CONTENT

S.NO. Particulars Page no.

1 Declaration 1
2 Acknowledgement 2
3 Overview of Biotech Park 3-8
4 Introduction 9-10
5 Review of Literature 11-14
6 Aim and Objectives 15
7 Instruments used in laboratory 16-22
8 Materials and methods 23-26
9 Experiments 27-43
10 Observation and Results 44-51
11 Conclusion 52
11 References 53

DECLARATION
I, Tanya Jain, hereby declare that this report entitled “Physicochemical and Microbiological
analysis of collected Gomti River Water” is submitted by me at Biotech Park, Lucknow as a
part of winter traning as a student of B.Sc (Biotechnology) Dr. Bhimrao Ambedkar
University. The data mentioned in this report is obtained during genuine work done by me and
none of the findings and observations pertaining to this work has been concealed. I hereby also
declare that the data is true to best of my knowledge and is a record of original work done by me
during three month training from 15th January to 14th April, 2019 at Biotech Park, Lucknow.

DATE: 14th April, 2019

(Signature of Student)
Tanya Jain

ACKNOWLEDGEMENT
I would like to express my sincere thanks of gratitude towards Prof. Pramod Tandon, CEO
Biotech Park, Lucknow for giving me an unmatched opportunity to undergo the training in the
field of Microbiology in this prestigious institution of national repute.
Heartiest thanks to my mentor Mr. Mohd Kashif Khan for being a constant backbone. His
uninterrupted guidance has made this training highly successful. He has been a constant source
of inspiration, provocation and knowledge throughout the tenure. I do sincerely acknowledge the
freedom rendered by him in the laboratory for independent thinking, planning and execution of
the research. I revere his lessons on perfection, skills and many more to germinate and grow up a
researcher in me.
I find the immense pleasure in be expressing my good gratitude towards Dr. Shashank Mishra,
Senior Scientist, Biotech Park for instilling the quality of dedication, punctuality and hard work
in me. His constant support, encouragement and positive criticism had been a major driving force
throughout my project work.
I would also likely to say thanks to Mr. Manish Verma for their ceaseless support and good
encouragement.
I would like to extend my sincere thanks to Dr. Ashok Kumar Upadhyay (Head of
department, Biotechnology) for giving me an opportunity to join Biotech Park, Lucknow. I
would also like to thank the teachers of Biotech Department of Agra College, Agra for their help
and support.
At last, I owe my special thanks to my parents who were behind me throughout my project
encouraging me to perform my best, their unwavering faith and confidence which made my
training a success.

DATE: 14th April 2019

TANYA JAIN

OVERVIEW OF BIOTECH PARK

 Biotech Park, Lucknow
The State of Uttar Pradesh, a vital hub of scientific activities endowed with rich biological
resources and biodiversity has the unique distinction of having a number of research institutions,
which have expertise and capabilities in biotechnology. In view of this, Lucknow was declared
as the Biotechnology City of India during the 89 th session of the Indian Science Congress on
January 3, 2002. These institutions are providing a great impetus and support in the development
of Biotech Park.
The Biotech Park has been set up on 8 acres of land provided by the Department of Science and
Technology, Government of Uttar Pradesh. The thrust areas identified for the initial phase of the
Park are:
1. Health Care
2. Agriculture
3. Environment
BIOTECH PARK FACILITIES:

BIO-BUSINESS BLOCK
The Bio-Business Block houses the following: Business Support Facilities
1. Bioinformatics Centre
2. Conference Hall
3. Cafeteria
4. Laboratory Space
BIOINFORMATICS CENTRE
A Bioinformatics Centre has been setup to establish a close network with various institutions,
provide information to industries regarding technological advancements and training facility.
SOLVENT EXTRACTION BLOCK
The extraction unit comprises of solid liquid solvent extraction system with solvent recovery
system for extraction of Photochemical / Lead molecules from high value medicinal plants and
multipurpose reaction cum hydrolysis and solvent recovery unit along with chromatography.
Extraction unit has the following facilities:
1. Hot air tray dryer, High capacity hammer mill and vacuum oven for drying, grinding/ Oil
fired steam boiler, evaporation capacity: 500 kg/hr and 150 psi steam pressure for basic
heating requirements to main extraction unit.
2. Pulverizing of raw materials / finished herbs / medicinal plants.
3. Refrigerated brine chilling circulation unit for carrying out reactions and chilling of
condenser water.
4. Solid liquid extraction unit consisting of two drug holders with agitators of 125 - 150
kg/batch capacity depending on plant bulk density with efficient solvent recovery system
from extract and spent marc.
5. Heavy-duty spray dryer for drying of herbal / aqueous extracts, capacity 10 – 20 kg.
6. Falling film evaporator for concentrating herbal / aqueous extracts for separation of the
extracts from the plant biomass.
7. Notch type vacuum filtration unit for filtration of aqueous solvent extracts for finer
filtration of the extracts for removal of particulate impurities.
8. Liquid - liquid counter current extraction equipment for the purification of crude extracts
by liquid partitioning.
9. Minor equipments like vacuum pumps, metering pumps, weighing machine, weighing
balance, trolleys etc for auxiliary infrastructure, process and material handling etc.
10. .Capacity- 250 kg biomass/batch
BIO-FERTILIZER, TISSUE CULTURE & CENTRE SUPPORT BLOCK
1. Bio-fertilizer unit – Ground floor
2. Plant Tissue culture unit – First floor
3. Central instrumentation facility –Second floor
BIOFERTILIZER UNIT
This unit has facilities for perfecting the technology and production of bacterial fertilizers and
pesticides, phosphate solubilizing bacteria (PSB), Azotobacter (with a capacity of up to 240
tones / annum) Trichoderma (with a capacity of up to 500 tones / annum). The trials runs have
been completed in March 2007 and batch production has started.
TISSUE CULTURE AND HARDENING FACILITY
Tissue culture facility at Biotech Park, Lucknow is spread over 2000 sq ft. Area that possess the
capacity to raise and multiply Banana, Potato, Jatropha seedlings. The culture media used in the
unit is completely biodegradable and non-toxic.
ADVANTAGES OF PROPAGATION BY TISSUE CULTURE-
1. The elimination of diseases & the production of disease free plantlets.
2. The rapid production of large no. of genetically identical plantlets.
3. Introduction of new varieties & or genotypes.
4. Preservation of germplasm.
5. Production of haploid plants which can be used for plant breeding.
6. Production of plantlets from species in which plant development from seed is difficult.
CAPACITY: 10000 to 100000 plants / batch & it can produce about 2 million plants / year.
ANALYTICAL & MOLECULAR BIOLOGY FACILITY:

