Title of Invention

"A METHOD FOR DETERMINATION OF BIO-POTENCY BY LETHAL DOSE TITRE-50 (LDt 50)OF LIVE ANTAGONIST AGAINST PATHOGEN"

Abstract

A method is proposed for determining the lethal dose titre value of 50% killing of live pathogen by using live form of biopesticides. The method consists of first determining the titre value of starter/seed culture by preparing their serial dilutions (101 to 1010) in nutrient broth, after incubation at 37°C±7°C. A control antagonist set of live antagonist titre, antagonist-pathogen interaction set and a control pathogen tube are prepared separately and their optical densities are measured using a colorimeter. The differences in optical densities of interaction set (z) and control antagonist set (y) are recorded. That antagonist titre dilution in the respective tube whose (z-y) value equals x/2 where x is optical density of control pathogen tube, determines LDtso. Alternately LDt50 is determined by preparing antagonist-pathogen interaction set and control pathogen set on agar petriplates. That antagonist titre dilution on the interaction petriplate showing a/2 number of pathogen colonies where 'a' is total number of pathogen colonies in control pathogen plate, deterfines LDt50.

Full Text

FIELD OF INVENTION This invention relates to a method for determination of bio-potency by lethal dose Titre-50 (LDt50) of live antagonist against pathogen. BACKGROUND OF INVENTION The live form of microbial biopesticides (antagonist cultures) before its use indiscriminatley, should first be evaluated for its accurate antagonistic efficacy against fish pathogens and associated human enteric pathogens. The existing LD so method for determining lethal potency of chemical form of pesticides against pest cannot be adopted for determining lethal efficacy of live biopesticides against live form of pathogens. OBJECTS OF PRESENT INVENTION The main object of the present invention is to propose a method for determination of bio-potency by lethal dose Titre-50 of live antagonist against pathogen. DESCRIPTION OF FIGURES The invention is illustrated with accompanying figures wherein:- Fig.l:- shows the flow chart for determination of titre value of starter/seed culture equivalent to 100 number of colonies. Fig.2:- shows the flow chart for preparation of final serial dilutions of live antagonist titre for determination of LDt 50 by cell density method. Fig.3- shows the flow chart for preparation of final serial dilutions of live antagonist titre for determination of LDtso by colony count method. STATEMENT OF INVENTION This invention proposes a method for determination of bio-botency by lethal dose Titre 50 (LDtso) of live antagonist against pathogen. The method consists of firstly preparing a starter seed culture of both live antagonist as well as pathogen by incubating at 37°C ± 7° for 20-40 hours on nutrient agar plates, separately and finding out the delution which gives 100 well-isolated colonies. Thereafter the LD t so is determined either by cell density method or by colony count method. In cell density method, final serial dilutions of live antagonist titre are first prepared in test tubes, one containing undiluted antagonist titre and remaining containing ten different serial dilutions 101 to 1010 of antagonist titre in nutrient broth. Control antagonist set is prepared by transferring 0.1ml aliquote from each of above tubes to another set of tubes containing 9.9ml sterile nutrient broth and incubating at 37°C±7° for 24 hours. This is followed by preparing antagonist-pathogen interaction set by mixing aliquotes from above antagonist titre dilution tubes, with undiluted pathogen titre and allowing cell-cell interaction for 30minutes. Further a control pathogen tube is prepared by incubating 0.1ml of undiluted pathogen for 24 hours at 37°C±7°. Thereafter optical density of control antagonist set, interaction set and control pathogen tube are measured using a colorimeter. The optical density of pathogen is determined by finding the difference between optical density of interaction (2) and that of antagonist tube (y). LDtso is determined by finding that antagonist titre dilution whose z-y is equal to half of the optical density of control pathogen. In colony count method, serial dilutions are prepared in the same way as above while preparation of control antagonist set is not necessary in this method as only pathogen colonies are to be counted. Interaction set is prepared in same way except that 0.1ml of antagonist titre and 0.1ml undiluted pathogen titre are asceptically transferred on agar petriplates and then incubated at 37°C±7°C for 24 hours and total number of pathogen colonies are counted. Control pathogen plate is prepared separately by incubating one agar petriplate which is inoculated with 0.1ml undiluted pathogen titre. LDt so is determined by finding that antagonist-titre dilution on the interaction petriplate which shows half the number of pathogen colonies in control pathogen plate. DESCRIPTION OF INVENTION According to this invention, the method for determination of bio-potency by lethal Dose titre 50 of live antagonist-against pathogen, comprises of the following steps.