The present invention relates to a water testing kit and method thereof. Particularly, the invention provides enzyme substrate medium based water testing kit and a simple method for the detection of microorganisms namely E. coli. in a water sample.
Background of the invention:
India suffers from problems in water quality and potable water availability. Water from large-scale centralized treatment plants run by municipalities or private water supply agencies, and water directly drawn from borewells are commonly used for drinking and household purposes. However, many water sources have bacteriological contamination due to release of untreated waste from industries, agriculture, and human settlements. Even piped water can be unreliable because of contamination from service lines. Currently in India, household water purification systems use methods such as reverse osmosis (RO), UV disinfection, membrane-based gravity filters and sediment filters. However, water purification systems can be expensive and unaffordable for most Indians. Therefore, majority of the Indian population who cannot afford an expensive water purification system lacks appropriate methods to treat their water and, as a consequence, faces severe long-term health risks. Furthermore, measurement of bacteriological contamination levels is difficult, and the lack of water quality data further exacerbates the problem. As a result, there is a need to develop low-cost, reliable, no-maintenance, on site, quick, and accurate bacteriological test kit to detect the presence or absence of bacteriological contamination in drinking water.

The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Bacteria are common single-celled organisms and are a natural component of lakes, rivers, and streams. Most of these bacteria are harmless to humans; however, certain bacteria, some of which normally inhabit the intestinal tract of warm-blooded animals, have the potential to cause sickness and disease in humans. High numbers of these harmless bacteria often indicate high numbers of harmful bacteria as well as other disease-causing organisms such as viruses and protozoans.
Escherichia coli (E. coli.) is a rod-shaped bacterium commonly found in the gastrointestinal tract and faeces of warm-blooded animals. Consumption of or contact with water contaminated with faeces of warm-blooded animals can cause a variety of illnesses. Minor gastrointestinal discomfort is probably the most common symptom; however, pathogens that may cause only minor sickness in some people may cause serious conditions or death in others, especially in the very young, old, or those with weakened immunological systems.

One method of determining bacteria counts is to count the number of bacteria colonies that grow on a prepared medium or through a culture method. In this method, when the medium multiplies, the color is changed. Some of the products available to detect E.coli. in drinking water includes Portable Lab Kits that includes membrane filtration, growth medium specific for E.coli., and a portable electric incubator; Fluorogenic presence or absence test that uses pre-prepared bottles, tubes, and incubation step, and the fluorescence under a UV light indicates presence of E.coli.; and pre-prepared plates with dehydrated growth media, which creates a gel when the sample is added. Once incubated for 24 hours, quantification is possible combining with membrane filtration. However, these methods requires technical skills and ancillary components, and hence expensive.
In addition to these methods, H2S presence or absence test kits that detect the presence of H2S producing bacteria including E.coli. Similarly, Most Probable Number (MPN) test involves a sterile collection bag, multi-well tray, and uses MPN method to quantify E.coli. Both these methods determine H2S production from bacteria rather than more specific markers of E.coli., hence the test yield false positive due to natural presence of many bacteria other than E.coli. in tropical soils (UNICEF TPP: Rapid E.coli Detection v 3.0).
In addition to these methods, PCR based methods also exists (Edberg, S., Rice, E., Karlin, R. and Allen, M. (2000), Escherichia coli: the best biological drinking water indicator for public health protection. Journal of Applied Microbiology, 88: 106S-116S. https://doi.org/10.1111/j.1365-2672.2000.tb05338.x; Rompré A, Servais P, Baudart J, de-Roubin MR, Laurent P. Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Methods. 2002 Mar;49(1):31-54. doi: 10.1016/s0167-7012(01)00351-7. PMID: 11777581.).
There is also enzyme substrate medium based method (S. K. Mitra, "Do-it-Yourself Kit for the detection of E. coli and total coliform in water", US non-provisional patent Application No. 17/347,793; S. K. Mitra, "Detection of water borne pathogens with paper strips", US non-provisional patent Application No. 17/245,052; Manafi M, Kneifel W, Bascomb S. Fluorogenic and Chromogenic substrate used in bacterial diagnostics, Microbiological Reviews, Sept. 1991, pages 335-348¬¬; Gunda NS, Chavali R, Mitra SK. A hydrogel based rapid test method for detection of Escherichia coli (E. coli) in contaminated water samples. Analyst. 2016 May 10;141(10):2920-9. doi: 10.1039/c6an00400h. PMID: 27137782)