The facility is equipped with High Performance Liquid Chromatography (HPLC), Gas
chromatography (GC), High Performance Thin Layer Chromatography (HPTLC), Atomic
absorption spectrophotometer (AAS), Nanodrop-spectrophotometer, Gel Electrophoresis, ELISA,
Polari meter, and other supportive equipments.
DISTILLATION UNIT
The distillation unit has been set up for obtaining essential oil from aromatic plants such as
Menthaarvensis, Menthapiperata, Menthacardiaca, Lemongrass, Palmarosa, Citronella, Basil,
Vetiver, and Geranium etc. The recovery of essential oil from different aromatic plants has been
found to be relatively high as compared to conventional distillation unit used by the farmers at
their site. About 1000 kg fresh herbs per batch can be distilled in the unit.
VERMICOMPOSTING UNIT
This is the fastest and effective way of recycling of organic waste with the help of earthworms
for the production of useful compost. To utilize the large quantity of agro-waste generated from
distillation and solvent extraction units the vermicomposting unit has been set up at the Park. The
unit will serve as demonstration-cum-training facility for the farmers.

Place: LUCKNOW

INTRODUCTION
Microbiology is the study of all living organisms that are too small to be visible with the naked
eyes. This includes bacteria, archaea, viruses, fungi, prions, protozoa and algae, collectively
known as microbes. These microbes play very important roles in the nutrient cycling,
biodegradation/climate changes and food spoilage. The causes and control of disease and
biotechnology thanks to their versatility microbes can be put to work in many ways ,making the
life saving drugs ,the manufacture of bio fuels ,cleaning up pollution ,and been producing
/processing foods and drinks. Bacteriological water analysis is common method of analyzing
water to estimate the total number of bacteria present to found out what sort of bacteria they are
it also represents the water quality aspect it is a microbiological analytical procedures in which
uses samples of water and from these samples determine the total average concentration of
certain bacteria or bacterial colonies. Analysis is usually performed using the culture,
biochemical and some optical methods. When indicator organism levels exceed specific analysis
for pathogens may then be undertaken and these can be quickly detected or suspected using
specific culture techniques.
The scope and relevance of microbiology
In the 20th century microbiology contributed greatly to the field of Biochemistry and genetics. It
also helped stimulate the rise of Molecular Biology. There is a wide variety of fields in
microbiology and many have a great impact on society which include the more applied
disciplines such as medical, Public Health, industry, food and dairy microbiology.M
Microbiology, equality, physiology, biochemistry and genetics are examples of basic
microbiology research fields.
The future of microbiology
Microbiologist will be faced with many exciting and important future challenges such as finding
new ways to combat disease, reduce pollution, and feed the world’s population. As the study of
gene structure, the control of diseases and the industrial processes based on the phenomenal
ability of microorganisms to decompose and synthesise complex organic molecules.
Microbiology is one of the most rewarding of profession because it gives its practitioners the
opportunity to be in contact with all the other natural Sciences and does to contribute in many
different ways to the betterment of human life.

Application of microbiology
Whilst there are undoubtedly some affair all my closed due to the association of some microbes
with various human illness, many microbes are also responsible for numerous beneficial
processes such as industrial fertilization (example the production of alcohol vinegar and dairy
products), antibiotic production and as vehicles for cloning in more complex organisms such as
plants. Scientist have also exploited the knowledgeof microbes to produce biotechnological
important enzymes such as Taq polymerase, reporter genes for use in other genetic System and
novel molecular Biology techniques such as the yeast two-hybrid system.
Bacteria can be used for the industrial production of amino acids. Corynebacteriumglutamicum
is one of the most important bacterial species with an annual production of more than 2 million
tonnes of amino acids, mainly L-glutamate and L- lysine.
A variety of biopolymers, such as polysaccharides, polyesters and polyamides are produced by
microorganism. Microorganisms are used for the biotechnological production of biopolymers
with tailored properties suitable for high value Medical application suggests tissue engineering
and drug delivery. Microorganisms are used for the biosynthesis of xanthan, alginate, cellulose,
cyanophycin, poly (Gamma glutamic acid), levan, hyaluronic acid, organic acids,
oligosaccharides and polysaccharides and polyhydroxyalkanoates.
Microorganisms are beneficial for microbial biodegradation or bioremediation of domestic,
agricultural and industrial waste and subsurface pollution in soil, sediments and Marine
environment. The ability of each microorganism to degrade toxic waste depends on the nature of
each contaminant. Since sites typically have multiple pollutant types, the most effective
approaches to microbial biodegradation is to use a mixture of bacterial species and strength, a
specific to a biodegradation of one or more types of contaminants.

REVIEW OF LITERATURE
Microbiological contamination of water has long been a concern to the public from the 1920 to
1960 The Bachelors which causes Typhoid fever was considered a major problem in the water
supply once it was eradicated new microbes were present to take its place in parts of the United
States, concern is increasing due to outbreaks of coliform bacteria, giardiasis, cryptosporidiosis
and hepatitis A some of these are Bacteria, while others are viruses or Protozoa for a public
water supply system, the water company incharge of the checking and innovation of microbial
contamination if the water supply is privately owned a reputable lab should be engage to check
the purity of the water treatment do exist for microbial contamination, but it is important to know
what is present before treatment is begin.
Coliform bacteria
Coliform bacteria live in soil or vegetation and in the gastrointestinal tract of animals perform
Centre water supplies from the direct disposal of waste into streams or lakes or from runoff from
wooded areas, pastures, feedlots, septic tanks, and sewage plant in two streams or groundwater in
addition, call forms can enter and individual house wire bag flow of water from a contaminated
source, carbon filters, or leaking well cabs that allow dirt and dead organisms to fall into the
water.
Coliforms and not a single type of bacteria, but a grouping of bacteria that includes many
strange, such as equal they are of ubiquitous and nature, and many types are harmless therefore it
is not definite that coliform bacteria will cause fatness many variables such as the specific type
of bacteria present, and your own immune systems effectiveness will determine if you will get
sick in fact, many people become immune to bacteria that are present in their own . Guests on the
other hand, may not have developed immunity to the water and may experience some
gastrointestinal distress such as diarrhoea and gastroenteritis.
Total coliform Sa the standard by which microbial contamination is measured columns will be
one of the first bacteria present in the water should contamination occur and they will be in much
larger quantities then some pathogenic microbes that may be present therefore, coliform act as
indicator of possible contamination the presence of coliform bacteria does not necessarily mean
that pathogenic microbes are also present however if large quantities are detected the presence of
other micro should be checked for generally testing is done it may also be wise to test the
following reasons:
1. A new well or pump has been installed
2. An old well or pipe has been repaired or replaced
3. Family or guest have reoccurring gastrointestinal distress
4. An infant is living in the home
5. A new home is being purchased, and the quality of water needs to be determined
6. The effectiveness of a water treatment system needs to be tested
7. The water has had a change in taste, colour or odour
Testing of your water can be done by a local testing laboratory, or by a county or State Health
laboratory. If your water is found to be contaminated, the best treatment is generally disinfection
of filtration. Other options involve UV radiation and ozonization. A water professional can help
you select the best treatment.

