A

PROJECT REPORT
ON
“

Submitted in Partial Fulfillment for the award of

Bachelor of Science.
In
BIOTECHNOLOGY
(2016-2017)
Submitted by
ANAMIKA AGRAWAL
B.Sc. Biotechnology
(VI Semester)

Under the supervision of Dr. Kamlesh Choure, Head,

Department of Biotechnology,
AKS University Satna (M.P.)

(ANAMIKA AGRAWAL)
1. Introduction.
2. Review Of Literature.
3. Objective.
4. Materials And Methodology.
5. Result And Discussion.
6. Conclusion.
7.References.
 INTRODUCTION
E.coli , was first described by Theodor Escherich in 1885, is a member of family
enteriobacteriaeace. It is a gram negative, motile, non-sporing bacillus , produces rose
pink colonies on MacConkey Agar. The species can be differentiated from other
members of enterobacteria by biochemical reaction, being a member of
enterobacteriaeace it is present as normal flora in the lower intestine of both humans
and animals; However, some strains can cause gastrointestinal illness ranging from mild
to cholera-like diarrhoea and may lead to potentially fatal complications, such as
haemolytic uremic syndrome & thrombotic thrombocytopenic purpurea in human
beings.

E. coli is a genetically and phenotypically diverse species whose strains are

identified on the basis of ‘O’, ‘H’ and sometimes ‘K’ antigens,biochemically &
serologically based on toxigenicity , On the basis of intestinal diseases, there are six
categories: enteroaggregative E. coli diffusely adherent E. coli enteroinvasive E. Coli
enteropathogenic enterohaemorrhagic E. coli and enterotoxigenic E. coli Over 700
serotypes of E. coli are recognized on the basis of O, H, and K antigen
 Several highly adapted clones have acquired specific virulence
factors that are accountable for a variety of intestinal and extra intestinal
diseases including diarrhea, acute inflammation, hemorrhagic colitis,
urinary tract infections, septicemia, and neonatal meningitis (Kaper et al.,
2004; Levine et al., 1987; Nataro et al. , 1998; Orskov et al. 1992). In
rare cases, virulent strains are also responsible for haemolytic-uremic
syndrome, peritonitis, mastitis, septicaemia and Gram-negative
pneumonia.

E. coli bacteria escape the intestinal tract through a perforation (for

example from an ulcer, a ruptured appendix, or due to a surgical error)
and enter the abdomen, they usually cause peritonitis that can be fatal
without prompt treatment. However, E. coli are extremely sensitive to
such antibiotics as streptomycin or gentamicin
 REVIEW OF LITERATURE
Escherichia coli Scientific classification (Brenner et al., 2005):
Domain: Bacteria
Kingdom: Eubacteria
Phylum: Proteobacteria
Class III: Gammaproteobacteria
Order XIII: Enterobacteriales
Family I: Enterobacteriaceae
Genus I: Escherichia
Species: E. coli
 They are a part of the normal gastrointestinal microflora
where they provide vitamin K2 that is required for blood
coagulation and are recognized as a protective agent against
pathogenic bacteria.
They are non-pathogenic flora of the human and animal
intestine, but in immunocompromised host, E. coli and other
members of the gut microflora may act as opportunistic
pathogens. Several strains of E. coli have developed the ability
to cause diarrhoea and extra intestinal infections in humans
and animals, as they possess different molecules called
virulence factors, which determine the pathogenicity of the
bacterium in different hosts as well as different host tissues
Due to their inability to survive outside the host for long
periods, they are considered ideal indicator organisms to test
for faecal contamination in environmental samples
 Pathogenic E. coli
Pathogenic E. coli include those types, which cause enteric infections and those, which
cause extraintestinal infections. The virulence factors of pathogenic E. coli are encoded by
genetic elements like plasmids, transposons and bacteriophages. These factors mostly promote
colonization of the host, adherence or invasion of cells, evasion of host defences and disruption
of host cell signalling pathways and include cell surface receptors, secreted enzymes and toxins
3. Routes of transmission to humans The different routes of transmission of pathogenic E.
coli to humans are via water, food, or other items that are contaminated either directly or
indirectly by faecal matter. E. coli poses a significant risk to human health as they can freely
propagate in the gastrointestinal tract of animals, and may be shed in large numbers in animal
faeces
Food-borne infection- Contaminated foods of animal origin such as unprocessed meat,
undercooked hamburgers, sausages, un-pasteurized milk, yoghurt and cheese are common
sources of E. coli infections. Investigation of an outbreak of E. coli in Washington and California,
due to dry cured salami estimated the infectious dose of bacteria to be lesser 14 than 50
organisms studied some outbreaks of EHEC associated with consumption of mayonnaise.
Simango (1995) isolated 126 strains of E. coli from different foods and drinks consumed in
a rural community in Harare, Zimbabwe and 7.5% of them were reported to be of the EPEC
serogroup. Michino et al. (1999) studied a large outbreak of E. coli O157 in Sakai, Japan in 1996
in which raw radish sprouts served in school lunch caused illness in more than 6,000 primary
school children. Cowden et al. (2001) investigated an outbreak of E. coli O157 in Scotland in
1996 in which 522 people were infected and 22 died after eating contaminated meat from a
butcher‟s shop
 Materials and Methodology:

