Rapid detection kit for water pollution indicator bacteria without incubator

Abstract

This invention relates to a portable kit for rapid detection of microbial contamination of water, which includes two vials containing lactose broth culture medium and bromothymol blue color indicator, a dropper for sample collection, and an activatable heating pad. The main innovation of this invention is the use of an insulated box covered with a foil layer and, together with the heating pad, acting as an independent incubator. The vials are fixed inside a cylindrical foam and the sample, after addition, is placed at the appropriate growth temperature without the need for an incubator. This system allows for rapid and field detection of water-polluting bacteria, especially coliforms and Escherichia coli.

Description

Description of the invention Title of the invention Rapid detection kit for water pollution indicator bacteria without incubator Technical background of the relevant invention This invention falls into the fields of water microbiology, sanitary quality control, rapid microbial detection technologies, colorimetric kits, and environmental monitoring tools. The purpose of this invention is to provide a portable, rapid, low-cost, and laboratory-free kit for the detection of Escherichia coli and coliforms in water samples. Technical problem and stating the objectives of the invention Common methods for detecting microbial contamination of water include: Classical culture in LST, BGLB and EMB environments Membrane filter Multi-step methods These methods have the following problems: 1. Long time (24 to 48 hours) 2. Need for incubators, test tubes, autoclaves and multiple environments 3. Need for a specialized expert 4. Possibility of secondary contamination 5. High cost and limited use in areas without laboratories Objectives of the invention: 1. Providing a rapid kit with a detection time of 4 to 8 hours 2. Possibility of visual detection without tools 3. Completely eliminating the need for an incubator with a portable heat box 4. Reducing costs and enabling domestic production 5. Suitable for field monitoring, crises, villages and areas without facilities 6. Increasing accuracy by using a control vial to prevent errors A description of the state of the prior art and the history of developments related to the claimed invention. Detection of microbial contamination of water, especially the identification of indicator bacteria such as coliforms and Escherichia coli, has long been one of the main indicators for assessing the hygienic quality of drinking water and water resources. Conventional methods for detecting these bacteria are mainly based on microbial cultivation in dedicated media and incubation under controlled temperature conditions. Among the well-known methods, the maximum probable number (MPN) method can be mentioned, which is based on the use of lactose broth culture medium and observation of acid and gas production by lactose-fermenting bacteria. This method has been widely approved in international standards, including the Standard Methods for the Examination of Water and Wastewater published by APHA, as well as the guidelines of the World Health Organization (WHO). However, the implementation of this method requires laboratory equipment such as an electric incubator, sterile glassware, appropriate laboratory space, and a relatively long incubation time (usually 24 to 48 hours).In recent years, ready-to-use kits for faster detection of microbial contamination in water have also been introduced, including colorimetric and fluorescence-based kits that indicate the presence of coliforms and E. coli by changing the color of the medium or producing a light signal (such as ONPG-based and MUG-based kits). Although these kits have simplified the testing process, they are often still dependent on a laboratory incubator, a stable power supply, and controlled environmental conditions, and their use in completely field conditions, remote areas, or critical situations (such as natural disasters) is limited. Some portable solutions have also been presented in the form of semi-automatic devices or warming chambers, but these systems usually have a complex structure, high cost, or dependence on batteries and electronic equipment, and are not suitable for widespread and simple use by non-specialist users.Therefore, despite the advances made in the field of microbial water contamination detection, there is still a need for a rapid, low-cost, fully portable, and independent detection system from laboratory equipment and municipal electricity that can provide the process of culturing and growing indicator microorganisms directly at the sampling site and in field conditions. Non-inventive sources: 1. APHA, AWWA, WEF Standard Methods for the Examination of Water and Wastewater, Latest Edition. 2. World Health Organization (WHO) Guidelines for Drinking-water Quality. 3. ISO 9308-1 Water quality — Enumeration of Escherichia coli and coliform bacteria. 4. Edberg et al., 2000 Escherichia coli: The best biological drinking water indicator for public health protection. Journal of Applied Microbiology Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention General structure of the kit: The kit includes the following components: 1. Control vial containing lactose broth + BTB, without adding a sample, its color remains green throughout the entire experiment and is used for comparison. 2. Test vial containing the same culture medium (lactose broth + BTB), a water sample is added to it. In the presence of Escherichia coli and coliforms: Bacteria ferment lactose, acid is produced, pH decreases, BTB color changes from green to yellow, this change occurs within 4 to 8 hours. 3. Portable thermal incubation box This box has the following features: Thermal insulation to maintain temperature Equipped with a heat pad Creating a temperature of 35 to 37 degrees without electricity and without an incubator Suitable for the growth of Escherichia coli Can be used in environments without a laboratory 5.. Scientific mechanism of detection: 1. Coliform and Escherichia coli bacteria have the ability to rapidly ferment lactose. 