura, et al., discloses use of fluorescence analysis using umbelliferone derivatives, including phosphates and galactosides. That reference also teaches use of lactose as an inducer. The organisms are first inoculated into broth for growth. There is no inhibitor in the media, and the reference indicates, at column 3, lines 40-45 that the test also picks up Erwinia, Proteus, and Salmonella—gram negative organisms not usually classified among the coliform bacteria. The media recommended by Koumura can also promote the growth of many other types of organisms such as gram positive bacteria, yeasts and fungi that may also be present in the samples. (See Table 2 of that reference.) Some of the non-coliform organisms are able to inhibit the growth of coliforms.
Hence, the tests of Koumura are not appropriate for use wherein there is a desire to find the total coliform populations.
Babelona, et al., (J. Micro. Meth. 12: 235-245) discloses use of MU-glucuronide complexes in testing for E. coli as does much of the prior art. There is no test using a non-fluorescing chromogen-glucuronide to test for E. coli, nor is there any MU-galactoside test for total coliforms.
US Patent No. 6,670, 145, (Brenner et al) discloses a method for the detection of coliforms and E. coli using a chromogenic and glucurogenic substates. an ingredient that will encourage growth and repair of injured coliforms, buffers to maintain a pH in the range of 6.5-8, at least one agent that suppresses growth of gram positive cocci and spore-forming organisms, at least one active agent that will suppress growth of non-coliform gram negative bacteria, and at least one chromogen or fluorogen has been used effectively and is cost effective. In the preferred embodiment, both a fluorogen and chromogen were used. Preferred methods include use of filter and/or plates containing the growth-promoting ingredients and the indicators.
Results are obtained in 16-24 hrs. Antibiotics are also used in the medium to prevent the growth of other bacteria. Since it is membrane filter technology, it can be performed by skilled personnel in a laboratory only. The medium is particularly useful for routine testing of drinking water.
US Patent No. 5,861270, discloses a two stage enzymatic method for the detection of coliform bacteria or E. coli, in a liquid or liquefied sample in a shorter time, for TC for example within a detection time of 7 hours wherein bacteria are concentrated on a membrane filter. This filter is placed on a growth medium containing nutrients, including preferably minerals, a protein hydrolysate and a sugar, preferably maltose or a polyalcohol, preferably mannitol, an inducer of a marker enzyme, in particular .beta.-galactosidase or .beta.-glucuronidase and inhibitors of the growth of competing bacteria. After a pre incubation step, the filter is placed on an assay medium containing a fluorogenic or chemiluminogenic enzyme substrate and a membrane permeabilizer. The membrane filter and the
assay medium are incubated to allow cleavage of the enzyme substrate producing fluorescent or chemiluminescent microcolonies on the membrane filter after triggering of light emission.
Bacteriological Analytical Manual (BAM) ONLINE January 2001 and September 2002 respectively described various media and methods for determining /enumerating coliforms and E.coli by LST-MUG method in shellfish and different microorganism which requires laboratory facilities.
The existing technologies, as can be seen from the disclosure herein above, suffer from one or the other disadvantages given below:
1. Require laboratory facility.
2. Require skilled personnel.
3. Not suitable for field conditions.
4. Involve more than one step.
5. Time consuming. Requires minimum 4 -6 hrs and maximum of 48 hrs.
6. Cost extensive.
7. Require additional ingredients or confirmatory test.
8. Poor shelf life and require special storing infrastructure.
9. Recommend high MUG concentration (50 to lOOmg/L)
It may be worthwhile to mention here that none of these methods are suitable for field conditions and shelf life over 1 year at ambient temperature. Thus there is an imperative need to develop a tool for rapid detection and enumeration of E.coli, which is simple, suitable for field conditions, highly stable, can obviate the disadvantages associated with the existing methods, and can help ascertaining quality of product for consumption or further processing before entering in to Commerce.
After continuous and painstaking research for prolonged period the applicant is successful in developing a tool and method for rapid detection of E. coli that can be used in turn to asses the quality of goods as well as preparing products thereof for consumption. Further the tool has diverse applicability.
SUMMARY OF THE INVENTION
The main object of the present invention is to provide a tool and method for rapid
detection of E. coli obviating the disadvantages associated with the existing
processes.
The other object is to provide a tool for qualitative as well as semi-quantitative detection of coliform and E. coli from raw materials such as meat vegetable, food products, as well as water samples.
