WO2019064307A1

WO2019064307A1 - Process for preparation of a chromogen based tool

Info

Publication number: WO2019064307A1
Application number: PCT/IN2017/000120
Authority: WO
Inventors: Priti WARKE
Original assignee: Himedia Laboratories Pvt Ltd
Priority date: 2017-09-27
Filing date: 2017-09-27
Publication date: 2019-04-04

Abstract

A process for preparing a tool for finding identity of microorganisms using reaction demonstrating realease of chromophore from a specific chromogenic substrate by action of specific enzymes released during growth of that microorganism. This tool is useful for speedily identification of the microorganism class from various samples such as clinical samples, water samples, food samples, pharmaceuticals, environmental samples, etc.

Description

Process for Preparation of a Chromogen Based Tool

FIELD OF INVENTION

The present invention relates to a process of preparing a tool using substrate for finding speedily identity of the microorganisms from clinical samples, water samples, food samples, pharmaceuticals and, environmental samples.

BACKGROUND OF THE INVENTION

Detection and finding identity of microorganism is necessity of various sectors such as pharma, clinical, water, dairy, food, environment, etc. The conventional method for finding identity of microorganism involves plating the sample on the agar medium, incubating the medium with sample for 24-48 hrs for bacterial growth and for 5-7days for yeasts and molds. The colonies grown are then inoculated on selective diagnostic media for finding identity of microorganisms. The media have to be incubated for further 24-48 hrs. Further, biochemical characterization of the organism is performed for confirmation. Thus current practices of microbial identification consume time and achieving results involve long time and manual labor. The other factor is high cost of growth medium and availability of certain reagents.

To ease the identification of microorganisms, recent practice is to use chromogenic, fluorogenic and non-chromogenic enzyme indicators in the medium. A chromogen or chromogenic substrate is a substance that is acted upon by an enzyme to produce a pigment or a dye. A chromophore is a group on a chromogen that produces a colour when a chromogen is cleaved by an enzyme. A fluorogen or fluorogenic substrate is a non- fluorescent material that is acted upon by an enzyme to produce a fluorescent compound. A fluorophore is a group on a fluorogen that is responsible for the fluorescence when a fluorogen is cleaved by an enzyme.

US patent No. 7141387 discloses a culture medium with chromogen for finding identity of yeasts and molds. US patent No. 8008059 discloses use of fluorogenic or chromogenic enzyme substrates for finding identity of microorganisms. The present invention discloses a process for speedy, reliable, simple and economical way for finding identity of microorganisms. The invention reduces the process time of identifying microorganism to 1-4 hrs and saves time, labor and expenses involved as compared to conventional finding identity methods. SUMMARY OF THE INVENTION

The present invention relates to a process for preparing a tool and to a tool with a reaction demonstrating substrate such as chiomogen, fluorogen or dye for finding speedily identification of the microorganisms. The said tool can be of any absorbing or adsorbing material on which substrate aiding for finding identity of microorganisms is absorbed or adsorbed. The use of the tool reduces time for finding identity of microorganism to 1-4 hrs.

DETAILED DESCRIPTION OF THE INVENTION

The finding identity of microorganisms is being accomplished using various selective media and biochemical tests. Identifying a microorganism involves use of enriched and selective media. The isolate is often presumptively identified morphologically by special staining methods and plated out on diagnostic media. Elaborate biochemical tests are required for confirmation of the isolate recovered. Thus, the entire process is time consuming, expensive and laborious. The instance invention gives solution for these problems by providing a tool for rapid reliable, simple and economical diagnosis.

The invention disclosed here is based on the utilization of specific substrates that can be chromogenic and fluorogenic for detection of activities of specific enzymes of microorganism. These sensitive methods have led to improved accuracy and faster detection and may be performed by using primary isolation media thus bypassing the need for time-consuming and laborious isolation procedures prior to finding identity. The tool described herein can be sterilized after incubation and the same can be preserved as permanent records.

The invention comprises of absorbent or adsorbent material used as a tool with chromogens. The tool is placed on the agar plate with microbial colonies. The agar plate with the tool can be incubated or the tool can be removed and incubated further for 1-4 hrs colour development. The organism can be identified from the colours developed on the tool.

The tool can be prepared from any material having the absorbing or adsorbing capacity such as textile or fabric material, non-woven substrate, cotton wool glass fiber, range of available filter papers, membrane filter paper, millipore filter paper, cotton, sponge, paper, cellulose ester sheet, fiber based material, synthetic material, polypropylene material, perforated cloth or plastic material having porous structure. It can be designed as a circle, square, rectangle or any other shape.