NAM (Nutrient agar media) LB media (Luria Bernini media)

Name of Amount s.no Name of Amount

s. chemical chemical
no
1 K2Hpo4 7gm
Peptone
F 5gm
1. 2. (NH4)2po4 1gm
Beef
2 extract 3gm
3. Kh2PO4 3gm
2.
Agar
3 3.0gm 4. MgSo47H2O 0.3gm
3.
Nacl
4 3.0gm 5. Agar 22gm
4.
6. D/W 1000ml
D/W
5 1000ml
5.
 Indol test

S.No Name of Amount

Chemical
1. Peptone 5gm
S.No Name of Amount
Chemical
2. Beef 3gm
extract

3. Nacl 3.0gm 1. Gram iodine 1ml

2. Cristal violet 1ml

4. D/W 1000ml

3. Seffaranin 1ml

4. Ethanol 50ml

S.No. Name of Chemical Amount

1. Tryptone broth 10gm

2. D/W 1000ml
 Methodology

•Collection of Water sample

•Media Preparation
•Isolation of Bacteria from Water sample(Serial
dilution method)
•Sub culturing of isolated Bacteria(Streaking)
•Microscopic Examination(Staining)Cell size,
Cell shape
•Biochemical Characterization
•Collection of water sample
Samples of water for bacteriological testing must be collected in sterile bottles, and care
must be taken to prevent accidental contamination of the water during its collection and
transportation to the water testing laboratory.

Sampling Bottles

Glass or plastic bottles used for water sampling should have a capacity of at least 200 ml. They
should be fitted with ground glass stoppers or screw caps. The stopper or cap and neck of the
bottle should be protected from contamination by a suitable cover either of paper or thin
aluminium foil. We can supply sterilized water bottles for sampling. Information to be supplied
with water samples
This should include:
a) Code number of the sample
b) Reasons for examination, for example whether a routine sample or otherwise.
c) Source from where the water has been collected, for example whether from a well, spring,
lake, reservoir, or piped supply. Mention also the exact place from where the water was taken. If
the sample was collected from a house-tap, mention whether the water was drawn from a cistern
or direct from the main.
d) Whether the water has been filtered, chlorinated, or treated in some
other way.
e) If the water is from a well, give details of its depth, whether covered
or uncovered, and whether recently constructed or altered.
f) If the sample is spring water, describe the stratum from which it issued
and whether the sample was taken directly from spring or from a
collecting chamber.
g) If the water is from a river or stream, mention the depth at which the
sample was collected, whether from the side or the middle of the stream,
h) whether the water level was above or below average, and whether
there had been heavy rainfall or flooding.
i) If the water is from a lake or reservoir, give the exact position and the
depth at which it was collected.
j) Temperature of the source of the sample.
k) Mention any possible sources of pollution in the area and their
approximate distance from the sampling point.
l) Date and time when the sample was taken and dispatched.
Aseptic collection of a water sample
The sterile bottle should be held by the base in one hand while the other hand is used
to remove the stopper and cover together. The stopper and cover should be retained in
the hand while the bottle is filled and then they should be replaced together. To
prevent contamination, the person collecting the water must not touch, or allow any
surface to touch, the screw thread of the bottle neck or the inside of the cap. If the
bottle becomes contaminated, it must not be used.
Collecting a sample from a tap.
a) Remove any external fittings from the tap, such as anti-splash nozzle or rubber tube.
b) Clean carefully the outside nozzle of the tap, especially any grease which has
collected.
c) Turn the tap on full, and allow the water to run to waste for 1 minute. This allows
time for the nozzle of the tap to be flushed and any stagnant water in the service pipe to
be discharged.
d) Sterilize the tap using the flame of a blowlamp or gas torch, or by igniting a piece of
cotton wool soaked in methylated spirit and holding it with a pair of tongs close to the
nozzle until the whole tap is unbearably hot to the touch.
e) Allow the tap to cool by running the water to waste for a few seconds.
f) Fill the sample bottle from a gentle flow of water, and replace the cap of the bottle.
g) Using a waterproof marker or grease pencil, number the bottle with the sample code
number.
Collecting a sample from a river, stream or other surface water
a) Aseptically remove the cap and cover of the sterile sample bottle, and face the
mouth of the bottle upstream.
Collecting a sample from a tube well
a) Continuously operate the hand pump for 5 minutes.
b) Heat the mouth of the pump, preferably by means of a blowlamp or gas torch, and pump several gallons of
water to waste.
c) Aseptically collect a sample of water by allowing the water from the pump to flow directly into the sterile
bottle. Carefully replace the bottle cap and cover.
d) Label the bottle with the sample code number. Collecting a sample from an open well
If the well is one from which water can be raised only by means of a bucket or can, use a weighted bottle to
collect the sample as follows:
a) Tie a sterile sample bottle on to a weighted length of rope or strong string. Use a stone or weight and attach
the bottle just above the weight.
b) Aseptically remove the cap from the bottle, and lower the bottle into the well to a depth of about 1 meter.
c) When no more air bubbles rise to the surface, raise the bottle out of the well and carefully replace the cap.
d) Label the bottle with the sample code number.
Media preaparation
Complex media are rich in nutrients, they contain water soluble extracts of plant or animal tissue
(e.g., enzymatically digested animal proteins such as peptone and tryptone). Usually a sugar, often
glucose is added to serve as the main carbon and energy source. The combination of extracts and
sugar creates a medium which is rich in minerals and organic nutrients, but since the exact
composition is unknown, the medium is called complex.
nitrogen source, various mineral salts and if necessary growth factors (purified amino acids,
vitamins, purines and pyrimidines).
Membrane Filters
Act by screening out particles. Their effectiveness depends on the size of
the membrane pores and the electrostatic attractions present. The most
commonly used filters in microbiology are usually made of cellulose
acetate or cellulose nitrate. Size of filter pores required to screen out:
Yeast0.45-1.2µmBacteria0.2µmVirusesandmycoplasmas0.01-0.1µm
Membrane filtration is usually employed for heat-sensitive substances,
e.g. vitamin solutions; the filters are heat-sterilised b
Microscopic examination
Gram staining method,
The most important procedure in Microbiology was developed by Danish Physician Hans
Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification
and taxonomic division.
This differential staining procedure separates most bacteria into two groups on the basis
of cell wall composition:
1.Gram positive bacteria (thick layer of peptidoglycan-90% of cell wall)- stains purple
2.Gram negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid
content) –stains red/pink
Nearly all clinically important bacteria can be detected using this method the only
exceptions being those organisms;

