shallow water of the lake Yellanalappa chetty lake located at Bangalore- Chennai road, (NH4) using a sterile conical flask, sealed and transported.

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1 ISOLATION OF A BACTERIAL SPECIES IN THE INSECTICIDE POLLUTED AREAS D.N.Saaswathi Raman, Pofesso in Botany, St.Joseph s College, Bangaloe Savitha.K, Lectue in Biotechnology, Don Bosco College, Bangaloe SYNOPSIS Bacteial species is isolated fom the soil at an agicultual fam and wate fom a lake. Chemical analysis of the soil and wate showed vey high concentation of chemicals i.e., Insecticides. Cultual, micoscopical and biochemical techniques showed that Bacillus fomed the majo biomass followed by Staphylococcus and Micococcus. The cultual techniques involved the seial dilution of the soil and wate samples followed by the plating of the sample in the cultue media and also the study of cultual chaachteistics. The micoscopical tests included the Gam s staining of the bacteia, endospoe staining and motility study. The biochemical activity of the Bacteia was confimed by a set of tests that included IMViC, stach hydolysis, oxidase test etc., Among the Bacillus species isolated, Bacillus steaothemophilus was found to show stess toleance against vaious factos such as high tempeatue, high salinity and also high concentation of insecticide such as chlopyifos due to which this species of Bacillus was selected fo futhe studies. INTRODUCTION The name Bacillus steaothemophilus is pesumably intended to mean fat and heat loving. The most distinctive chaactes ae the capacity to gow at 65 0 c. Bacillus stains capable of gowing at tempeatue of 65 0 c and above do not belong to a single species, it is howeve a useful diagnostic chaacte. Bacillus steaothemophilus occus in soil, hot spings, deset, actic wates, ocean sediments, foods and compost. This paticula stain isolated fom wate and soil sample is found to gow in the pesence of high pecentage of insecticide, chlopyifos. Chlopyifos is one of about 100 oganophosphate insecticides in the maket today which is widely used. It is used to kill insect pests by disupting thei nevous system. Chlopyifos has an advantage ove othe poducts in that it is effective against a wide ange of plant eating pests. MATERIALS AND METHODS The samples wee obtained fom two diffeent aeas of Bangaloe. The soil sample was collected fom an agicultual fam, R.K. Fams, hosu oad, Bangaloe. The soil sample was collected at a depth of 15cm and tansfeed to a clean containe (N.S.Subba Rao). The wate sample was collected fom the

2 shallow wate of the lake Yellanalappa chetty lake located at Bangaloe- Chennai oad, (NH4) using a steile conical flask, sealed and tanspoted. Cultuing and isolation was done by using Hi-media. A volume of 1ml of the sample was used and successively diluted and a fixed aliquot of 1ml fom each dilution was mixed with molten, cooled, cultue media and plated( K.R.Aneja,) Plates wee incubate fo 24 hs, cultual chaachteistics of the esultant micobial colonies wee noted. Both Bacteial and fungal foms wee detected fom which Bacteial foms alone wee selected and subcultued.(micheal J. Pelca, E.C.S. Chan, Noel R. Kieg,) Gam s staining was pefomed by commecially available chemicals to detect the Gam s eaction of the bacteium(haold J Benson,). Enospoe staining was also pefomed by using the commecially available stains to detect the pesence of spoes and the position of spoe whethe teminal o cente.( Haold J Benson,). The motility of the bacteia was studied by hanging dop technique (Haold J Benson,). Biochemical tests such as Indole poduction test, Methyl ed test, Vogesposkaue test, citate utilization test, nitate eduction test, uease test, catalase test, oxidase test, amylase test, suga-fementation test wee caied out using the espective Hi-media and eagents. Indole poduction test was caied out by using peptone wate both and Kovac s eagent, while Methyl ed and Voges-poskaue test was conducted with glucose phosphate both and the eagents ae Methyl ed and Baitt s eagent espectively Citate utilization test was pefomed by using Simmon s Citate aga. Pesence of catalase enzyme is detected by using 3% Hydogen peoxide as the eagent, while oxidase test was conducted using oxidase eagent soaked in a filte pape followed by steaking of the cultue onto the filte pape. Nitate eduction test is conducted by inoculating the bacteia into nitate both and the eagents used wee naphthlamine and sulphanilamide Amylase test was conducted using stach aga media plates that ae late flooded with Gam s iodine which is used as a eagent to detect the pesence of amylase in bacteial cultue while the suga fementation tests wee conducted to detect the fementation of sugas that wee taken in the desied media and also by using Duham s tube fo detection of gas poduction by the oganism. (John.G.Holt,) The insecticide toleance was detemined by commecially available Lethal TC- Chlopyifos (20%) manufactued by Insecticides, India, Ltd, while the salinity toleance was detected by using diffeent concentation of sodium chloide. Conical flasks with fixed volumes of nutient both wee taken and the stess agents (chlopyifos and sodium chloide) wee added to each flask in diffeent concentations. All the flasks wee inoculated with 2% v/v inoculum fom 24 h both cultue, uninoculated both was used as contol. Flasks wee placed in a shaking incubato at a tempeatue of 55 0 c fo 24 hs at 60 pm. The cultue afte incubation was gently votexed and then allowed to stand fo 10min. The optical density of each inoculated both was ead at 600 nm with uninoculated both as blank fo efeence by using UV-Visible spectophotomete. Tempeatue toleance was studied using cultue media onto which a 24 hs old cultue was steaked and incubated at diffeent tempeatues such as 37 0 c, 45 0 c,55 0 c, 60 0 c and 65 0 c and obseved fo gowth afte 24 hs. the plates wee

