“TO INVESTIGATE THE PRESENCE OF PSEUDOMONAS STRAIN (BACTERIA) FROM SAMPLE

OF DIFFERENT LOCATION.”

NASHIK CAMBRIDGE SCHOOL

(Affiliated to CBSE, Delhi Up to Senior Sec Level)Nasik

ACADEMIC YEAR: 2012-2013

Project Report submitted to

Nasik Cambridge School

CBSE, Delhi up to Sr. Sec Board

BY

TEJASWINI AHIRE

Under the Guidance of Mr. A. SHANKAR RAO (PGT Biology)

Dept. of Biology,Nasik Cambridge School

ACKNOWLEDGEMENT

This project report is the result of a solo effort and

combined efforts of my guide, friends and my

family. I would like to express my gratitude to all

those who have helped me during my project work.

I am greatly indebted to Prof. Ramchandran Sir and

Mrs. Bharti Ramchandran Mam (Trustees of NCS).

I take this opportunity to thank our Principal Sir Mr.

C. Somu.

I sincerely thank Mr. A. Shankar Rao (PGT-Biology)

who guided me through the project for helping me

to learn the techniques involved in the project.

I would like to thank all the teachers, non – teaching

staff of NCS for their support and motivation.

E- MAIL: nashikcambridge2004@rediffmail.com TELEPHONE NO. : 0253-2377638\2377639

CERTIFICATEThis is to certify that, Tejaswini Ahire 12th grade Student of NASHIK CAMBRIDGE SCHOOL (AFFILIATED TO CBSE BOARD, DELHI) has worked in dept. of biology on a project- “To investigate the presence of pseudomonas strain (bacteria) from sample of different location.”

During this period, she got herself acquainted with some techniques in biology- sterilization, media preparation, weighing, identification of colonies, isolation of bacteria, spreading and staining.

Mr. C. SOMU Mr. A.SHANKAR RAO

(Principal Sir) (Internal Examiner)

External Examiner

INDEX

CONTENTS

ABBREVIATIONS

ABSTRACT

1. Introduction

2. Materials and Methods

3. Observations

4. Results

5. References

LEGENDS

METHODOLOGIES

APPENDIX

ABBREVIATIONS

NAM: - Nutrient Agar Medium

NB : - Nutrient Broth

oC : - Celsius

PH : - Hydrogen ion concentration

Mo’s :- microorganisms

Ps : - Pseudomonas strain

Abstract:

The soil collected from oil refineries, petrol bunks, mechanical sheds having many toxic compound/ xenobiotics compounds which inhibits/ kills the soil microbial flora. Based on recent scientific investigation some soil micro-organism like pseudomonas strain present in soil of oil refineries convert the toxic compound into non toxic forms. This is called biotransformation/ detoxification. NB was prepared under sterilized condition and NB was inoculated with soil sample of oil refineries to culture pseudomonas strain in an incubator at 37 oC to 24 hours. From NB 2-3 drops of pseudomonas strain were collected & added on the middle of NAM plates in front of spirit lamp & spreaded with sterilized spreader kept for incubation in incubator for 24 hours at 37 oC . After 24 hrs of incubation agar medium shows the growth of pseudomonas strain indicates the biodegradation of toxic compounds in soil of oil refineries.

INTRODUCTION

PSEUDOMONAS

Pseudomonas is a genus of Gram-negative aerobic gammaproteobacteria, belonging to the family Pseudomonadaceae . The members of the genus demonstrate a great deal of metabolic diversity, and consequently are able to colonize a wide range of niches.[3] Their ease of culture in vitro and availability of an increasing number of Pseudomonas strain genome sequences has made the genus an excellent focus for scientific research; the best studied species include P. aeruginosa in its role as an opportunistic human pathogen, the plant pathogen P. syringae, the soil bacterium P. putida, and the plant growth promoting P. fluorescens.

Characteristics

Members of the genus display the following defining characteristics: [13]

Rod shaped Gram-negative One or more polar flagella, providing motility

Aerobic Non–spore forming positive catalase test Positive oxidase test.