Yet other object is to provide a tool having stability over one year at ambient temperature.
Another object is to provide a tool for rapid detection of E. coli, that is user friendly can be used in field conditions as well as under laboratory conditions.
Yet other object is to provide a tool that does not require any sophisticated infrastructure and /or skilled trained personnel to operate while maintaining precision.
Yet another object is to provide a tool that is cost effective. The tool makes use of pre-dispensed media matrix.
Further, the invention intends to provide a method for rapid detection of coliforms in general and E. coli in particular using the said tool.
Still other object is to provide a single step method, which is rapid takes up to 13 hours only.
Still another object is to provide a method that is sensitive, easy to operate even at field conditions, does not require special laboratory facilities such as sterile/aseptic atmosphere, sophisticated infrastructure for enumeration or counting of colonies, and skilled personnel, cost effective, time saving.
Yet other object is to provide a method that employs chemiluminescent agent to
detect change in colour for presence of coliforms and appearance of fluorescence for presence of E. coll.
Yet another object is to provide a method capable of eliminating false positive as well as negative effects.
The invention also aims to provide a process for preparation of the tool as herein above described and a sterile MUG based medium for detecting/enumerating E.coli, using easily available ingredients.
STATEMENT OF INVENTION
Accordingly the present invention provides a tool for rapid detection of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development.
According to one of the embodiments of this invention the test container may be glass or plastic bottle or tube wherein the adaptability of opening and resealing is imparted by providing stopper/ cap.
According to other embodiment the test container may be transparent or semitransparent having ability to allow the light transmission and airtight.
According to another embodiment the biologically inert matrix may be sand/ plastic and or glass beads of 10 to 30 mesh.
According to yet other embodiment the source of nitrogen and other conventional nutrients may be proteose peptone and yeast extract; buffering agent may be phosphate buffer; inhibitor may be tergitol 7; chemiluminescent agent may be 4-methylumbelliferyl p-D glucuronoid (MUG).
According to yet another embodiment the test composition may be MUG broth consisting of proteose peptone in the range of — 3 to 5 g, yeast extract ~ 3g, buffer to adjust pH to 6.8 to 7.2, tergitol 7 O.lml MUG in the range of 20 to 100mg/l, and water 200ml.
In accordance with other aspect pf this invention there is provide a method for rapid detection and enumeration of E. coli comprising combining thoroughly in a test container, water, where water does not constitute a test sample, and a test sample and observing fluorescence detection time (FDT) and enumerating E. coli using standard graph.
The method as disclosed herein above may employ the matrix ranging from 4 to 5 times of MUG broth and pre-dispensation may be conducted by coating the matrix with the MUG broth.
Further, the water used for combining may ranges from 4 to 80 ml and test sample ranges from 1 to 20g.
The FDT may be performed under long wavelength UV (366mm) at hourly intervals against water as blank.
The enumeration may be carried out comparing the FDT with corresponding colony count on the graph.
In accordance with the other aspect of this invention there is provided a method for preparing tool consisting the following steps:
(i) preparing test composition (MUG broth),
(ii) contacting the said composition with the inert matrix in a
conventional manner such as here in described to get
predispensed test composition matrix, (iii) dispensing the said matrix in test container, sterilizing in a
known manner to obtain tool and storing at ambient
temperature.
DETAILED DESCRIPTION OF THE INVENTION
As required detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.
The following examples are included solely to aid in a more complete understanding of the invention described and claimed herein. The examples do not limit the scope of the claimed invention in any fashion. However, one of the ordinary skilled in the art appreciates the modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below.
The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or a solution to occur or become more pronounced are not to be construed as critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the dependency of this application and all equivalents of those claims as issued.
EXAMPLES Working example I
Bacto grade microbiological media ingredients- proteose peptone, tergitol 7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade) and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable, transparent, screw capped glass bottles/tubes were procured from reputed manufacturers.
MUG broth was prepared using (i) proteose peptone No. 3 — 5g, (ii) yeast extract — 3g, (iii) dipotassium hydrogen orthophosphate — 0.3g, (iv) tergitol 7 — 0.1 ml, MUG — 0.03g, water — 200ml. pH was adjusted to 7.2
 MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating.
 Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per
standard procedures.
 Then MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.
 Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be
dispensed in clean individual glass tubes/bottles and mixed thoroughly.
 Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap.
 After the tubes/bottles were cooled the caps were tightened and stored at room temperature.
 To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.
 l-20g of the test sample was added to the bottle and shaken well.
 To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.
 Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.
 Tubes/bottles were observed for blue fluorescence under long wave length (366mm) UV at hourly intervals till the detection of fluorescence or for 13 h which ever is early and FDT was recorded.
 Control tubes with no fluorescence serve as negative control.
 Absence of visible fluorescence after 13 h of incubation indicated the absence of E.coli in the sample.
 Concentration of E.coli was determined by comparing the FDT with corresponding cell count on the standard graph.
Working example II
 Bacto grade microbiological media ingredients- proteose peptone, tergitol
7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade)
and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable,
transparent, screw capped glass bottles/tubes were procured. »«« MUG broth was prepared using proteose peptone No. 3-5g, yeast extract-
3g, dipotassium hydrogen orthophosphate-0.3g, tergitol 7-0.1 ml, MUG-
0.04g, water 200ml. pH was adjusted to 7.2.
 MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating.
 Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per standard procedures.
 The MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.
 Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be dispensed in clean individual glass tubes/bottles and mixed thoroughly.
 Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap.
 After the tubes/bottles were cooled the caps were tightened and stored at room temperature. To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.
 l-20g of the test sample was added to the bottle and shaken well.
 To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.
 Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.
 Tubes/bottles were observed for blue fluorescence under long wave length (366mm) UV at hourly intervals till the detection of fluorescence or for 13 h which ever is early and FDT was recorded.
 Control tubes with no fluorescence serve as negative control. Absence of visible fluorescence after 13 h of incubation indicated the absence of E.coli in the sample.
 Concentration of E.coli was determined by comparing the FDT with corresponding cell count on the standard graph.
Working example III
 Bacto grade microbiological media ingredients- proteose peptone, tergitol 7, MUG, yeast extract, dipotassium hydrogen orthophosphate (AR grade) and clean (alkali and acid washed) river sand (10-30 mesh), autoclavable, transparent, screw capped glass bottles/tubes were procured from reputed manufacturers.
 MUG broth was prepared using proteose peptone No. 3-5g, yeast extract-3g, dipotassium hydrogen orthophosphate-0.3g, tergitol 7-0.1 ml, MUG-0.05g, water 200ml. pH was adjusted to 7.2.
 MUG broth then added to the clean sand in the ratio of 1:4 (v/w) and mixed thoroughly to get uniform coating. Screw capped glass tubes/bottles (20-200 ml capacity) were cleaned as per standard procedures. Then MUG broth coated sand (4-80g) was dispensed in cleaned glass tubes/bottles.
 Alternately 4-80g of cleaned sand and l-20ml of MUG broth could be dispensed in clean individual glass tubes/bottles and mixed thoroughly.
 Tubes/bottles were sterilized using moist heat under pressure in autoclave at 121° C for 15 minutes after replacing the cap. After the tubes/bottles were cooled the caps were tightened and stored at room temperature.
 To conduct the test 4-80ml of sterile/boiled and cooled potable water was added into the bottle containing MUG broth coated sand.
 l-20g of the test sample was added to the bottle and shaken well. To test water, 5-100ml of the water sample was added to the tubes/bottles and shaken well.
 Tubes/bottles were incubated at 25-40°C along with control containing only sterile/boiled water.
 Tubes/bottles were observed for blue fluorescence under long wave length
(366mm) UV at hourly intervals till the detection of fluorescence or for 13
h which ever is early and FDT was recorded.
 Control tubes with no fluorescence serve as negative control. Absence of
visible fluorescence after 13 h of incubation indicated the absence of E.coli
in the sample.
 Concentration of E.coli was determined by comparing the FDT with
corresponding cell count on the standard graph.
MUG concentration
A minimum of 20mg/L of MUG in the medium is required to get just detectable fluorescence and 30mg/L is effective contrary to lOOmg/L as described in prior art.
Table-1

