2016

-2017

BIOLOGY
PROJECT

PRAGYA JAIN
CLASS – XII-A
ROLL NO. 14
ADMISSION NO. 74523
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Aim
To study the effect of antibiotics on the
microbes present in the soil sample.
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Contents
S. No. Topic Page No.
1. Acknowledgement 4
2. Certificate 5
3. Definitions 6
4. Some examples of bacteria found in soil 8
5. About Antibiotics 9
6. Types of Antibiotics 10
7. How Do They Work? 11
8. Uses and Limitations of Antibiotics 13
9. Some Clinically Important Antibiotics 16
10. Experiment Apparatus 17
11. Procedure 18
12. Observations 23
13. Conclusion 25
14. Bibliography 26
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Acknowledgement
I would like to express my wholehearted
gratitude towards my teachers- Dr. Rita
sharma, and Ms. Akshita bej, for their
constant support and guidance and for all
facilities that they provided me for this
project work.
I am highly obliged to my family and my
friends for their endless encouragement and
continuous support during my project work.

Pragya Jain
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Certificate
This is to certify that Pragya Jain of class
XII A, Chowgule Public School, Delhi has
completed this project in the stipulated
time period as given by CBSE Board in the
year 2016-17 under my supervision.

Dr. Rita Sharma

Biology Teacher
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Definitions
I. Flora
It refers to the population of commensal bacteria normally present in
the intensive, body openings, and on the skin.

II. Food borne Pathogens

They are infectious organisms associated with livestock that can
cause diseases in humans through intake of contaminated food. They
include Salmonella, Campylobacter, and Listeria.

III. Gene
 It is a segment of DNA that carries the direction for the
structure of a given protein.
 Antibiotics resistance genes direct the synthesis of antibiotic
resistance proteins.

IV. Incidence
It is the frequency of new occurrences of a disease within a defined
time interval.
Number of new cases of a disease
Incidence Rate =
Total population over a period time (Usually 1 year)

V. Micro-organisms
They are minute, microscopic or sub-microscopic living organisms
including bacteria, fungi, and protozoa.
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Viruses are often included in this category, although, they are incapable
of growth and reproduction outside the host cells.

VI. Multiple Drug Resistance

It is a condition where some bacteria are resistant to more than one
type of antibiotics.

VII. Narrow Spectrum Antibiotic

It is an antibiotic which is effective against a limited number of
bacterial species.

VIII. Broad Spectrum Antibiotic

It is an antibiotic which is effective against a large number of
bacterial species.

IX. Antibacterial
It refers to a drug that kills or inhibits the growth of bacteria.

X. Antibiotics
 They are a class of substances that kill or inhibit the growth of
bacteria.
 Example- Penicillin, Tetracycline, Fluoroquinolones.
 Originally, antibiotics were derived from natural sources like
Penicillin was derived from moulds but many currently used
antibiotics are semi-synthetic and modified with additions of
synthetic chemical components.
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 Some scientists reserve the term “antibiotics” for naturally

produced substances and use antimicrobial to encompass both
synthetic and natural forms.

Examples of Some Bacteria Found in

Soil
1. Decomposers
They serve as decomposers, digesting organic materials and breaking
them down into soil and compost. They typically take simple
compounds like roots and plant litter and convert it into forms that
plants can use. Some decomposers can break down pesticides or
herbicides, and are very good at retaining nutrients within their cells,
preventing essential nutrient loss in the soil.
E.g. Bacillus subtilis, Pseudomonas fluorescens, etc.

2. Nitrogen Fixers
They extract nitrogen gas from the air and converting it into mineral
form that the plant can take up. These bacteria often live in root
nodules of plants like alfalfa, clover and legumes.
E.g. Rhizobium, etc.

3. Actinomycetes
These bacteria are decomposers that specialize in decomposing
tough materials like cellulose and chitin.
They are responsible for giving the freshly turned earth its
recognizable “earthy” scent.

4. Aerobic Bacteria
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They are most common in well drained sols where there is no

standing water. These bacteria make up the majority of the species
found in most soils, and also play a vital role in nitrification. These
bacteria turn ammonium into nitrates which are then used by the
plants to turn into proteins.
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About Antibiotics
What are they?
The standard definition states that an antibiotic is a substance
produced by micro-organisms that kill or inhibit other micro-
organisms.
That is, antibiotics are strong medicines that can cure many bacterial
illnesses and infections.
Chemically antibiotics are complex organic compounds usually
aromatic in nature and nowadays, minerals, metals and other
compounds (e.g.- HCl, Sulphur, etc.) are added to the pure forms to
increase their utility.