Classic Gram staining techniques involves following steps:

1.Fixation of clinical materials to the surface of the microscope slide either by heating or by using
methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is
especially useful for examining bloody specimen material).
2.Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple
3.Application of Mordant: The iodine solution (mordant) is added to form a crystal violet iodine
(CV-I) complex; all cells continue to appear blue.
4.Decolorization Step: The decolorization step distinguishes gram-positive from gram-negative
cells.
The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich,
thin walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, gram-
positive bacteria. The gram negative bacteria appear colorless and gram positive bacteria remain
blue.
5.Application of counter stain (safranin): The red dye safranin is stains the decolorized gram-
Gram Staining Procedure/Protocol
Smear preparation
Fix material on slide with methanol or heat. If slide is heat fixed,
allow it to cool to the touch before applying stain.
•Results and discussion
Growth kinetics and expression of recombinant proteins in E. coli cells E. coli
cells grow in three phases; lag phase, log phase and stationary phase. Initial
lag phase of growth is due to sudden exposure of the bacterial cells to new
environment. Cells take time to restore osmotic balance across membranes.
Log phase is due to exponential division of the cells.
After some time, growth rate declines due to scarcity of nutrients in media and
accumulation of toxic metabolites. Growth kinetics of E. coli cells expressing
seven different proteins were studied (hGH, oGH, PKS, PyK, aldolase, enolase
and SOD).

Growth kinetics for hGH, oGH, PKS, PyK, aldolase, enolase and SOD are
shown in Fig. 3.1a to 3.7a respectively. There was a significant decrease in cell
growth after induction with IPTG. Expression of recombinant proteins inside E.
coli was driven by a strong promoter. Induction with IPTG (1 mM final
concentration) transformed whole E. coli into a live protein translation factory
thereby resulted in a sharp decline of cell growth. Reduction in cell growth due
to metabolic burden was associated with gene expression
6. Conclusions
From the isolation, purification and compositional analysis of seven inclusion body
proteins, following conclusion were made:
1. High level expression of recombinant proteins as inclusion bodies was associated
with decline in cell growth. Expression of the proteins plateaued after 4 hrs of IPTG
induction.
2. Inclusion bodies were mostly made up of the recombinant protein of interest. Other
contaminating proteins in inclusion bodies are mostly cell membrane associated
proteins. These proteins get into pellet while centrifugation after cell lysis.
3. Contaminants associated with inclusion bodies can be removed by optimal detergent
(DOC) washings.
4. Contaminants were lesser dense (62 % of sucrose) as compared with inclusion bodies
(66-70 % of sucrose). This enables removal of contaminants by sucrose density gradient
ultracentrifugation.
5. Density of inclusion bodies has no relation with mol. wt. of expressed recombinant
protein. Inclusion bodies were dense at core and loose at periphery.
6. Inclusion bodies were ovoid in shape. They had a size in the range of 0.3-0.7
Inclusion bodies of all proteins were negatively charged at surface.
7. Quality of inclusion bodies prepared by induction in log phase was better as
compared with induction in lag phase.