MICROORGANISMS PRESENT IN WATER

1. Pseudomonas
Members of the genus display the following defining characteristics:
 Rod shaped
 Gram negative
 One or more polar flagella, providing motility
 Aerobic
 Non-spore forming
 Positive catalyst test
 Positive oxidase test
Other characteristics which tend to be associated with Pseudomonasspecies (with some) include
secretion of pyoverdine, a fluorescent yellow-green siderophore under iron-limiting conditions.
Certain Pseudomonas species may also produce additional types of siderophore, such as
pyocyanin by Pseudominas aeruginosa and thioquinolobactin by Pseudimonas fluorescence.
Pseudomonas species also typically give a positive result to the oxidase test, the absence of gas
formation from glucose, glucose is oxidized in oxidation/fermentation test using Hugh and
Leifson O/F test, beta haemolytic (on blood Agar), indole negative, methyl red negative test, and
citrate positive. Pseudomonas is a Gram Negative rod that belongs to the family
Pseudomonaceae. More than half of all clinical isolates produce the blue green pigment
pyocyanin. Pseudomonas often has a characteristic sweet odour.
These pathogens are widespread in nature, inhabiting soil, water, plants, and animals (including
humans).Pseudomonas aeruginosa has become an important cause of infection, especially in
patients with compromised host defense mechanism. It is the most common pathogen isolated
from patients who have been hospitalized longer than one week. It is a frequent cause of
nosocomial infections such aspneumonia, urinary tract infections, and bacteraemia.
Pseudomonas infection are complicated and can be life threatening.
2. Shigella
Cell structure: Shigella’a structural characteristics follow that of Gram Negative bacteria. Most
research would agree that Shigella is non motile but some evidence suggests that they do in fact
have flagella, although motility is not necessary for infection of intestine. The flagella tend to be
on one pole of the cell and about 10 microns in length and 12 to 14 nm in diameter. The genes
coding for the flagella in S. dysenteriae, S. flexneri, S. boydii and S. sonnei were found to be
different and attribute to the genetic diversity among the species.
Metabolism: S. boydii, when found in the intestine, go through anaerobic metabolic pathways
but can survive outside of the body due to its ability to utilise aerobic pathways. More
specifically, S. boydii, typically does not have oxidase enzymes but rather catalase enzyme.
Methyl red testing is positive, meaning that the bacteria use a mixed acid fermentation pathway.
Voges-Proskaeur and Simmons’ citrate reactions are negative, meaning that this organism does
not utilise the butylene glycol pathway or produce acetoin. Lysine decarboxylase, arginine
dihydrolase and ornithine decarboxylase are not present. S. boydii does not produce H2S, does
not hydrolyse urea and does not grow in KCN broth. Carbohydrates are usually fermented and
these include glucose (in the absence of gas production), d-mannitol, arabinose, trehalose and
mannose
3. Salmonella
Cell structure: Salmonella bacteria are rod shaped Gram Negative bacteria. Gram Negative
bacteria do not retain the Crystal Violet dye used in the Gram staining method due to the fact that
they have cell walls that are thin. Gram Negative bacteria are often harmful to a host, which is
the case for many of the Salmonella bacteria.
Salmonella bacteria are between 2 and 5 micrometres long and 0.7 to 1.5 micrometres in
diameter. They have flagella, which are tail-like projections made of proteins that help the
bacteria to move.
Metabolism: The Gram test determines to the composition of a bacterium’s cell wall.
Salmonella is Gram Negative, which signifies high amount of peptidoglycan, a mesh like
substance that provides structure and strength.
On MacConkey Agar, Salmonella colonies appear colourless and transparent, though they
sometimes have dark centres. A colony is a group of bacteria that are growing together.

AIM AND OBJECTIVES

1. To learn the basic microbiological techniques

2. To determine the microbial count and microbiological test of water sample.
3. To determine the total Dissolved Oxygen concentration (DO) of waste water sample.
4. Physico-chemical analysis of bacteria

INSTRUMENTS USED IN THE LABORATORY

1. AUTOCLAVE:
 Make & model: NAT steel equipmentsAutoclave is sterilization equipment used
to sterilize glassware, culture media, culture waste, etc. It works on the principle of moist heat
sterilization in which the article to be sterilized is subjected to the high pressured steam (15psi)
at 1210C, for about 15–20 minutes. Such extreme temperature and pressure eliminates any
living organism that can cause contamination. Depending upon its orientation, autoclaves can
either be horizontal or vertical.

Vertical Autoclave Horizontal Autoclave

2. GLASS ELECTRODE pH METER:

Make &Model: Eutech 510
A pH meter is one of the most important equipments in a microbiology laboratory & is used for
measurement as well as adjustment of the pH of a liquid, mostly a culture media.
A glass electrode pH meter consists of a glass electrode (which itself consists of AgCl wire
suspended in KCl solution in a glass bulb made up of Na &Ca salts) & a reference electrode
(itself consisting of KCl wire suspended in KCl solution).When both the electrodes are dipped in
a solution, the H+ ions from solution replace metal ions from the glass bulb of glass electrode.
Similarly the H+ ions in KCl solution replace Ag+ ions from AgCl wire. Since the two solutions
on either side of the glass bulb have different acidity, the amount of H + ions exchanged will also
be different. This results in development of a potential difference between two sides of glass
which in turn results in a potential difference glass & reference electrodes which is amplified and
displayed in the form of a pH value.

3. VORTEX MIXER:
Make & model:It is a relatively simpler device which is used to decrease the time required to
dissolve a substance in a liquid by creating a small vortex within the container of the liquid.
The device consists of a rubber end which moves in a circular motion at very high speeds. When
the container of a liquid comes in contact with it, the motion is transferred to it, creating a vortex
within, thus facilitating rapid mixing.
4. HOT AIR OVEN:
Make & model: JSGW hot air ovens are also drying equipment used to sterilize as well as dry
various laboratory apparatuses. Contrary to an autoclave, it works on the principle of dry heat
sterilization where the article to be sterilized or dried is subjected to temperatures ranging from
50-300 °C. Since it can operate at such high temperatures, it is primarily used for glass & metal
articles. A thermostat keeps temperatures within set limits while a fan fitted inside the oven
ensures uniform distribution of heat within.