History of Antibiotics
Sir Alexander Fleming, who observed the inhibition of Staphylococci
on a plate contaminated by a Penicillin mould, discovered the first
antibiotic, Penicillin in 1929.
By the mid-1940s, antibiotics were available for treatment against
many bacterial infections including strep throat, pneumonia, skin
infections, wound infections, scarlet fever, toxic shock syndrome,
and other bacterial infections.
By the mid-1950s, the discovery and introduction of streptomycin,
tetracycline, and other antibiotics led to effective treatment of a vast
array of formerly life-threatening infections, illnesses and diseases.
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Types of Antibiotics
Even though many antibiotics are known to us today, each antibiotic
doesn’t have an effect against all bacteria rather than antibiotics are
selective.
Most bacteria can be classified as Gram-Positive Bacteria (G+) and
Gram-Negative Bacteria (G-) depending on their response to the
gram stain. HANS GRAM, bacteriologist of the 1800s, developed this
classification method. According to Gram’s system, many bacterial
infections are either G+ or G- depending on the type of bacteria that
causes them.

LIMITED SPECTRUM BROAD SPECTRUM ANTIBIOTICS

ANTIBIOTICS
i. When an antibiotic is i. These antibiotics are
effective against some effective against a large
micro-organisms, i.e., a range of micro-organisms
specific spectrum or range (G+ and G-).
and generally not effective
against other bacteria
which are not present in
that spectrum, then such
antibiotics are called
limited spectrum
antibiotics.

ii. Examples - Clindamycin, ii. Examples- Tetracycline,

Eythromycin, Gentomycin, and Chloramphenicol
and Golistin
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How do Antibiotics Work?

Antibiotics fight pathogenic micro-organisms and cancer cells by
interfering with their normal cell process.
This occurs in three main ways:
1. Prevention of Cell Wall Formation
2. Disruption of Cell Membrane
3. Disruption of Chemical Processes

Prevention of Cell Wall Formation

 Bacterial cells are enclosed in a membrane that is surrounded
by a rigid wall that prevents the cells from splitting open.
 Penicillin and some other antibiotics destroy pathogenic
microbes by hindering the formation of this wall.
 Peptidoglycan is one of the major wall targets because it is
found only in bacteria.
 Human cells don’t have nor need rigid cell walls, and so are not
damaged by these antibiotics.

Disruption of Cell Membrane

 Some antibiotics including amphotericin B and nystatin,
disrupt the cell membrane of certain microbes.
 This membrane controls the movement of materials in and out
of the cell. If the cell membrane is disrupted, vital nutrients
may escape from the cell or poisonous substances may enter
the cell, and kill the cell.
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 However, the membranes of human cells are not affected

because these antibiotics disrupt cell membranes that contain
elements found only in the cells of microbes.

Disruption of Chemical processes

 All cells produce proteins. Some of the antibiotics target
bacterial protein synthesis, because bacterial ribosomes (70S)
are different from the ribosomes (80S) of humans, and other
eukaryotes.
 Similarly, griseofulvin (antifungal agent) binds specifically to the
tubulin proteins that make up the microtubules of fungal cells,
these tubulins are somewhat different from the tubulins of
humans.
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Uses and Limitations of Antibiotics

Uses
1. Therapeutic Drugs
They are used to fight bacterial infections and thus, are prescribed
widely by doctors.

2. Treatment of Cancer
A small number of antibiotics were developed to attack human
cells; they are selectively toxic as they mostly damage cells that
undergo unlimited mitosis. These types of antibiotics interact with
DNA.

3. Treatment and prevention of diseases in

animals
Antibiotics are added to livestock feed to stimulate the animal’s
growth and prevent the occurrence of diseases. If the animal has
already been infected with a bacterial disease, antibiotics are then
used by veterinarians to cure the animal.

4. Prevention of damage to fruits and grains

Certain antibiotics are sprayed on the standing crops to prevent
them from being spoiling.