3 examined fo diffeential gowth density. The maximum gowth tempeatue of the isolates was thus detemined. Expeiment was conducted in tiplicates. The esults of the above studies ae epesented in tables and figues. RESULTS AND DISCUSSION Table 1: cultual chaacteistics of wate micofloa Plate diluti on Size Shape Magi n 10-1 Pin point Cicula Medium Cicula Medium Cicula Lage Iegul a Small Cicula 10-2 Pinpoint Cicula Small Cicula Lage Cicula 10-3 Pinpoint Cicula Lage Cicula Elevation Pigmentat ion Opacity Textue Gam s natue Iegul a Raised White Opaque Smooth - ods Entie Raised Pinkish Tansluce Smooth + ods nt Entie Convex White Opaque Smooth + ods Seate Raised White Opaque smooth + ods Wavy Umbonate White Tansluce nt smooth + ods Iegul Raised White Opaque Smooth + ods a Wavy Umbonate White Tansluce Smooth + ods nt Entie Convex White Opaque Smooth + ods Iegul Raised White Opaque smooth - ods a Entie Convex White Opaque Smooth + ods

4 FIGURE:1 Bacteial colonies of wate micofloa Table 2: cultual chaacteistics of soil micofloa Plate diluti on Size Shape Magin Elevatio n 10-1 Small Cicula Medium Cicula Medium Cicula Lage Iegul a Lage Cicula 10-2 Pinpoint Cicula Small Cicula Pinpoint Cicula 10-3 Lage Cicula Pin Cicula point Pigment ation Opacity Textu e Wavy Umbona te White Tansluce nt smoot h Entie Raised Pinkish Tansluce Smoo nt th Entie Convex White Opaque Smoo th Seate Raised White Opaque smoot h Entie Convex White Opaque Smoo th Iegula Raised White Opaque Smoo th Wavy Umbona White Tansluce Smoo te nt th Iegula Raised White Opaque smoot h Entie Convex Off Opaque Smoo white th Iegula Raised White Opaque Smoo th Gam s natue + ods + ods + ods + ods + ods + ods + ods - ods + ods - ods

5 Table 3: Mophological chaachteistics of the isolate Gam s eaction Mophology Motility Endospoe Gam positive Rods Motile teminal Based on the esults of the table:3, the genea of the isolate is concluded as Bacillus Table 4: Biochemical tests Test Result Indole + Methyl ed +++ Voges-poskaue - ph in VP medium <6 + Citate utilization - Nitate eduction + Uease test - Suga fementation Glucose ++ Lactose - Mannitol - Catalase test +++ Oxidase test ++ Amylase +++ Based on the esults of the table:4, the isolate is concluded steaothemophilus as Bacillus Table 5: Studies on tempeatue toleance pofile of the isolate Tempeatue Incubation peiod(hs) Gowth on aga Degee of gowth Gowth on both Degee of gowth 37 0 c c c c c efes to pesence of gowth - efes to absence of gowth 3+ efes to high degee of gowth

6 FIGURE:2 BACTERIAL COLONIES AT VARYING TEMPARATURES Table 6: Results of tubidometic studies on stess toleance pofile Stess agent Concentation Incubation O.D. at 660nm tempeatue Sodium chloide 2% 55 0 c 0.05(+0.01) 6% 55 0 c 0.09(+0.03) 8% 55 0 c 0.01(+0.02) 10% 55 0 c 0.01(+0.02) chlopyifos 0.001% 55 0 c 0.03(+0.03) 0.005% 55 0 c 0.05(+0.02) contol 2%v/v 37 0 c 0.05(+0.01) 2%v/v 55 0 c 0.09(+0.03) Figues in the paenthesis epesent the standad deviation.

7 BIBLIIOGRAPHY John G Holt, Neol R Kieg, Pete H A Sneath, James T Stanley, Staneley T Williams, 9 th edition, Begey s manual of Deteminative Bacteiology. K R Aneja,2003,(157,192,102,110,75) 4 th edition, Expeiments in Micobiology, plant pathology and Biotechnology. Haold J Benson, Micobiological application N S Subba ao,,1986,(63), 4 th edition of soil micooganisms and plant gowth. Micheal J Pelcza, E C S Chan, Neol R Kieg, 1993, (144), 5 th edition, Micobiology. BIODATA OF THE AUTHORS 1) D. D.N.Saaswathi Raman is cuently woking as Pofesso in St.Joseph s college since She stated the eseach as JRF and SRF (CSIR) and continued with Post doctoal fellowship (DST). She is cuently involved in eseach elated to biotechnology, molecula biology, biochemisty and physiology. In addition to PG teaching, she is involved in guiding students fo M.Phil and Ph.D of vaious univesities. 2) Ms. Savitha.K is woking as Lectue in Biotechnology in Don Bosco College fom August 2002 till date. She has done he Post gaduation in Micobiology and pesently pusuing Ph.D degee in Biotechnology unde the guidance of D. D.N.Saaswathi Raman.

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