5. COLONY COUNTER
A colony counter is an instrument used to count colonies of bacteria or other microorganisms
growing on an agar plate. Early counters were merely lighted surfaces on which the plate was
placed, with the colonies marked off with a felt-tipped pen on the outer surface of the plate while
the operator kept the count manually. More recent counters attempt to count the colonies
electronically, by identifying individual areas of dark and light according to automatic or user-set
thresholds, and counting the resulting contrasting spots.
6. BOD INCUBATOR:
Make & model: LABCO A BOD incubator refers to Biochemical Oxygen Demand Incubator. It
is specifically made to maintain at 200C during the incubation period so that BOD tests can be
conducted. In such an incubator, the samples are kept saturated with oxygen and the temp is kept
constant by a compressor and a heater working against each other.

7. HORIZONTAL LAMINAR AIR FLOW CABINET:

Make & model:It is a device which creates a sterile environment within its working area by the
virtue of laminar flow of air. Laminar flowis the motion of a medium (fluid or a gas)in parallel
layers, with no disruption between the layers. Due to parallel flow, a unidirectional layer of
medium is created which takes the contaminants along with itself all the way out. In addition to
it, laminar air flow cabinet also deploys HEPA Filters (High Efficiency Particulate Air Filters) to
filter out dust particles & contaminants from air.

8. BRIGHT FIELD COMPOUND MICROSCOPE:

It is commonly used for generally laboratory operations. It consists of a series of two lens
systems between the eye and the object and a direct light source (sun and mirror or lamp) where
light shines directly on the specimen and dark object in a bright field is observable.
Principle:A typical bright field microscope consists of four basis parts:
1. A base to provide firm support and stability to the microscope.
2. The stage, which is a flat platform and holds the slide in position.
3. The arm which is movable is used for carrying the microscope and can be set in a
comfortable position for the worker.
4. The body tube for transmitting the magnified image.

The optical system consists of two series of lenses, the objective and ocular lens (eye piece). The
upper end of the tube holds the ocular lens (10x-15x) through which the image is viewed. The
lower end which is lodged close to the object being examined is fitted with a rotating nose piece
holding generally three objective lenses, which provide magnification powers of 10x, 40x, and
100x. The greatest magnification achieved by most standard bright field microscope is 1000x.
The resolution of such a microscope is 0.00027mm (0.27um).The coarse adjustment which raises
or lowers the base tube, is used for focusing the low power objective and fine adjustment is used
for focusing high power and oil immersion objectives. The area seen through microscope is
called field of vision.
Uses and application: A compound microscope is now-a-day used in several fields of sciences
like the microbiology, Botany, Geology, Genetics and the like. Forensic experts and scientists
can also find out the country from which the drug has come by viewing its particles under a
compound microscope as the shape of the crystals can give a reference as to which country the
opium was grown in. Microscope also help in looking at the minute details of the human cells
and determine the presence or absence of minerals, identify the presence of metals, thus solve
crimes and discover medicines.

MATERIALS AND METHOD

1. NUTRIENT AGAR MEDIA
Nutrient agar is a microbiological growth medium commonly used for the routine cultivation of
the non-fastidious bacteria. It is useful because it remains solid even at relatively high
temperature. Also bacteria grow in nutrient agar on the surface and are clearly visible as small
colonies. In nutrient broth, the bacteria grow in liquid, and are seen as a soupy substance, not as
clearly distinguishable clumps.
Requirement:
1. Conical flask
2. Non-absorbent cotton
3. Distilled water
4. NA media (2.8g for 100 ml)

Procedure:
1. 100 ml distilled water was poured in the flask.
2. A weighed amount of Nutrient agar was added to 100 ml distilled water.
3. It was made sure that all the contents dissolve properly.
4. pH of the medium was adjusted to 7.0 using pH meter, by adding either acid or alkali, as
the case may be.
5. The medium was sterilized in an autoclave at 121C temperature and 15 lbsp.s.i. pressure
for 15 minutes.
6. The autoclave was allowed to cool.
7. The prepared NA medium was placed in L.A.F. chamber.

2. SYNTHETIC POTATO DEXTROSE MEDIUM

Requirement:
1. Conical flask
2. Non-absorbent cotton
3. Distilled water
4. PDA powder ( 3.9g for 100 ml)

Procedure:
1. 3.9g PDA media was weighed.
2. PDA medium was dissolved in flask containing 50 ml distilled water.
3. The solution was well mixed and volume was made upto 100 ml by adding distilled
water.
4. The solution was heated on the heating plate between 40-50C.
5. The medium was sterilized by autoclaving at 121C temperature, 15 lbsp.s.i. pressure for
15 minutes.
6. The autoclave was allowed to cool.
7. The prepared PDA medium was placed in L.A.F. chamber.

1. PREPARATION OF PDA PLATES

Requirement:
 PDA media
 Aluminium foil
 70% ethanol
 Petridish
 Conical flask
Procedure:
1. All glasswares and media were sterilized in autoclave at temperature 121 C and 15 lbs
pressure for 15-20 minutes.
2. When temperature cools down, it was transferred into the cabinet of the laminar air flow
or inoculation chamber built for inoculation work. However, these must be sterilized by
UV light for 15 minutes before the start of work.
3. Take the flask containing PDA medium and pour about 15-20 ml medium aseptically into
the bottom half of the Petri plates separately when temperature remains to about 40 C.
4. Leave the plates in the laminar flow chamber to solidify for about 25-30 minutes.
5. These plates containing the solidify medium are called as PDA plates.

2. TO PERFORM STREAK PLATE TECHNIQUE:

A streak plate technique is used to isolate a single species from a mixed species population.
Sample of the mixed species on a sterile loop and streak on agar medium into four zones, reflame
the loop. After incubation, single species colonies should be visible within the fourth zone.
 Requirements:
1. Culture of isolated bacterial strain
2. Sterile petridish with appropriate bacterial media
3. Inoculating loop
4. Bunsen burner
5. Laminar air flow cabinet

Procedure:
1. Labeling of all the plates with the bacterial strain to be inoculated was done with the
marker.
2. The flame sterilized loop was taken in the right hand and introduced into the culture plate
and one loopful culture was withdrawn.
3. Inoculums were placed on media surface at the edge
4. Loop was reflamed and allowed to cool. Loop was allowed to touch the inoculum placed
on the plate then loop was dragged in zig zag manner over the surface of media.
5. The lids of the petridish were closed after completing the streaking and sterilized the loop
on flame
6. All the plates were incubated at 350C in an inverted position for 48hours to 72 hours.