- STEP 1- PREPARATION OF STARTER/SEED CULTURE: For this purpose, the cultures of both i.e. live antagonist as well as pathogen from stock cultures are streaked on nutrient agar plates and incubated at 37°C±7°C for 20-40 hrs. These 24 hrs fresh cultures are transferred to nutrient broth tubes and incubated for growth of bacteria 37°C±7°C for 20-40 hrs. This subculturing in nutrient broth tubes is repeated 2-3 times and used as starter/seed cultures of an antagonist and pathogen. While transferring an inoculum most precautiously, a care is taken to maintain the microbial load per inoculum as uniform as possible by applying same pressure on the inoculation needle, covering the same area of microbial lawn, using the same volume of the culture medium etc. STEP-II - DETERMINATION OF TITRE VALUE OF STARTER/SEED CULTURE EQUIVALENT TO 100 NUMBER OF COLONIES: Using the standard procedure of serial dilution technique, ten different serial dilutions of the starter seed cultures of both antagonist and respective pathogen are prepared in nutrient broth and plated (0.1 ml) on nutrient agar plates separately. After incubation at 37°C±7°C for 20-40 hours, the plates which showed around 100 well isolated colonies are selected and respective dilution is considered as titre value of that organism giving nearly 100 well isolated colonies. STEP-IH- DETERMINATION OF BIO-POTENCY AS LDt5o BY MEASURING CELL DENSITY USING COLORIMETER fie. CELL DENSITY METHOD): This step comprises of following:- (i) Preparation of final serial dilutions of live antagonist titre:- Eleven test tubes, one containing undiluted antagonist titre and the remaining containing ten different serial dilutions (101 to 1010) of the antagonist titre in nutrient broth are prepared. (ii) Control antagonist set:- A 0.1 ml aliquote from each of the above tubes is separately transferred asceptically into another similarly labelled eleven tubes respectively, each containing 9.9 ml sterile nutrient broth. All the tubes are incubated at 37°C for 24 hrs, in an incubator-cum shaker. (iii) Antagonist-Pathogen Interaction set:- In an another set of similarly labelled eleven test tubes, aliquotes from each of the above antagonist titre dilution tubes are separately mixed with the undiluted pathogen titre on a volume per volume basis. The microbial cells (antagonists and Pathogens) are allowed to interact with each other at room temperature for exactly 30 min with constant shaking on a rotary shaker. After the cell-cell interaction for 30 min, 0.2 ml aliquotes from each of these eleven interaction tubes (which contains 0.1ml antagonist titre dilution and 0.1ml undiluted pathogen titre since equal volumes of both antagonist and pathogen cultures are mixed) are aseptically inoculated separately into a fresh lot of similarly labelled eleven tubes respectively, each containing 9.8 ml nutrient broth. All these eleven tubes after inoculation are incubated at 37°C for 24 hours in an incubator-cum shaker. (iv) Control Pathogen Tuber-One test tube containing 9.9 ml nutrient broth is inoculated with 0.1 ml of undiluted pathogen titre (equivalent to 100 colonies) and is incubated at 37°C for 24 hours in an incubator with contant shaking. This tube is used as control pathogen tube. (v) Control Blank Tube:- One test tube containing 10ml nutrient broth only is incubated (uninoculated) at 37°C for 24 hours in an incubator-cum shaker and is used as control blank to zero the colorimeter. (vi) Calculation of Lethal dose titre- 50(LDt)50 of live antagonist against pathogen by cell density method:- After an incubation of 24hours, the optical densities (ODs) of all the tubes of control live antagonist set, interaction set, and control pathogen tube are measured at 640 nm, using a colorimeter. The O.D. is taken as a unit measure of cell-density. For calculating the final dilution titre of antagonist giving lethality/mortality in 50% of the pathogen population (i.e. LDt so), the O.D. of control pathogen tube is taken as the base (control) value. In each interaction tube, the live antagonist will kill proportionate number of pathogen cells during the incubation period. The measured O.D. of such an interaction tube will be due to both the live antagonist and the live pathogen as well. Whereas in each tube of the control antagonist set, the number of antagonist will decrease with increase in dilution. Thus, the O.D. (cell density) in each tube will vary accordingly and correspond to live antagonist cells only. In this backdrop, the LDt so value is finally calculated as follows: - Let the O.D. of control pathogen tube be "x", therefore, the O.D. of 50% pathogen population could be "x/2". Let the O.D. of control antagonist tube be "y" and the O.D. of respective interaction tube be "z". Therefore, the O.D. of only pathogen in that respective interaction tube would be (z-y). By comparing these "z-y" values, that "z-y" value is found which is near or equal to x/2. That antagonist titre dilution in the respective tube whose "z-y" value is near or equal to x/2 is taken as LDt50 of live antagonist against live pathogen. (B) DETERMINATION OF BIOPOTENCY AS LDt50 BY COUNTING NUMBER OF PATHOGEN COLONIES (i.e. COLONY COUNT METHOD): (i) Preparation of final serial dilutions of live antagonist titre:- Same as described in STEP- III (i) above. (ii) Control antagonist