In this method the sample is cultured in the presence of a substrate for enzymes specifically contained in E.coli. or E.coli. group, followed by analyzing the color. The intensity of the color can be correlated with E.coli. concentration. The most commonly used biomarker for E.coli. is ß-D-galactosidase (GAL). There are several substrates that produce color change in the presence of GAL enzyme, the most prominent out of them is 6-Chloro-3-indolyl-ß-D-galactopyranoside (also called Rose Gal, Salmon Gal or Red-Gal) (Gunda et al., Mobile Water Kit (MWK): a smartphone compatible low-cost water monitoring system for rapid detection of total coliform and E. coli, Anal. Methods, 2014,6, 6236-6246 ). The GAL enzyme produced by coliform bacteria hydrolyses the Red-Gal molecule producing red colour 6-Chloro-3-indolyl molecule. There are many enzymatic reaction-based detection methods that are robust (Gunda NS, Chavali R, Mitra SK. A hydrogel based rapid test method for detection of Escherichia coli (E. coli) in contaminated water samples. Analyst. 2016 May 10;141(10):2920-9. doi: 10.1039/c6an00400h. PMID: 27137782; Gunda et al., Mobile Water Kit (MWK): a smartphone compatible low-cost water monitoring system for rapid detection of total coliform and E. coli, Anal. Methods, 2014,6, 6236-6246).
However, the above conventional methods are not efficient and has problems, for example, in the culture method, in addition to the time until the bacteria grow by culture, a determination method of whether the bacteria that have grown is Escherichia coli or Escherichia coli is complicated. In other words, the culturing method requires one day only by estimating the presence of E. coli. or E. coli. group, and it takes two days to determine whether E. coli. or E. coli. group is mixed. Special dyeing and other tests need to be repeated, so it takes time to make a decision.
A gel based rapid test method for detection of Escherichia coli (E. coli.) in contaminated water samples. Analyst 141, 2920–2929 (2016) discloses Beta-galactosidase substrate-based method for detecting E. coli. It includes lysis buffer, so sensitivity is limited since bacteria cannot multiply too much. The detection E. coli limit of the method can be as high as 4 × 106 CFU/mL to 4 × 105 CFU/mL within 5 min and 4 × 104 CFU/mL to 400 CFU/mL within 60 minutes using the integrated plunger-tube assembly containing the gel matrix.

Without bacterial culture, it is unlikely to perform much better than 1-100 CFU/100 mL. It is to be noted that the World Health Organization deems water undrinkable if even one single E. coli. is present. Hence, such inventions are not very sensitive to low levels of contamination.
Hence, there is a need of an easy to use, on site, smart, quick, highly sensitive to up to 1 CFU/mL and cost-effective water testing kit for the detection of microorganisms in a water sample. Particularly, there is needed a kit and method to detect E. coli. in water samples which is on-site and which performs testing through smart, easy, quick, and accurate at low level of few CFU/mL. As E. coli. appears to provide the best bacterial indication of faecal contamination in drinking water, which further indicates the possibility of the presence of other waterborne pathogens having much more detrimental health impact.
There is also a need for a water testing kit based on smart-phone application for recording test results, instant time-stamps and geo-locations.

Object/s of invention:
Primary object of the present invention is to overcome the limitation of prior art and provide a robust E.coli. detection kit and method that is sensitive 1-100 CFU/100 mL range, does not require ancillary components such as portable incubator, plunger-tubes, autoclave for sterilizing equipment, plunger-tube assembly, UV lamps, and specific to E.coli.
Another object of the present invention is to provide kit and method to detect E. coli. in water samples on-site through smart, quick, and does not require any training to perform the test.
Another object of the present invention is to provide water testing kit based on smart-phone application for recording test results, instant time-stamps and geo-locations along with storing data in a cloud server.
Another object of the present invention is to provide kit which are highly efficient and cost effective for the detection of source-level water contamination.