3. TO PERFOR M GRAM STAINING

Materials:
 Bacterial culture
 Glass slides
 Cover slips
 Blotting paper
 Microscope
 Crystal violet dye
 Gram’s iodine
 95% alcohol
 Saffranin
Procedure:
 Microscope slides were cleaned thoroughly.
 Surface of the slide and inoculation loop was flamed to remove contamination.
 Smear was made on the slide with the help of the loop and was allowed to dry thoroughly
and heat fixed.
 The smear was stained by flooding with crystal violet for 30 seconds and then was
washed with distilled water for few seconds.
 The smear was then covered with Gram’s iodine for 60 seconds.
 The iodine solution was then washed off using 95% alcohol drop by drop until no more
colour flowed from the smear.
 The slide was washed with the distilled water and drained.
 Saffranin was applied to the smear for 30 seconds.
 At the end of the designated time, the excess stained was washed off slowly by running
water.
 The back of the slide was whipped off and the stain was bolt using blotting paper.
 The stained smear was placed on the microscopic stage, smears side up and focused at
100X objectives.

PHYSICO-CHEMICAL ANALYSIS OF SAMPLE

EXPERIMENT N0.1
DETERMINATION OF TOTAL DISSOLVED SOLID
The dissolved solid of water can be measured manually and mechanically which displays the
total number of solids dissolved in water sample.
Aim: To determine the total dissolved solids in water sample.
Materials required:
1. Beaker
2. Hot plate
3. Water sample
4. Weighing machine
5. Dessicators
Procedure:
1. An empty beaker was taken and its weight was recorded.
2. 100 ml of sample was poured in the beaker and then it was kept on hot plate till it dries
completely.
3. After that beaker was placed in dessicators to cool down the temperature.
4. The weight of the beaker was noted again. (containing dried water sample residue)
5. The following values were recorded:
Constant weight before evaporation(A)
Constant weight after evaporation(B)
Volume of sample(V)
Therefore,
TDS(mg/l) = (B-A)*1000/ V mg/l

EXPERIMENT N0. 2
ESTIMATION OF TOTAL SUSPENDED SOLIDS
The suspended solid of water can be measured manually and mechanically which displays the
total number of suspended solids in water sample.
Materials required
1. Conical flask
2. Hot air oven
3. Water sample
4. Weighing balance
5. Funnel
6. Whatman filter paper
Procedure:
1. A Whatman filter paper was taken and its weight was noted down.
2. The filter paper was placed in funnel and 100 ml of water sample was passed through it.
3. The funnel having filter paper was dried in hot air oven at 1800C.
4. The weight of filter paper was taken again. (containing dried residue)

EXPERIMENT N0. 3
ESTIMATION OF TOTAL CHLORIDES IN WATER SAMPLE
Principle:
Chlorides are widely distributed as salts of calcium, sodium and potassium in water. Chlorides
are responsible for salty taste in water. The amount of chlorides present in water can be easily
determined by titrating the water sample with silver nitrate solution.

Reagents:
Standard sodium chloride solution- 1.648g of sodium chloride was dissolved in 100 ml of
distilled water in a volumetric flask.
Standard silver nitrate solution (0.0282N)- 4.791g of silver nitrate was dissolved in distilled
water and diluted to 100 ml in a volumetric flask.
Potassium chromate indicator: 2.5g of potassium was dissolved in 25 ml of distilled water and
diluted to 100 ml with distilled water in a volumetric flask.

Procedure:
1. 20 ml of sample was taken in a clean 250 ml flask.
2. 1 ml potassium chromate was added to get light yellow colour.
3. Then sample was titrated against silver nitrate solution till the yellow colour changes to
brick red at end point.
4. The value of silver nitrate consumed in titration is recorded.
5. A blank of 20 ml distilled water was also titrated with the same procedure and the value
was noted down.

Calculation:
Volume of silver nitrate consumed in sample (V1)
Volume of silver nitrate consumed in distilled water (V2)
Normality of silver nitrate (N)
Equivalent weight of chlorine= 35.5
Total chlorides (mg/l) = (V1-V2)* N * 35.45 * 1000/ Volume of sample

EXPERIMENT N0. 4
DETERMINATION OF DISSOLVED OXYGEN IN WATER
Some amount of oxygen is dissolved in water which is used by the aquatic plants and animals.
The source of dissolved oxygen in water is the autotrophic aquatic plant which as a result of
photosynthesis evolves oxygen and air where from oxygen is dissolved in water depending on
salinity, temperature and water movement. Moreover in an oligotrophic lake the amount of
dissolved nutrient salts remains low, therefore , its support sparse plants and animal lives .This
result in high dissolved oxygen gradually increasing with depth, In addition in atrophic water
reservoirs e.g. lakes , ponds, pool etc. the organic nutrient accumulate abundantly which in turn
are subjected to microbial decomposition . More growth of microorganism, plants and animals
depletes oxygen. This depletion increases with increase with water depth
Dissolved oxygen is measured by titration methods. The theory behind this is that the dissolved
oxygen combines with mangoes hydroxide, which in turn librates iodine (equivalent that of
oxygen fixed) after Acidification with H2SO4 the iodine can be titrated with sodium thiosulphate
solution by using starch indicator.

Requirements:
 BOD Bottle (250 ml capacity)
 Water sample from a water body, sewage or treated sewage water, etc.
 Alkaline iodine azide solution
 Sodium thiosulphate
 Manganesesulphate solution
 H2SO4(conc.)
 Pipette
 Titration set

Reagents:

Alkaline KI Solution: Dissolve 100 gm of KOH and 50 gm of KI in 200ml of Distilled Water.

Manganese sulphate (MgSO4.4H2O) Solution: Dissolve 100 gm of Manganese sulphate in

200ml of Distilled Water. Filtered and keep it in stopper bottle.
Starch Indicator: Dissolve 5g of starch in 100ml of hot distilled water and few drops of
formaldehyde (HCHO).