Any substance which is known to exhibit specific activity through which target microorganisms can be identified can be used as substrate and impregnated on the too! for the finding identity of microorganisms. The substance used can be an acidic dye, basic dye, pH indicator, chemical compound, chromogenic or fluorogenic substance, carbohydrates, amino acids, nutrients and all other substrates that serves as a base in the finding identity. The substance can be used individually or as a mixture of the compounds. The substrates are selected on the basis of their ability to detect specific organisms.

Substrates like indoxyl- -D-glucuronide (IBG) or 5-bromo-4-chloro-3-indolyl-p-D- glucuronide (X-GLR) are used for specific detection of E. coli. E coli metabolizes IBG/ X- GLR to release chromophores. Metabolism of IBG by E. coli via glucuronidase activity releases characteristic blue colour which is easily detectable by unaided eyes. When X- GLR is used, colonies with enzymatic activity show purple colour. Indoxyl butyrate greyhound can be used as substrate for finding identity of Pseudomonas and Klebsiella. The coliform group of organisms within the family Enterobacteriaceae can be detected by using chromogenic substrates such as o-nitrophenyl- -D-galactopyranoside (ONPG), p- nitrophenyl-p-D-galactopyranoside or 6-bromo-2-napthyl-P-D-galactopyranoside (BNGAL).

The tool for finding identity of the microorganisms of interest is prepared by treating the base material with the specific substrate and other excipients. The material serving as a base for the tool and the substrate being used along with the excipients are sterilized separately. Alternatively, the base material may be sterilized after the substrate mixture is incorporated into the base material. Method of sterilization can be either dry heat sterilization, moist heat sterilization, radiation sterilization such as gamma irradiation

The solvent used for the dissolution of the individual substrate or the mixture of different substrates can be water, methanol, ethanol, ether, NaOH, HCI, acetone dimethylforaiamide, alcohol, organic solvents or inorganic solvents or a mixture thereof depending upon the type of the substrates used. The substrates can either be individually dissolved and the solutions can be used individually for loading on the base material or then solutions of the individual substrates can be mixed and used for loading or the mixture of the substrates may be dissolved in specified solvent. The pH of the solution is adjusted such that it falls within the range of the respective substrate/ dye/ chromogen. The pH adjustment may be done before or after the sterilization of the substrate solution. The individual substrate solution can be used for soaking the base material/ impregnation on the base material/ for dipping the base material or for spraying on the base material one followed by the other or the mixture solution of the substrates can be used for soaking the base material/ impregnation on the base material/ for dipping the base material or for spraying on the base material. The base material may be treated with other excipients to retain viability of microorganisms.

The base material with the substrate is dried under controlled conditions to retain the potency of the substrates. Humidity is controlled below 45RH. The temperature used for drying could be from 20°C to 90°C under controlled conditions. The material once dried should be preserved at temperature of -20°C to 25°C away from bright light and in a dry and cool place. The tool thus manufactured and stored at recommended conditions has an optimum performance within a period of one year (Not to mention or more than 1 year).

The standardization of the substrate for the desired target organism is carried out using standard microbial cultures from ATCC.

Example 1

The tool for finding identity of the microorganisms of interest is prepared by treating the base material with the specific substrate. Finding identity tool for E. coli: Any material with adsorbing or absorbing potential can be used as base material. Cellulose ester sheet is used as base material. The base material- cellulose ester sheet is treated with the substrate indoxyl^-D-glucuronide (IBG) by soaking. The substrate treated base material is sterilized. The base material is then dried at 40-120°C, preferably 40-70°C. The tools thus prepared are stored at temperature of -20°C to 30°C away from bright light and in a dry and cool place.

Example 2

The tool for finding identity for Salmonella: Cellulose based Membrane filter paper is used as a base material. The base material is treated with the substrate 5-bromo-4-chloro-3- indolyl- -D-galactopyranoside (X-GAL) by soaking. The substrate treated base material is sterilized. The base material is then dried at 40-120°C, preferably 40-70°C. The tools thus prepared are stored at temperature of -20°C to 30°C. away from bright light and in a dry place.

Example 3 The sample from which microorganisms are to be identified/ the microorganisms to be identified are inoculated on general purpose media such as Nutrient Agar, Soyabean Casein Digest Agar, Plate count agar etc. Any of the surface plating methods: Four quadrant streak pattern, T streak method or Spread plate method may be adapted for inoculation. The inoculated medium in the plate is incubated at 20-40 °C (preferably 35- 37°C) for 18-24 hrs. The plate is checked for growth of microorganism.