P. putida has the ability to degrade organic solvents such as toluene.[32] At least one strain of this bacterium is able to convert morphine in aqueous solution into the stronger and somewhat expensive to manufacture.Pseudomonas bacteria can be found in soil, marshes, coastal marine habitats, and plant and animal tissue; generally, these bacteria can tolerate a variety of physical conditions.

TOXIC COMPOUNDS PRESENT IN OIL REFINERIES, GAS STATIONS & MECHANICAL SHEDS

As a result of the increased use of automobiles, theDemand on gasoline/diesel stations is increasing. In suchStations, fuel oil, which is classified as hazardous waste(Bartha & Bossert 1984) is spilled during transfer andServicing operations. The accidental spillage of hydrocarbonsOn the soil may result in a selective increase inHydrocarbon-utilizing microorganisms (VenkateswaranEt al. 1995; Ferrari et al. 1996). The enhancement or

Reduction will depend upon the chemical composition ofThe contaminating hydrocarbons and on the species ofMicroorganism present within the microbial communityOf the particular ecosystem (Atlas 1995). The distributionOf the type and the number of microorganisms at aSite may help to characterize the site with respect to theConcentration and age of the contaminant.The widespread distribution of members of the genusPseudomonas in all hydrocarbon-polluted soils of thisStudy as well as of the other investigations confirms theirPrevalence and reflects their potential in utilizing theseHydrocarbon contaminants for growth and thus cleanThese polluted sites (Cork & Krueger 1991). Therefore,The different Pseudomonas spp. of this study wereEvaluated for such potential using the modified methodOf Jacobs et al. (1983) through transforming alkanes toTheir corresponding alcohols. Detection of alcoholFormation by microorganisms may be an applicableApproach for evaluating their activity on the simpleAlkanes. Saadoun (2002) reported that this techniqueCan be applied for screening of organisms for theirAbility to degrade hydrocarbons.

Bioremediation& biodegradationBioremediation of polluted soils through the introduction

Of exogenous microbial isolates into the polluted environment(Bioaugmentation) has gain increasing interest as an alternative methodTo biostimulation in situation where the indigenous species cannot cope with their environmental pollutants. However, many factors including predation, competition with indigenous microorganisms, nutrient availability, physicochemical parameters and other environmental factors necessary for growth has been found to influence the survival of bioaugmented microbes as well as their bioremediation efficiencies Many of the previous studies reported the use of activated contaminated soils or sludge as inoculants for bioaugmentation but the fair of transferring more hazardous contaminants through the activated soil or sludge is also seen as a concern hence, the report of possible usage of uncontaminated soil with potential contaminant degrader as inoculants

Materials and Methods

Material Required:

I) Glassware :

1) Beaker

2) Conical flask

3) Petri plates

4) Glass rods

5) Syringes

6) Test tubes

7) Measuring cylinder

8) Spirit lamp

9) Spreader

II) Electronic Devices:

1) Electronic Balancer

2) PH meter

3) Auto clave

4) Hot air oven

5) Incubator

6) Hot plate

III) Chemicals:

1) De ionized water

2) Surgical Spirit

3) Nutrient agar medium

4) Nutrient broth

IV) Other requirement:

1) Cotton plug

METHODS

Preparation of Nutrient Agar Medium

Take a 250ml of distilled water in 500ml of beaker

To this add 9.25gm of agar medium

Mix thoroughly by using a sterilized glass rod

Adjust the pH of nutrient agar medium in between 7.2 to

7.4

Transfer the medium into conical flask

Add 3 to 4gm of agar-agar and mix thoroughly by using

sterilized glass rod

To liquefy agar-agar, boil the medium with the help of

Bunsen burner or hot plate

Close the conical flask with the help of non adsorbent

cotton plug

Sterilize the NAM in an autoclave at 121o c temperature,

16lbs pressure for 15 minutes

After autoclaving collect the NAM

Pour the NAM into the Petri plates in sterilized

condition and keep at room temperature for 10-15

minutes to solidify the nutrient agar medium.