STANDARD PROTOCOL
1. Collect water sample in a BOD glass bottle (300ml) in such a way that water bubble
should not come out.
2. Pipette separately 2 ml of Manganese Sulphate and 2 ml of alkaline iodine azide solution.
3. Add these solutions in succession at the bottom of bottle and place the stopper of bottle.
4. Shake the bottle upside down for about 6-8 times. There develops brown precipitate
5. Leave the bottle for few minutes, precipitate settle down
6. Add 2 ml concentration H2SO4 in the bottle; shake properly so that brown precipitate may
dissolve
7. Take a clean flask and pour 200 ml of this water sample. Titrate it against 0.025 N
sodium thiosulphate solution taking in a burette until pale straw colours develops.
8. Add 2 drops of starch solution to the flask. Colour of contents change from pale to blue.
9. Again titrate against thiosulphate solution until the blue colour disappears. Note the
volume of sodium thiosulphate solution used in titration.

EXPERIMENT N0. 5
DETERMINATION OF BIOLOGICAL OXYGEN DEMAND IN WATER
The BOD test is used to measure waste loads to treatment plants, determine plant efficiency (in
terms of BOD removal), and control plant processes. It is also used to determine the effects of
discharges on receiving waters. A major disadvantage of the BOD test is the amount of time( 3
or 5 days) required to obtain the results.
Aim: To calculate BOD of given water sample.
Reagents:
Phosphate buffer solution: 0.85g potassium di-hydrogen phosphate (KH2PO4), 2.175g
potassium hydrogen phosphate (K2HPO4), 3.34g disodium hydrogen phosphate (Na2HPO4.7H2O)
and 0.17g ammonium chloride (NH4Cl) were dissolved in about 50 ml of distilled water and
diluted to 100 ml. pH of the solution was kept 7.2.
Magnesium Sulphate solution: 2.25g magnesium sulphate ( MgSO4.7H2O) was dissolved in
distilled water and diluted to 100 ml.
Calcium chloride solution: 2.75g calcium chloride was dissolved in distilled water and diluted
to 100 ml.
Ferric Chloride Solution: 0.025g hydrated ferric chloride (FeCl3.6H2O) dissolved in distilled
water and diluted to 100 ml.
Other reagents for dissolved oxygen
Alkaline KI Solution: Dissolve 100 gm of KOH and 50 gm of KI in 200ml of Distilled water.
Manganoussulphate (MnSO4.4H2O) Solution: Dissolve 100 gm of Manganese sulphate in
200ml of Distilled Water. Filtered and keep it in stopper bottle.
Starch Indicator: Dissolve 5g of starch in 100ml of hot distilled water and few drops of
formaldehyde (HCHO).
Sodium thiosulphate stock solution: approximately 2.4 g of sodium thiosulphite
(Na2S2O3.5H2O) was dissolved in boiled distilled water and volume made up to 100ml. 1g of
sodium hydroxide was added to preserve it.
Standard sodium thiosulphate solution: 25.0ml of stock solution was mixed in boiled water
and volume was made up to 100ml.

Procedure:
1. 10 ml of water sample was taken in a flask and the volume was made up to 1 L.
2. At the time of use, 1 ml each of phosphate buffer, magnesium sulphate, calcium chloride
and ferric chloride were added for 1 litre of dilution water.
3. Two BOD bottles were rinsed and filled with 300 ml of dilution water.
4. Initial DO was determined for one bottle and one bottle was kept for incubation at
270Cfor 3 days.
5. The value of initial DO was recorded.
6. After 3 days, incubation at 270C, the final DO was obtained and recorded.
7. Thus,
BOD (mg/l)= [(initial DO-Final DO)×1000]/ml sample

MICROBIOLOGICAL ANALYSIS OF SAMPLE

 EXPERIMENT NO.6
Aim-: Microbial count for Gomti River Water Sample through Serial Dilution Method

Principle: The serial dilution method is based upon the principle that when material containing
microorganism is been cultured, each viable microorganism well develops into a colony.Hence,
the number of colonies present on the plate represents the number of living microorganisms
present in the sample.
In the serial dilution agar -plate method, a known amount of material is been suspended or
agitated in a much known volume of sterile water blank to make a microbial suspension. Serial
dilution is made. Finally 1 ml of aliquot of various dilutions isadded to the sterile petri plates to
which are been added 15ml of the sterile, cool, molten media or spreaded on the Agar surface.

Materials Required:
1. Water Sample
2. Test tubes
3. Petri dishes
4. Sterile tips and micropipette
5. Nutrient agar medium and potato dextrose agar meduium
Procedure:
1. 6 blanks were prepared in test tubes filled with 9ml distilled water.
2. 100 ml Nutrient agar and 100ml PDA media was prepared according to the composition.
3. The water blanks and mediums were sterilized in autoclave at 121 0C, 15 psi pressure for
15- 20 minutes.
4. 100ml Nutrient agar media and 100 ml PDA media was also poured in sterile petri plates.
The plates were kept for solidifying.
5. Then 5 test tubes were taken which were filled by the 9ml sterile distilled water and
marked them as 10-1, 10-2, 10-3, 10-4 and 10-5 dilutions.
6. 1ml of the sample was mixed properly in 1st test tube.
7. 1ml of aliquot was taken out with the help of the micropipette from the test tube and was
mixed to the test tube which was been marked as 10-2, containing the 9ml sterile water.
8. This process was repeated for the other test tubes like 10-3, 10-4, 10-5dilutions
9. Then 100ml of aliquot of dilutions 10-3, 10-4, 10-5 were spread on the respective NA plates
and 10-2 spread down on the PDA plate.

EXPERIMENT N0. 7
Aim: Bacteriological identification and confirmation of Salmonella, Pseudomonas and
Staphylococcus.
 For the presence of Salmonella.
1. Preparation of Salmonella broth
2. Inoculation of the enriched water sample into the Salmonella broth in the laminar air
flow.
3. Incubated in the BOD incubator shaker for 48 hours at 42 0C for the growth of the micro-
organisms.
4. Observed for growth and characteristics.
Materials required:
1. Distilled water
2. 3 conical flask
3. Micropipette
4. Salmonella broth
Procedure:
1. 50 ml distilled water was poured in 3 flasks.
2. The distilled water was autoclaved at 1210C temperature at 15 lbs p.s.i pressure for 15
minutes.
3. In 50 ml of distilled water 1.35g of Salmonella broth was added in each flask.
4. The medium was boiled and allowed to cool.
5. The Salmonellabroth flasks were placed in laminar air flow and inoculated with 2.5 ml of
water sample in each flask.
6. After inoculation the flasks were kept in BOD incubator for 48 hours at 420C.
 For the presence of Pseudomonas.
1. Preparation of the Pseudomonas aeruginosa presumptive broth and its sterilization in the
autoclave at 1210C and 15 lbs p.s.i. pressure for15 minutes.
2. Inoculation of water sample in Pseudomonas presumptive broth.
3. Incubation in the BOD incubator for 48 hours for the growth of micro-organisms.
4. Then they were observed for the growth.
5. The positive ones should be further streaked on skim milk agar plates and incubate in the
BOD incubator at 480C.
6. These were then observed for growth.