Finding identity of E. coli: The tool for finding identity of microorganism prepared as in example 1 is placed on the medium with microbial growth. The orientation of the finding identity tool is marked in correspondence to the microbial growth medium plate. The time for tracing the microbial growth from the medium onto the finding identity tool is 20 to 120 sec, preferably 30-40 sec. The said tool with microbial growth traced on it is incubated at 20-40°C for maximum period of 4 hrs. The incubated tool is observed for blue colonies of E. coli. Standard run of E. coli is also carried out along with the sample. Finding identity of Salmonella: The tool for finding identity prepared as in example 2 is placed on the medium with microbial growth. The orientation of the said tool is marked in correspondence to the microbial growth medium plate. The time for tracing the microbial growth from the medium onto the finding identity tool is 20 to 120 sec, preferably 30-40 sec. The tool with microbial growth traced on it is incubated at 20-40°C for maximum period of 4 hrs. The incubated tool is observed for purple blue colonies of Salmonella. Standard run of Salmonella is also carried out along with the sample.

Claims

What is claimed is:

1. A process for the manufacture of a tool for finding identity of microorganisms wherein the base material for the tool is treated with the substrate known to exhibit specific activity through which target microorganisms can be identified.

2. A process for the manufacture of a tool for finding identity of microorganism as claimed in claim 1 wherein the base material for the tool can be any material having the absorbing or adsorbing capacity such as textile or fabric material, non- woven substrate, cotton wool glass fiber, range of available filter papers, membrane filter paper, Millipore filter paper, cotton, sponge, paper, cellulose ester sheet, fiber based material, synthetic material, polypropylene material, perforated cloth or plastic material having porous structure.

3. A process for the manufacture of a tool for finding identity of microorganisms as claimed in claim 1 wherein the substrate for the treatment of the base material can be an acidic dye, basic dye, pH indicator, chemical compound, chromogenic or fluorogenic substance, carbohydrates, amino acids, nutrients and all other substrates that serves as an base in the finding identity

4. A process for the manufacture of a tool for finding identity of microorganisms as claimed in claim 1 wherein the pH of the substrate solution is adjusted such that it falls within the range of the respective substrate

5. A process for the manufacture of a tool for finding identity of microorganisms as claimed in claim 1 wherein the base material and substrate are sterilized separately and then the base material is treated with the substrate or the base material is sterilized after treating with the substrate.

6. A process for the manufacture of a tool for finding identity of microorganisms as claimed in claim 1 wherein the treatment of the base material with the substrate solutions is carried out by soaking the base material/ impregnation on the base material/ dipping the base material or by spraying the substrate solution on the base material one substrate solution followed by the other or the mixture solution of the substrates can be used for soaking the base material/ impregnation on the base material/ for dipping the base material or for spraying on the base material.

7. A process for the manufacture of a tool for finding identity of microorganisms as claimed in claim 5 wherein the base material is then dried at temperature from 20°C to 120°C

8. A tool for finding identity of microorganisms wherein the base material for the tool is treated with the substrate known to exhibit specific activity through which target microorganisms can be identified.

9. A tool for finding identity of microorganisms as claimed in claim 7 wherein the base material for the tool can be any material having the absorbing or adsorbing capacity such as textile or fabric material, non-woven substrate, cotton wool glass fiber, range of available filter papers, membrane filter paper, mUlipore filter paper, cotton, sponge, paper, cellulose ester sheet, fiber based material, synthetic material, polypropylene material, perforated cloth or plastic material having porous structure.

10. A tool for finding identity of microorganisms as claimed in claim 7 wherein the substrate for the treatment of the base material can be an acidic dye, basic dye, pH indicator, chemical compound, chromogenic or fluorogenic substance, carbohydrates, amino acids, nutrients and all other substrates that serves as an base in the finding identity

1 1. A process for the finding identity of microorganisms with the help of a tool for finding identity of microorganisms comprising of the base material treated with the substrate known to exhibit specific activity through which target microorganisms can be identified wherein a] The sample/ organisms from the sample are inoculated on general purpose media such as Nutrient Agar, Soyabean Casein Digest Agar, Plate count agar etc. by adapting any of the surface plating methods - Four quadrant streak pattern, T streak method or Spread plate method b] Incubating the sample/ organisms from the sample with medium at 20-40° C for 18-48 hrs depending upon the species and checking for the growth of microorganisms c] Placing the tool for finding identity of microorganisms (treated with the substrate respective to the microorganism of interest) with marked orientation on the surface of the agar plate having bacterial growth for minimum 20 sec and maximum 40 sees to trace the growth on the tool. d] Incubating the tool having traced microorganisms in empty sterile Petri dish at 20-40^c C for 1-4 hrs. e] Observing the colour development after incubation and establish identification of the organism from the colour developed from the substrate.