PREPARATION OF NUTRIENT BROTH

Take a 250ml of distilled water in 500ml of beaker

To this add 9.25gm of agar medium

Mix thoroughly by using a sterilized glass rod

Adjust the pH of nutrient agar medium in between 7.2 to

7.4

Transfer the medium into conical flask

Close the conical flask with the help of non adsorbent

cotton plug

Sterilize the NAM in an autoclave at 121o c temperature,

16lbs pressure for 15 minutes

After autoclaving collect the NAM

SpreadingCollect 2-3 drops of the pseudomonas culture and spread it thoroughly on NAM plates with the help of a sterilized spreader and incubate it in an incubator in an inverted position. Preparation of test sample: 1 gm of soil from different location is added to 9 ml distill water.

Inoculation:Test sample is inoculated into NB.

Incubation:Incubate the NAM plates in an incubator for 24 hrs at 37oc.the growth of pseudomonas in NB is observed by increase in turbidity of NB.

Observation:

Fig: 1.1The Photograph showing the growth of pseudomonas

strain in control experiments.

Fig- 1.2The photograph showing the zone of inhibition.

Result:

Tiny colonies of pseudomonas strain is observed on NAM plates that indicates the biodegradation of toxic compound in soils collected from oil refineries, petrol bunks & mechanical shed.

REFERENCES

▪ Comprehensive practical biology class-XII

▪ Ananthanarayana-Microbiology

▪ Prescott-Microbiology

▪ Microbiology-Laboratory Manual – S M Reddy &

S Ram Reddy

▪ Vikas publication-Botany

▪ www.wikipedia.com

LEGENDS

▪ Fig.1.1 – Photograph showing growth of mouth

microbial flora in control experiment.

▪ Fig1.2 - Photograph showing zone of inhibition for

different toothpastes.

Fig 1.3 – Observation table showing size of zone of

inhibition.

▪ Fig 1.4 – Graph showing zone of inhibition for different toothpastes against mouth microbial flora.

METHODOLOGIES

AUTOCLAVING

Autoclave is a common and most essential

instrument in every biology laboratory. It is used

for sterilization of media (solid and liquid), glass-

wares and rubber products. It is based on principle

that saturated steam heats an object many times

more efficiently than hot air at the same

temperature. This apparatus, when switched on

generates a saturated steam under pressure and

then brings about sterilization. The increased

pressure results in the elevation of boiling point of

water and produces steam with high temperature.

However it is important to note that it is not

pressure that kills the organisms but the high

temperature of the steam. The boiling point of

water at 15 lbs pressure is 121 ° C.

HOT AIR OVEN

Oven is another common instrument of microbiological laboratories and is used for sterilization of glassware. It is based on the principle whether sterilization is accomplished by dry heat or hot air. An oven consists of an insulated, heat proof cabinet which maintains a desired constant temperature with the help of electric heating mechanism and a thermostat. Normally, all the routine glassware is sterilized by keeping at 160 ° C for 2 hours.

INCUBATOR An incubator is very similar to oven in construction.

However, its low thermostat is designed in such a

way to maintain low temperature i.e. below 80 °C. It

is used for incubating, maintaining the cultures at

constant desired temperatures or above the

ambient temperature. Like the oven, an incubator

consists of a heating element at the bottom, a

thermostat, temperature probe and devices for

regulating the temperature. Some incubators are

provided with light arrangements to provide light to

microorganisms which require light for their

growth and sporulation.

APPENDIX

NAM COMPOSITION : 1000ml

▪ NaCl 5.0grams▪ Beef extract 3.0 grams▪ Peptone 5.0 grams▪ Agar-Agar 15 grams

N.B COMPOSITION : 1000ml▪ NaCl 5.0 grams▪ Peptone 5.0 grams▪ Beef extract 3.0 grams

Soil sample preparation: Distilled water 9 ml Soil 1 grams