Preparation of Pseudomonas presumptive broth:

1. DL Asparagine 0.1g
2. L Proline 0.05g
3. Anhydrous dipotassium hydrogen phosphate 0.05g
4. Magnesium sulphate heptahydrate 0.24g
5. Anhydrous potassium sulphate 0.5g
6. Ethanol 1.25 ml
7. Distilled water 50 ml

Materials required:
1. Distilled water
2. 3 conical flask
3. Micropipette
4. Measuring cylinder

Procedure:
 50 ml distilled water was poured in flask.
 Dissolve all the weighed amount of chemicals into the flasks except ethanol.
 The medium was autoclaved at 1210C temperature at 15 lbs p.s.i pressure for 15 minutes.
 Sterilise ethanol by filtration and add required volume into the flask.
 Take the autoclaved Pseudomonas broth in the LAF chamber.
 Add 5 ml of water sample into each flask.
 After inoculation the flasks were kept in BOD incubator for 48 hours at 320C.

 For the presence of Staphylococcus.

a) MANNITOL SALT AGAR MEDIA
a sodium chloride, concentration of 75% serves to inhibit most organisms except Staphylococci
in mixed flora specimens. The beef extract and peptones supply the essential elements carbon,
nitrogen and sulphur. Mannitol is added to show the fermentation capabilities of the organisms.
Acid production as the result of fermentation of this sugar results in the formation of colonies
with a yellow zone. Those Staphylococci that do not ferment mannitol show a purple or red zone
around the colonies. Mannitol fermentors such as S.aureus appear as yellow colonies with
yellow zones in the media. Non- mannitol fermentors such as S.epidermis, if present, will have
clear pink to red colonies with no yellow colour change in the medium.
Materials:
 Mannitol salt agar- 6.6 g
 Distilled water- 60 ml
Procedure:
 Mannitol salt agar was weighed and was made sure that all the above components
dissolve properly.
 The medium was sterilized in autoclave at 1210C and 15 lbsp.s.i pressure for 15 minutes.
 Autoclave was allowed to cool.
 The prepared M.S.A medium was placed in the L.A.F chamber.
 The medium was poured into 4 petriplates.
 Then bacterial culture was streaked on the M.S.A plates.
 After streaking the petriplates were kept in BOD incubator for 48 hours at 320C.

BIOCHEMICAL TEST OF BACTERIA

1. MANNITOL FERMENTATION TEST
Principle:
Some of the bacteria and known to use many tall as a substrate and convert it into acidic by
products such as acetic acid and other acidic by-products phenol red has a property to show
yellow colour in acidic medium and red colour in basic medium since birth is basic in nature it
shows red colour but bacteria producing acidic product shows yellow colour.
Requirements:
1. Mannitol fermenting broth
2. test tubes
3. inoculating loop
4. master plates
5. burner
Preparation of mannitol fermenting broth (for 100 ml of broth):
1. Mannitol-1 gram
2. Peptone-1 gram
3. NaCl-0.5 grams
4. phenol red-600 microlitre
5. pH 7.4
Procedure:
1. Mannitol, peptone and NaCl were added in hundred ml distilled water and pH is
maintained at 7.4.
2. Then 600 microlitre phenol red was added to the broth.
3. 20 ml of broth was transferred to each of the 4 test tubes.
4. Then was autoclaved for 20 minutes at 1210C and 15 psi.
5. Three of them were inoculated for master plates.
6. Label it accordingly to the master plates from which inoculation was done.
7. One of the test tubes was remained uninoculated and used as control.
8. Close the mouth of the test tubes with the help of cotton plugs.
9. Kept in incubator for 24 hours at 350C.
2. AMYLAZE PRODUCTION TEST ( STARCH HYDROLYSIS)
Principle:
The purpose is to see if the microbe can use a starch, a complex carbohydrate made from
glucose, as a source of carbon and energy for growth use of starch is accomplished by an enzyme
called Alpha amylase a medium containing starch is used after inoculation and overnight
incubation iodine Reagent is added to detect the presence of starch iodine Reagent reacts with
starch to form blue black colour in the culture medium clear Hallows surrounding colonies is
indicative of their ability to digest the starch in the medium due to the presence of Alpha amylase
the medium used in starch test is nutrient Agar to which starch is added iodine reagent is added
after incubation to flood the surface of the plate.

Procedure:
The starch Agar 60 ml was prepared from the following composition:
 Starch 1.2 grams
 Beef extract 0.18g
 Peptone 0.30g
 Agar 0.90 g

1. The media autoclaved.

2. The media was poured in Petri dishes and allowed to solidify.
3. With the help of sterile inoculating loop streaking work done on all the three plates by
different strain S1, S2 and S3.
4. Close the plates.
5. The plates were incubated for 24 hours at 300C.
6. After incubation the plates were flooded withiodinereagent.
7. If starch hydrolysis takes place then a clear zone surrounding the microbial Colony
appeared.

3. INDOLE TEST
Tryptophan is an essential amino acid, is oxidised by some bacteria by the enzyme tryptophanase
resulting in the formation of indole, pyruvic acid and Ammonia. The indole test is performed by
inoculating bacteria in tryptone broth. When Kovac’sreagent (p-dimethyl amino benzaldehyde)
is added to the broth with indole in it a cherry red ring develops.

Requirement:
 Tryptone broth 10 ml per tube
 Kovacs reagent
 Dropper
 Bunsen burner
 Inoculating needle
 Test tubes

Procedure:
1. 0.6 gram of tryptone broth was dissolved in 60 ml of distilled.
2. Broth was sterilized in the autoclave at 15 psi, 1210C for 15 minutes.
3. About 10 ml was transferred in 4 test tubes and then inoculate three test tubes with strain
S1, S2 and S3 and one is left as control.
4. Incubate inoculated test tubes at 350C for 48 hours.
5. After 48 hours of incubation, add 1 ml of Kovac’s reagent to each of the tube.
6. Shake the tube gently after intervals of 10 to 15 minutes.
7. Allowthe tubes to stand to permit the reagent to come to the top.
8. The positive indole test shows the formation of a pink to red colour in the reagent layer
on the top of the medium within seconds of adding the reagent.
9. If the culture is negative the reagent layer will remain Yellow or slightly cloudy.