2. The fermentation product is acid. 3. Lowering the pH causes the BTB color to change from green to yellow. 4. The healthy sample remains green. 5. The control vial serves as a reference for the color change. Explanation of shapes, maps and diagrams Figure (1): Schematic view of the components of the incubator-free water contamination indicator bacteria detection kit, which includes (1) insulated box, (2) inner foil cover of the box, (3) foam support, (4) test vial, (5) control vial, (6) heat-activated pad placed next to the vials, and (7) sample collection dropper. Figure (2): Block diagram showing the process of detecting microbial contamination of water using the provided kit. (8) Taking a suspicious water sample (9) Adding the sample to the test vial (10) Placing in the chamber (11) Creating the appropriate temperature (12) Observing the color change A clear and precise statement of the advantages of the claimed invention over prior inventions. Compared to methods, commercial kits, and prior art, the present invention provides a portable kit for rapid detection of microbial water contamination that operates without the need for an electric incubator. While non-patented sources and prior art rely mainly on laboratory incubators and stationary equipment, this invention provides suitable temperature conditions for bacterial growth independently by utilizing an insulated box covered with a foil layer and an activated thermal pad. In this invention, vials containing culture medium are stabilized inside a cylindrical foam and the sample is placed at the appropriate growth temperature after addition, without the need for specialized equipment. This feature enables rapid and field detection of water contamination indicator bacteria, including coliforms and Escherichia coli. Thus, the present invention has advantages over prior methods and inventions, including portability, ease of use, cost reduction, and elimination of dependence on laboratory equipment.Top of FormBottom of Form #x200fExplanation#x200f#x200e #x200e#x200fAt least#x200f#x200e #x200e#x200fA#x200f#x200e #x200e#x200fMethod#x200f#x200e #x200e#x200fExecution#x200f#x200e #x200e#x200fFor#x200f#x200e #x200e#x200fTo#x200f#x200e #x200e#x200fApplication#x200f#x200e #x200e#x200fInvention#x200f 1. The two vials in the kit include a control vial and a test vial, each containing lactose broth medium and bromothymol blue indicator. Before performing the test, the vials are visually inspected to ensure that the lid is intact, there is no leakage, and the medium is clear. 2. Adding the sample to the test vial A specific amount of water sample (usually 2 ml, or according to the instructions included in the kit) is added to the test vial using a dropper or sterile syringe. The control vial is left without adding the sample. 3. Mixing and closing the vials After adding the sample, the vial is gently shaken to homogenize the contents. Both vials are completely closed and ready for incubation. 4. Placing the vials in the kit's thermal box The control vial and the test vial are placed in a thermally insulated box. The heating pad in the activated box is activated by opening the lid and exposing it to air, creating and maintaining a temperature of 35 to 37 degrees Celsius. This temperature is suitable for the growth of coliform and Escherichia coli bacteria, acting as an incubator 5.Incubation and waiting for the reaction The vials remain in the box for 4 to 8 hours to allow the fermentation process to take place if lactose fermenting bacteria are present. 6. Observe the color change and interpret the result after the end of the incubation time: The control vial should remain green. The test vial changes from green to yellow if Escherichia coli or lactose fermenting coliforms are present. No color change means that the target bacteria are not present in the sample. 7. Record the result and safely dispose of the vials After observing the result, the vials are collected as low-risk biological waste and disposed of according to the kit instructions or local regulations. Explicit mention of the industrial application of the invention 1. Urban and rural water and wastewater companies for rapid assessment of microbial contamination of water sources, reservoirs, wells and distribution networks. 2. Water and wastewater treatment plants Continuous monitoring of the performance of chlorination, filtration and ensuring the removal of microbial contamination. 3. Water quality control laboratories Use as a rapid supplementary or screening method before conducting standard culture tests. 4. Food and beverage production plants Assessment of the quality of water consumed in the production, washing and processing process. 5. Livestock, poultry and aquaculture farms Monitoring of water pollution that directly affects animal health 6. Tourism and service industries including hotels, resorts, swimming pools and recreational centers for water hygiene control 7. Crisis management centers and field relief teams Possibility of monitoring water pollution in emergency situations without the need for a laboratory. 8. Use in environments without laboratory equipment, such as rural areas, small workshops, field projects, and environmental missions.

Claims

Complaint What is claimed: Claim 1) The rapid detection kit for microbial water contamination includes at least two vials containing lactose broth culture medium and bromothymol blue color indicator, a dropper for adding water samples, an activated heat pad, and an insulated box covered with a foil layer, so that the vials are placed inside the protective foam inside the box and the heat pad, by creating appropriate temperature conditions, allows bacteria to grow without using an incubator.