Summary of the Invention:
In an aspect of the Invention, there is provided a low-cost kit and easy to use sensitive method for the detection of microorganisms namely E. coli. in a water sample comprising:
a) atleast a gravity/syringe based filtration mechanism comprising 0.22 um pore size filters to pre-concentrate 10 mL of sample water from 100 mL of sample water;
b) atleast a first vial pre-filled with Lauryl Tryptose Broth powder to be filled with 10 mL of water pre-concentrated from 100 mL of sample water to be tested obtained after the pre-concentration step;
c) atleast a second vial pre-filled with a gel present in an amount ranging from 100-1000 µL wherein the gel comprises:
i) agar or its purified form in an amount ranging from 2-20 mg
ii) Triton X-100 in 10 – 50mM Tris-HCl having pH 7.5-8.5 or other similar cell lysing agent in an amount ranging from 25-100 uL;
iii) Lauryl Tryptose Broth solution, present in an amount ranging from 100-400 uL;
iv) chemical solution/mixture present in an amount 50-200 uL, wherein said chemical solution/mixture comprises 6-Chloro-3-indolyl-ß-D-galactopyranoside present in an amount ranging from 20-90 mg, N, N-Dimethylformamide (DMF) present in an amount ranging from 1000-2000 uL, deionized grade water present in an amount ranging from 1000-2000 uL.

In another aspect of the Invention, a method of testing a water sample for the detection of microorganisms namely E. coli by the kit as described above. The method comprises the steps of:
a) pre-concentration of E. coli. bacteria using simple gravity/syringe filtration technique wherein the sample volume of the water to be tested is filtered through a 0.22 um filter paper to obtain the concentrated volume of 10 mL of the water sample;
b) pouring the pre-concentrated water sample obtained from step (a) in the first vial pre-filled with 0.3-0.5 g of Lauryl Tryptose Broth powder;
c) shaking the tube thoroughly for approx. 1 minute followed by keeping the tube inside the heating pouch (temperature would be maintained around 370C) or in ambient room temperature ranging from 30 to 400C for 20 hours;
d) adding the solution to the second vial pre-filled with a chemical infused gel present in an amount ranging from 100-1000 µL followed by keeping the vial inside the heating pouch (temperature would be maintained around 370C) or ambient room temperature ranging from 30 to 400C for 4 hours;
e) reading the results after completion of step d wherein no color change indicates absence of E. coli. while the appearance of purple color indicates presence of E. coli.;
f) clicking the picture/image of the color of the water sample of second vial and uploading the captured image in a software application using the cellular network.
Detailed description of Drawings:
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:
Figure 1: illustrates method to carry out the test with the 10 mL water sample, in one of its embodiment.
Figure 2: illustrates results of the tests performed to validate/ confirm that the claimed kit / method is efficiently determining the presence of E. coli. in the water sample.

Detailed description of the Invention:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components.

The invention provides a water testing kit and method thereof. Particularly, the invention provides technology for the onsite detection of E. coli. in water samples.

Particularly, the invention provides enzyme substrate medium based water testing kit for the detection of microorganisms namely E. coli. in a water sample.

In an embodiment, the invention provides kit and method to detect E. coli in water samples on-site through smart, easy, quick, and accurate way.

The test is very simple to perform and do not require skilled manpower. The method is very fast and the result can be seen in 24 hours. The color change is only sensitive to presence and absence of E. coli. The results can be determined by color change (identified by naked eye as well as smartphones). The testing kit does not contain multiple reagents. It comes with pre-filled powder and gels, and the user has to pour water to obtain results.

In an embodiment, the method comprises the steps of:
a) pre-concentration of E. coli. bacteria using a gravity/syringe based filtration technique
b) optionally, incubation inside heating pouch
c) enzymatic reaction that produce colour in the presence of E. coli.
In another embodiment, the water testing kit comprises smart-phone application for recording test results, instant time-stamps and geo-locations along with storing all data in a cloud server. The kit is useful for monitoring water quality on-site incubation-based E. coli. detection, and rapid processing at cloud database for public access. The kit based on the software application maps the E. coli. water quality data, through the software application, in one common cloud database for public access. The technology has several advantages over the existing H2S test kits that produce false positive due the presence of naturally occurring bacteria in tropical soils. The kit is highly efficient and specific to detect only E. coli. and cost effective for the detection of source-level water contamination.
In an embodiment, the invention provides a single test kit that can detect E. coli. in water samples at a detection level of 1 number/100 mL of water sample.

In an embodiment, the invention provides low-cost kit which is easy to use and is highly sensitive for the detection of microorganisms namely E. coli. in a water sample comprising:
a) atleast a gravity/syringe based filtration mechanism comprising 0.22 um pore size filters to pre-concentrate 10 mL of sample water from 100 mL of sample water;
b) atleast a first vial pre-filled with Lauryl Tryptose Broth powder to be filled with 10 mL of water pre-concentrated from 100 mL of sample water to be tested obtained after the pre-concentration step;
c) atleast a second vial pre-filled with a gel present in an amount ranging from 100-1000 µL wherein the gel comprises:
i) agar or its purified form in an amount ranging from 2-20 mg
ii) Triton X-100 in 10 – 50mM Tris-HCl having pH 7.5-8.5 or other similar cell lysing agent in an amount ranging from 25-100 uL;
iii) Lauryl Tryptose Broth solution, present in an amount ranging from 100-400 uL;
iv) chemical solution/mixture present in an amount 50-200 uL, wherein said chemical solution/mixture comprises 6-Chloro-3-indolyl-ß-D-galactopyranoside present in an amount ranging from 20-90 mg, N, N-Dimethylformamide (DMF) present in an amount ranging from 1000-2000 uL, deionized grade water present in an amount ranging from 1000-2000 uL.

In an embodiment, the kit comprises Lauryl Tryptose Broth solution which is prepared by mixing 0.3 – 0.5 g of Lauryl Tryptose Broth powder in 10 mL of deionized water to obtain a solution (a).

In an embodiment, the gel is prepared through the steps of:
a) Mixing 1000 to 2000 µL of DMF solution in 1000 to 2000 uL deionized water solution, and 20-90 mg of 6-Chloro-3-indolyl-ß-D-galactopyranoside powder to obtain a solution (b);
b) dissolving 2-10 mg of agar in 100-400 uL of Lauryl Tryptose Broth solution (a), 25-100 uL of Triton X-100 in 10 – 50mM Tris-HCl (pH 7.5-8.5), and 50-200 uL of solution (b)
c) Microwaving the solution for 1-2 minutes to obtain the gel.

In another embodiment, the gel composition comprises the components:
1) Agar gel or similar
2) Triton X-100 in Tris-HCl buffer or similar cell lysing agent
3) Lauryl Tryptose Broth (LTB) solution
4) 6-Chloro-3-indolyl-ß-D-galactopyranoside (also called Rose Gal, Salmon Gal or Red-Gal)
5) N, N-Dimethylformamide (DMF)
6) Deionized grade water

In an embodiment, the gel volume is 100-1000 uL.

In an embodiment, the gel comprises following components:
• agar gel present in an amount ranging from 2-20 mg,
• Triton X-100 in Tris-HCl buffer present in an amount of approx. 25-100 uL,
• 100-400 uL of Lauryl Tryptose Broth (LTB) solution,
• 50-200 uL of chemical mixture made with 6-Chloro-3-indolyl-ß-D-galactopyranoside present in an amount ranging from 20-90 mg,
• N, N-Dimethylformamide (DMF) present in an amount ranging from 1000-2000 uL,
• Deionized grade water present in an amount ranging from 1000-2000 uL.
In an aspect, the gel composition is prepared through the steps of:
Step 1: 1000-2000 uL DMF (solution)+1000-2000 uL deionized grade water (solution)+20-90 mg Red Gal (powder) is mixed in a vial, hereafter known as Red Gal solution
Step 2: Mix 50-200 uL of Red Gal solution with 25-100 uL of Triton X-100 in 10 – 50mM Tris-HCl (pH 7.5-8.5), 100-400 uL of LTB solution (mixture of LTB 0.3-0.5 gm of LTB powder in 10 mL of deionized grade water) and 2-10 mg of agar. Microwave for 1-2 minutes. The gel is ready.

In an embodiment, the method of testing a water sample for the detection of microorganisms by the kit as described above comprises the steps of:
pre-concentration of E. coli. Bacteria using filtration technique, incubation inside vial kept inside a heating pouch, and enzymatic reaction that produce colour in the presence of E. coli. The smart-phone application also records test results, instant time-stamps and geo-locations and stores all data in a cloud server. The technology has the capability to map the E. coli. water quality data in one common cloud database for public access. The method has several advantages over the existing H2S test kits, and will empower the masses with a low-cost E. coli. water testing kit to deal with source-level water contamination.