4. CITRATE TEST
Principle:
Cigarette test is used to differentiate among bacteria on the basis of their ability to utilize/ferment
citrate as the soul carbon source. The utilization of it depends on the presence of an enzyme
citraze produced by the organism that breaks down the citrate to oxaloacetic acid and acetic acid.
These products are later converted to pyruvic acid and carbon dioxide. The citrate test is
performed by inoculating the microorganisms into an organic synthetic medium, Simmons citrate
Agar, where sodium citrate is the only source of carbon and energy. Bromothymol blue is used
as an indicator which is green when acidic and blue when alkaline. A change in colour of
indicator from Green to Blue constitutes its positive test.

Requirements:
 Nutrient broth cultures of all three strains S1,S2 and S3
 Simmons citrate Agar slant
 Bunsen burner
 Inoculating needle

Procedure:
Simmons’s citrate agar medium was prepared from the following composition :

1. Ammonium dihydrogen phosphate 0.05g

2. Dipotassium hydrogen phosphate 0.05g
3. Sodium chloride 0.25 g
4. Sodium citrate 0.1g
5. Magnesium sulphate 0.01g
6. Agar 0.75g
7. Bromothymol blue 0.04g
8. Distilled water 50ml
9. pH 6.8

Procedure:
1. 50 ml (synthetic media) was prepared and sterilized in autoclave at 121ºC temperature
and 15 lbs psi pressure for 15 minutes.
2. Then media was dispensed 5 to 6 ml into sterile slant tubes and were kept in a slanted
position (long slant shallow butt) under
3. Simmons’s citrate agar slants were inoculated with isolated strains by means of a stab and
streak and incubated at 37ºC for 48 hours. One kept inoculated as control.
 OBSERVATION AND RESULTS

1. GRAM STAINING

The strains found in the given sample are gram positive.

PHYSICO-CHEMICAL ANALYSIS OF SAMPLE

DETERMINATION OF TOTAL DISSOLVED SOLIDS

Initial weight of beaker: 40.9132 g
Final weight of beaker: 40.9227 g
Volume of the sample taken: 50 ml
Weight of residue in grams= final weight- initial weight
= 40.922-40.923
= 0.00095 g
Weight of residue in milligrams= 0.0095 x 1000
= 9.5 mg
TDS= (9.5 x 1000)/ 50
= 190 mg/l
Result: TDS of sample is 190 mg/ml.

DETEERMINATION OF TOTAL SUSPENDED SOLIDS

Initial weight of filter paper: 1.046 g
Weight of filter paper after filtering the sample: 1.075 g
Volume of sample taken: 50 ml
Weight of residue on filter paper: 1.075-1.046
= 0.029 g
Weight of residues in mg= 29 mg
TSS in sample= (29 x 1000)/50
Result: TSS in the sample is 580 mg/l.

ESTIMATION OF TOTAL CHLORIDES IN WATER SAMPLE

Volume of silver nitrate for sample, V1 = 2.2 ml
Volume of silver nitrate for blank, V2 = 0.4 ml
Normality = 0.014 N
Volume of sample in ml= 20 ml
Equivalent weight of chlorine = 35.45
Total chlorides= {(2.2-0.4) x 0.014 x 35.45 x 1000}/ 20 mg/l
Result: Total chloride in the sample was 44.67 mg/l.

DETERMINATION OF DISSOLVED OXYGEN IN WATER

The amount of sodium thiosulphate needed was 2.2 ml. Therefore, the dissolved oxygen in the
effluent sample at 27 degree Celsius is found to be 2.2 mg/l.

DETERMINATION OF BIOLOGICAL OXYGEN DEMAND IN WATER

The value of BOD in mg/l can be determined as,
BOD= {[initial DO-Final DO] x 1000}/ dilution factor of sample in ml
Initial DO of the sample= 7.0 mg/l
Final DO of the sample= 5.6 mg/l
Sample size=
BOD= [(7.0 – 5.6) x 1000]/
Result: The biological oxygen demand of the sample was mg/l.

MICROBIOLOGICAL ANALYSIS OF SAMPLE

MICROBIAL COUNT FOR GOMTI RIVER WATER
RESULT OF BACTERIAL COUNT
At 10-3 dilution, no. of colonies
C.F.U. = 5 x 103= 5.0 x 103 cfu/ml

At 10-4 dilution, no. of colonies

C.F.U. = 49 x 104 = 4.9 x 105 cfu/ml

At 10-5 dilution, no. of colonies

C.F.U. = 12 x 105 = 1.2 x 106 cfu/ml
RESULT OF FUNGAL COUNT

C.F.U= 7 X 10-1cfu/ml

 For the presence of Salmonella

No growth was seen in Salmonella broth.
  For the presence of Pseudomonas sps
Growth was observed in sample inoculated flask.

 For the presence of Staphylococcus.

Growth was observed on mannitol salt agar plates.
Further streaking was done on Staphylococcus agar plates.

Staphyllococcus was found but further confirmation is required.

BIOCHEMICAL TEST OF BACTERIA

2. MANNITOL FERMENTATION TEST
All the strains showed change in colour from red to yellow. Hence, giving a positive result.

5. AMYLAZE PRODUCTION TEST ( STARCH HYDROLYSIS)

After flooding of iodine:

A positive starch hydrolysis reaction was given by the strains.

6. INDOLE TEST
There was ring formation in test tubes from the strains and no ring formation in the control.
Hence it showed positive result.
7. CITRATE TEST

Test tube showed blue colour due to citric acid metabolization and CO2 generation. Hence result
is positive.

CONCLUSION
The Biotech Park gave me an immense support to pursue my training in Microbiology as a form
of essential laboratory knowledge, techniques such as Autoclaving, bacterial cell culture
isolation, spreading serial dilution was learnt during the course of training.
This study was aimed to confirm the presence of different bacterial species in Gomti river water
sample by using selective enrichment broth and selective media.

Prevention strategies include the use of hand washing sanitizer.

The practical knowledge gained through Biotech Park would be helpful in our future research
work in the field of microbiology.

REFERENCES
 Experiments in microbiology, plant pathology and biotechnology K.R. ANEJA.
 Angulo FJ et al. (1997) A community waterborne outbreak of salmonellosis and the
effectiveness of a boil water order. American journal of Public Health, 87:580-584.
 Bureu of Indian Standards
 Applied and environmental microbiology
 Dubey R.C. Maheshwari D.K., Practical microbiology
 Miller G Robbert, Kopler C Frederick and S Nancy. (1984): Ulmer the occurrence of
aluminium in water.
 Chakrabarty et. al.
 HardaloC, Edberg SC(1978) Pseudomonas aeroginosa: assessment of risk from drinking
Indian Pharmacopia 2007.