set:- Not necessary here since only pathogen colonies are to be counted. (iii) Antagonist- pathogen Interaction Set:- Same as described in STEP-in (iii) except that 0.2 ml aliquotes (comprising of 0.1 ml antagonist titre dilution and 0.1 ml undiluted pathogen titre) from each of the eleven interaction tubes are asceptically transferred separately on a fresh lot of similarly labelled eleven MacConkey agar petriplates and uniformly spread. All hese petriplates are then incubated at 37°C for 24 hours in an incubator and the total number of pathogen colonies are counted. (iv) Control Pathogen Plate:- One MacConkey agar peteriplate was inoculated with 0.1ml undiluted pathogen titre (equivalent to 100 colonies), spread uniformly and incubated at 37°C for 24 hours in an incubator. This plate was used as control pathogen plate. (v) Control Blank :- Not necessary for this experiment. (vi) Calculation of LDtso live antagonist against pathogen by counting pathogen colonies. The LDtso value is calculated as follows.- Let the Total Number of Pathogen colonies in control pathogen plate be "a". That antagonist titre dilution on the interaction pelriplate showing a/2 number of pathogen colonies is taken as LDtso of an antagonist against the pathogen. We claim; 1. A method for determination of biopotency by Lethal Dose titre-50, LDT 50 of live antagonist, naturally occurring bacterium Bacillus thuringiensis against pathogen comprising the steps of: (a) firstly preparing a starter seed culture of both Bacillus thuringiensis as well as pathogen by incubating at 37°C + 7° for 20-40 hours on nutrient agar plates separately and finding out the dilution which gives 100 well isolated colonies and taking dilution value of that plate as titre value of that organism (b) there after the LDt 50 is determined either by cell density method or by colony count method. 2. A method for determination of biopotency by Lethal Dose titre 50 of live antagonist Bacillus thuringiensis against pathogen by cell density method as claimed in claim 1 comprising the steps of: (i) preparation of final serial dilutions lie antagonist titre in nutrient broth in 11 tubes, taking one as undiluted and other dilutions varying from 101 to 1010 (ii) preparation of control antagonist set by asceptically transferring 0.1 ml aliquot of each of the above dilutions to the tubes containing 9.9 ml of sterile nutrient broth the incubating them (iii) preparation of antagonist pathogen interaction set by mixing aliquots from above antagonist titre dilutions with undiluted pathogen titre on a volume per volume bases, allowing the interaction for about 30 minutes at room temperature, asceptically inoculating aliquots from each of the eleven tubes to the fresh, similarly labeled, eleven tubes containing nutrient broth and incubating them (iv) preparation of control pathogen tube by incubating a test tube containing 0.1 ml of undiluted pathogen titre (v) preparation of control blank tube by incubating a test tube containing only 10ml of nutrient broth (vi) measuring optical densities, at 640nm using colorimeter, of all the tubes of control live antagonist set, interaction set and control pathogen tube (vii) computing the differences between optical densities of each of interaction tube (z) and control antagonist tube (y) and comparing (z-y) for each tube with the half of the value of optical density (x) of control pathogen tube (viii) taking LDtso as the antagonist titre of that tube whose (z-y) value defined as above is equal or is nearly equal to x/2 where-in x is optical density of control pathogen tube 2. A method for determination of biopotency by Lethal Dose titre 50 of live antagonist Bacillus thuringiensis against pathogen by colony count method as claimed in claim 1 comprising the steps of (i) preparation of serial dilutions of live antagonist titre as in cell density method such as herein described (ii) preparation of antagonist-pathogen interaction set by incubating 0.2ml aliquots on agar petriplates preferably for 24 hour (iii) preparation of control pathogen plate by incubating one agar petriplate which is inoculated with 0.1ml of undiluted pathogen titre and counting the number of pathogen colonies on the control pathogen plate i (iv) taking LDtso as the antagonist titre dilution on that interaction petriplate which shows half the number of pathogen colonies on the control pathogen plate 4. A method for determination of biopotency by Lethal Dose titre 50 of live antagonist B. thuringiensis against pathogen as claimed in claim 1 wherein the said incubation for preparation of control antagonist, Bacillus thuringiensis set as well as for preparation of antagonist pathogen interaction set and for preparation of control blank is carried out preferably for 24 hours.

Documents:

1148-del-2001-abstract.pdf

1148-del-2001-claims-(03-04-2008).pdf

1148-del-2001-claims.pdf

1148-del-2001-correspondence-others.pdf

1148-del-2001-description (complete)-03-04-2008.pdf

1148-del-2001-description (complete).pdf

1148-del-2001-drawings.pdf

1148-del-2001-form-1.pdf

1148-del-2001-form-18.pdf

1148-del-2001-form-2.pdf

1148-del-2001-form-3.pdf

1148-del-2001-form-4.pdf

1148-del-2001-form-5.pdf

1148-del-2001-gpa.pdf

1148-del-2001-petition-others.pdf