In another aspect, there is provided a method of testing a water sample for the detection of microorganisms by the kit as described above. The method comprises the steps of:
a) pre-concentration of E. coli. bacteria using simple gravity/syringe filtration technique wherein the sample volume of the water to be tested is filtered through a 0.22 um filter paper to obtain the concentrated volume of 10 mL of the water sample;
b) pouring the pre-concentrated water sample obtained from step (a) in the first vial pre-filled with 0.3-0.5 g of Lauryl Tryptose Broth powder;
c) shaking the tube thoroughly for approx. 1 minute followed by keeping the tube inside the heating pouch (temperature would be maintained around 370C) or in ambient room temperature ranging from 30 to 400C for 20 hours;
d) adding the solution to the second vial pre-filled with a chemical infused gel present in an amount ranging from 100-1000 µL followed by keeping the vial inside the heating pouch (temperature would be maintained around 370C) or ambient room temperature ranging from 30 to 400C for 4 hours;
e) reading the results after completion of step d wherein no color change indicates absence of E. coli. while the appearance of purple color indicates presence of E. coli.;
f) clicking the picture/image of the color of the water sample of second vial and uploading the captured image in a software application using the cellular network.
In an embodiment, the data is stored in cloud database for public access.
In an embodiment, the software application records test results, instant time-stamps and geo-locations along with storing the data in a cloud server wherein the software application based on the intensity of colour produced, quantifies the numbers of E. coli. present in the test samples.

The kit and the method of the present invention advantageously combines a pre-concentration step, optional incubation step inside heating pouch containing smart heat producing materials, and simple detection based in enzymatic reactions, to detect the presence of E. coli. in the water sample.

162 tests were performed with different type of water samples such as spiked E. coli samples, garden water, tap water, ground water. Deionized grade water, RO plant, laboratory water purification system. Results with 100% accuracy was found.

While this description has disclosed certain specific embodiments of the present invention for illustrative purposes, various modifications will be apparent to those skilled in the art which do not constitute departures from the spirit and scope of the invention as defined in the following claims, and it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

The Invention is further described with the help of non-limiting examples:

Example 1:
Pre-concentration step: A starting sample volume 100 mL to 500 mL that will be filtered through a 0.22 um filter paper and reduced to the 10 mL volume was taken for the testing. The filter will not allow the E. coli. to pass through, and as a result, all the E. coli. will be pre-concentrated in the 10 mL volume.

The kit comprises 2 vials and optionally a heating pouch/bag. A 15 mL vial that is pre-filled with Lauryl Tryptose Broth or similar powder. The vial needs to be filled with 10 mL water sample obtained from the pre-concentration step. Then we put it inside an inside a heated pouch, heating bag for 20 hours so that the temperature is maintained between 30-40oCelsius. Once 20 hours is over, the water sample from the 15 mL vial has to be poured into a 1.5 mL vial containing the gel. After 4 hours, if E. coli. is present, the colour changes to pink to purple confirming the presence of E. coli. In the sample. If there is no colour change, the sample do not contain any E. coli. bacteria.
Once the experiment is complete, a picture needs to be uploaded using a smart phone. The data will be stored in cloud database for public access

Example 2:
The gel composition comprises the components:
1) Agar gel or similar
2) Triton X-100 in Tris-HCl buffer or any other cell lysing agent
3) Lauryl Tryptose Broth (LTB)
4) 6-Chloro-3-indolyl-ß-D-galactopyranoside (also called Rose Gal, Salmon Gal or Red-Gal)
5) N, N-Dimethylformamide (DMF)
6) Deionized water

The gel volume is 100-1000 uL.

Example 3:
Preparation of the gel

The gel is prepared in 2 steps
Step 1: 1500 uL DMF (solution)+1200 uL deionized grade water (solution)+50mg Red Gal (powder) is mixed in a vial, hereafter known as Red Gal solution
Step 2: Mix 120 uL of Red Gal solution with 50 uL of Triton X-100 in 30mM Tris-HCl (pH 7.8), 200 uL of LTB (mixture of LTB 0.4 gm of LTB powder in 10 mL of deionized grade water) and 5 mg of agar. Microwave for 1-2 minutes. The gel is ready.

Example 4:
The Analysis of water sample was performed taking Sterilised water as Sample 1, contaminated Water as Sample 2 and Distilled water spiked with E.coli. from pure Culture as Sample 3. The following results were obtained. The results were found satisfactory.

Example 5:

REPEATABILITY TEST (E. coli.)
The experiments were performed to analyze the repeatability. Following results were obtained:

Interpretation:
Positive result: Media Color in small vial changed to Red
Negative result: No color change in small vial

Example 6:
Various tests were performed to assess the presence of E. coli. as per the claimed invention and the well-established methods in the market done through NABL accredited laboratories. The results validate/ confirms that the claimed kit / method is efficiently determining the presence of E. coli. in the water sample. The results are mentioned below and are also shown in Figure 2.

a)

b)

c)

d)

e)

Example 7:
Test Results of E. coli. spiked water samples at different levels of CFU.
E. coli. (BL21) cells were grown overnight (as primary culture) in LB or LT broth. Next day secondary inoculations were done with 1% primary inoculum in required volume of LT or LB broth. The cells were allowed to grow at 37°C till the OD 600~0.8-1. The cells were then pelleted and resuspended in equal volume of Mili-Q water. The resuspended cells were used in serial dilution for further gel assay. The concentrations are tentative.

Test Results of different types of water samples collected from various sources

We Claim:

1. A low-cost kit and easy to use sensitive method for the detection of microorganisms namely E. coli. in a water sample comprising:
d) atleast a gravity/syringe based filtration mechanism comprising 0.22 um pore size filters to pre-concentrate 10 mL of sample water from 100 mL of sample water;
e) atleast a first vial pre-filled with Lauryl Tryptose Broth powder to be filled with 10 mL of water pre-concentrated from 100 mL of sample water to be tested obtained after the pre-concentration step;
f) atleast a second vial pre-filled with a gel present in an amount ranging from 100-1000 µL wherein the gel comprises:
v) agar or its purified form in an amount ranging from 2-20 mg
vi) Triton X-100 in 10 – 50mM Tris-HCl having pH 7.5-8.5 or other similar cell lysing agent in an amount ranging from 25-100 uL;
vii) Lauryl Tryptose Broth solution, present in an amount ranging from 100-400 uL;
viii) chemical solution/mixture present in an amount 50-200 uL, wherein said chemical solution/mixture comprises 6-Chloro-3-indolyl-ß-D-galactopyranoside present in an amount ranging from 20-90 mg, N, N-Dimethylformamide (DMF) present in an amount ranging from 1000-2000 uL, deionized grade water present in an amount ranging from 1000-2000 uL.
2. The kit as claimed in claim 1, wherein Lauryl Tryptose Broth solution is prepared by mixing 0.3 – 0.5 g of Lauryl Tryptose Broth powder in 10 mL of deionized water to obtain a solution (a).
3. The kit as claimed in claim 1, wherein the gel is prepared through the steps of:
d) Mixing 1000 to 2000 µL of DMF solution in 1000 to 2000 uL deionized water solution, and 20-90 mg of 6-Chloro-3-indolyl-ß-D-galactopyranoside powder to obtain a solution (b);
e) dissolving 2-10 mg of agar in 100-400 uL of Lauryl Tryptose Broth solution (a), 25-100 uL of Triton X-100 in 10 – 50mM Tris-HCl (pH 7.5-8.5), and 50-200 uL of solution (b)
f) Microwaving the solution for 1-2 minutes to obtain the gel.

4. The kit as claimed in claim 1, wherein the microorganisms comprises E. coli.
5. A method of testing a water sample for the detection of microorganisms by the kit as claimed in claim 1, comprising the steps of:
g) pre-concentration of E. coli. bacteria using simple gravity/syringe filtration technique wherein the sample volume of the water to be tested is filtered through a 0.22 um filter paper to obtain the concentrated volume of 10 mL of the water sample;
h) pouring the pre-concentrated water sample obtained from step (a) in the first vial pre-filled with 0.3-0.5 g of Lauryl Tryptose Broth powder;
i) shaking the tube thoroughly for approx. 1 minute followed by keeping the tube inside the heating pouch (temperature would be maintained around 370C) or in ambient room temperature ranging from 30 to 400C for 20 hours;
j) adding the solution to the second vial pre-filled with a chemical infused gel present in an amount ranging from 100-1000 µL followed by keeping the vial inside the heating pouch (temperature would be maintained around 370C) or ambient room temperature ranging from 30 to 400C for 4 hours;
k) reading the results after completion of step d wherein no color change indicates absence of E. coli. while the appearance of purple color indicates presence of E. coli.;
l) clicking the picture/image of the color of the water sample of second vial and uploading the captured image in a software application using the cellular network;

6. The method as claimed in claim 5, wherein the data is stored in cloud database for public access.
7. The method as claimed in claim 5, wherein the software application records test results, instant time-stamps and geo-locations along with storing the data in a cloud server wherein the software application based on the intensity of colour produced, quantifies the numbers of E. coli. present in the test samples.
8. The method as claimed in claim 5, wherein the microorganism comprises E. coli.