(Table Removed)
ADVANTAGES
(i) The process and the test kit developed are simple and cost effective.
(ii) It is rapid and easy to perform and no skill/training required
(iii) It does not require laboratory facilities
(iv) Suitable for field conditions
(v) The test kit is stable for more than 1 year in room temperature
(vi) Concentration of E.coli in the sample can be obtained from the graph by
the determination of FDT. (vii) MUG concentration could be reduced to 30 mg/L contrary to lOOmg/L
as in conventional methods.

WE CLAIM:
1. A tool and method for rapid detection and enumeration of E. coli comprising sterile, non-fluorescent test container adaptable for opening for introduction of test sample and resealing after introduction of test sample having disposed therein a pre-dispensed test composition matrix, wherein the matrix is a biologically inert material, and test composition comprises of (i) source of nitrogen and other conventional micro nutrients, (ii) buffering agent, (iii) inhibitor capable of selectively inhibiting activity of gram positive bacteria, and a chemiluminescent agent capable of getting cleaved and activated by bacterial enzyme releasing substance to indicate presence of bacteria through colour/ fluorescence development.
2. A tool as claimed in claim 1, wherein the test container is transparent or
semitransparent glass or plastic airtight bottle or tube wherein the
adaptability of opening and resealing is imparted by providing stopper/ cap.
3. A tool as claimed in claims 1 to 3 wherein the biologically inert matrix is
sand/ plastic and or glass beads of 10 to 30 mesh.
4. A tool as claimed in claims 1 to 4 wherein the source of nitrogen and other
conventional nutrients is proteose peptone and yeast extract; buffering agent
is phosphate buffer; inhibitor is tergitol 7; chemiluminescent agent is 4-
methylumbelliferyl p-D glucuronoid (MUG).
5. A tool as claimed in claim 1 wherein test composition is MUG broth
consisting of proteose peptone in the range of -- 3 to 5 g, yeast extract — 3g,
buffer to adjust pH to 6.8 to 7.2, tergitol 7 O.lml MUG in the range of 20 to
1 OOmg/1, and water 200ml.
6. A method for rapid detection and enumeration of E. coli comprising
combining thoroughly in a test container, water, where water does not
constitute a test sample, and a test sample and observing fluorescence
detection time (FDT) and enumerating E. coli using standard graph.
7. A method as claimed in claim 6 wherein the matrix used is 4 to 5 times of
MUG broth and pre-dispensation is conducted by coating the matrix with
MUG broth.
8. A method as claimed in claims 7 & 8 wherein the water used for combining
ranges from 4 to 80 ml and test sample ranges from 1 to 20g.
9. A method as claimed in claims 7 to 9 wherein the FDT is performed under
long wavelength UV (366mm) at hourly intervals against water as blank and
enumeration is carried out comparing the FDT with corresponding colony
count on the graph.
10. A method for preparing tool consisting the following steps:
(i) preparing test composition (MUG broth),
(ii) contacting the said composition with the inert matrix in a
conventional manner such as here in described to get predispensed
test composition matrix, (iii) dispensing the said matrix in test container, sterilizing in a known
manner to obtain tool and storing at ambient temperature.
11. A tool and method for rapid detection and enumeration of E. coli and a
method therefor substantially as herein described.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=stuVqAiJA6f1igl/ZVkVvQ==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

278188

Indian Patent Application Number

1712/DEL/2006

PG Journal Number

53/2016

Publication Date

23-Dec-2016

Grant Date

16-Dec-2016

Date of Filing

26-Jul-2006

Name of Patentee

DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION, MINISTRY OF DEFENCE

Applicant Address

MINISTRY OF DEFENCE, GOVT. OF INDIA, WEST BLOCK-VII, WING-1, FIRST FLOOR, R.K. PURAM, SECTOR-1 NEW DELHI 110 066.

Inventors:

#	Inventor's Name	Inventor's Address
1	BHAGIRATHI BELMAN	DEFENCE FOOD RESEARCH LABORATORIES SIDDARTHANAGAR, MYSORE-570011
2	KOLPE RADHKRISHNA	DEFENCE FOOD RESEARCH LABORATORIES SIDDARTHANAGAR, MYSORE-570011
3	KADABA ANANTHARAMAN SRIHARI	DEFENCE FOOD RESEARCH LABORATORIES SIDDARTHANAGAR, MYSORE-570011
4	BAWA AMARINDER SINGH	DEFENCE FOOD RESEARCH LABORATORIES SIDDARTHANAGAR, MYSORE-570011

PCT International Classification Number

H01M 2/12

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA