1. “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

2. 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.

3. E- MAIL: nashikcambridge2004@rediffmail.com TELEPHONE NO . : 0253-23776382377639
CERTIFICATE
This is to certify that, Tejaswini Ahire 12th
grade Student of NASHIK
CAMBRIDGE SCHOOL (AFFILIATED TO CBSE BOARD, DELHI)
has workedin dept. of biologyon 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

4. INDEX
CONTENTS
ABBREVIATIONS
ABSTRACT
1. Introduction
2. Materials and Methods
3. Observations
4. Results
5. References
LEGENDS
METHODOLOGIES
APPENDIX

5. ABBREVIATIONS
NAM: - Nutrient Agar Medium
NB : - Nutrient Broth
oC : - Celsius
PH : - Hydrogen ion concentration
Mo’s :- microorganisms
Ps : - Pseudomonas strain

6. Abstract:
The soil collectedfrom oil refineries, petrol bunks,
mechanicalsheds having many toxic compound/
xenobiotics compounds which inhibits/ kills the soil
microbialflora. Based on recent scientific investigation
some soil micro-organismlike 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 biodegradationof
toxic compounds in soil of oil refineries.

7. INTRODUCTION
PSEUDOMONAS
Pseudomonas is a genus of Gram-
negative aerobicgammaproteobacteria,belonging to
the family Pseudomonadaceae . The members of the
genus demonstrate a great deal of metabolicdiversity,
and consequently are able to colonize a wide range of
niches.[3] Their ease of culture in vitro and availabilityof
an increasing number
of Pseudomonas strain genome sequences has made the
genus an excellentfocus 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 followingdefining
characteristics: [13]
 Rod shaped
 Gram-negative
 One or more polar flagella,providing motility

8.  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 convertmorphine 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, the
Demand on gasoline/dieselstations is increasing. In
such
Stations, fuel oil, which is classified as hazardous waste
(Bartha & Bossert 1984) is spilled during transfer and
Servicing operations. The accidentalspillage of
hydrocarbons
On the soil may result in a selective increase in
Hydrocarbon-utilizing microorganisms (Venkateswaran
Et al. 1995; Ferrari et al. 1996). The enhancement or
Reduction will depend upon the chemicalcomposition
of
The contaminating hydrocarbons and on the species of

9. Microorganismpresent within the microbialcommunity
Of the particular ecosystem (Atlas 1995). The
distribution
Of the type and the number of microorganisms at a
Site may help to characterizethe site with respect to the
Concentration and age of the contaminant.
The widespread distribution of members of the genus
Pseudomonas in all hydrocarbon-polluted soils of this
Study as well as of the other investigations confirms
their
Prevalenceand reflects their potential in utilizing these
Hydrocarbon contaminants for growth and thus clean
These polluted sites (Cork & Krueger 1991). Therefore,
The different Pseudomonas spp. of this study were
Evaluated for such potential using the modified method
Of Jacobs et al. (1983) through transforming alkanes to
Their corresponding alcohols. Detectionof alcohol
Formation by microorganisms may be an applicable
Approach for evaluating their activity on the simple
Alkanes. Saadoun (2002) reported that this technique
Can be applied for screening of organisms for their
Ability to degrade hydrocarbons.
Bioremediation& biodegradation
Bioremediationof polluted soils through the
introduction
Of exogenous microbialisolates into the polluted
environment
(Bioaugmentation) has gain increasing interest as an
alternative method

10. To biostimulation in situation where the indigenous
species cannot cope with their environmental
pollutants. However, many factors including predation,
competitionwith indigenous microorganisms, nutrient
availability,physicochemicalparameters and other
environmental factors necessary for growth has been
found to influence the survival of bioaugmented
microbes as wellas their bioremediationefficiencies
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

11. 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

12. 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) Cottonplug

13. 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

14. 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

15. 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
Spreading
Collect 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.
Preparationof test sample:
1 gm of soil from different locationis added to 9 ml
distill water.

16. 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.

17. Observation:
Fig: 1.1
The Photograph showing the growth of pseudomonas
strain in control experiments.

18. Fig- 1.2
The photograph showing the zone of inhibition.

19. Result:
Tiny colonies of pseudomonas strain is observed on
NAM plates that indicates the biodegradationof toxic
compound in soils collectedfrom oil refineries, petrol
bunks & mechanicalshed.

20. 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

21. 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.

22. 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.

23. HOT AIR OVEN
Oven is another common instrument of
microbiological laboratories and is used for
sterilizationof 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

24. microorganisms which require light for their
growth and sporulation.

25. 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