5. Food Preservation
Small quantities of antibiotics are added to packaged foodstuff to
prevent it from spoiling by bacterial action.
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Limitations
Many antibiotics are among the safest drugs when properly used but
they can also cause unpleasant side effects at times. Thus, one must
realize that these are only effective against bacterial diseases and
also some fungi and protozoa. But they are not effective against
viruses and hence, can’t be used to treat chickenpox, measles, cold,
and other viral infections.
The effectiveness of antibiotics is sometimes limited because of
pathogenic microorganisms and become resistant to them.
There are 4 main problems that might arise while using antibiotics:

1. Allergic Reactions
In most cases allergic reactions are mild and produce only a rash
or a mild fever. But a severe reaction to the drug can even result
in death.
Although, all antibiotics can produce allergic reactions, such
reactions occur most frequently with penicillin.

2. Destruction of helping organisms

Certain areas of the body commonly harbour both harmless and
pathogenic microorganisms. These two types microbes compete
for food and so the harmless microbes help in restricting the
growth of the pathogenic microbe.
Many antibiotics especially broad-spectrum drugs do not always
distinguish between harmless and dangerous microbes.
If a drug destroys too many harmless microbes, the pathogenic
ones will have a greater chance to multiply. This situation often
leads to the development of a new infection called supra
infection. Doctors usually prescribe a secondary drug to combat
supra infection.
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3. Damage to organs and tissues

It is rare in people using antibiotics that act only against the calls
of pathogenic microbes. However, extensive use of some of these
antibiotics may damage tissues and organs.

4. Resistance in bacteria
Overuse or improper use of antibiotic causes bacteria to become
resistant to the drug and hence, new drugs need in such cases.
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Some Clinically Important Antibiotics

S. Antibiotic Producer Type Site or
No. Organism Mode of
Action
1. Penicillin Penicillin Gram Positive Wall
chrysogenum synthesis
2. Cephalosporin Cephallosporium Broad Wall
acremonium Spectrum synthesis
3. Griseofulvin Penicillin Dermatophytic Microtubules
griseofulvum
4. Bacitracin Bacills subtilis Gram Positive Wall
synthesis
5. Polymyxin B Bacillus Gram Negative Cell
polymyxa membrane
6. AmphotericinB Streptomyces Fungi Cell
nodosus membrane
7. Erythromycin Streptomyces Gram Positive Protein
Erythreus synthesis
8. Neomycin Streptomyces Broad Protein
fradiae Spectrum synthesis
9. Streptomycin Streptomyces Gram Negative Protein
griseus synthesis
10. Vancomycin Streptomyces Gram Positive Protein
orientalis synthesis
11. Gentamycin Micromonospora Broad Protein
purpurea Spectrum synthesis
12. Tetracycline Streptomyces Broad Protein
rimosus Spectrum synthesis
13. Rifamycin Streptomyces Tuberculosis Protein
mediterranei treating synthesis
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Experiment

Aim
To show the effect of antibiotics on the microbes present in the soil
sample.

Apparatus
Laminar flow, spirit, pipettes, soil sample solution, eppendorfs,
distilled water, pestle and mortar, cycle-mixer, spreader, burner,
cork borer, petri plates, marker, camera, scale, screw gauge, and
autoclave.
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Procedure
This experiment involves six steps which are:
A. Pouring the plates
B. Preparation of the antibiotic solutions
C. Spreading of the bacteria
D. Formation of wells
E. Addition of antibiotic solutions
F. Recording observations

Before the experiment: Preparation of Luria and Bertani (LB) Medium

Composition:
1. Yeast Extract(0.5g/100ml)
2. Tryptone/Peptone(1.0mg/100ml)
3. NaCl(0.5g/100ml)
4. Agar(1.5g/100ml)

Preparation:
1. The materials needed for the medium are weighed
accurately on a butter paper using a digital weighing
machine.
2. The weighed substances are added to a clean conical flask.
3. Distilled water is added to this mixture to form a 100ml
solution.
4. The conical flask is put on a magnetic stirrer to mix the
contents properly.
5. A cotton plug is put onto the opening of the conical flask,
and the solution is autoclaved to melt agar which transforms
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into a jelly like substance, and also to kill any unwanted

microbes that might contaminate the medium.
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Prosecution of the steps:

A.Pouring of Plates
1. After autoclaving, procured around 25ml of LB medium in a
petri dish.
2. The medium is now allowed to solidify in the plate.

B. Preparation of Antibiotic Solution of

Different Concentration
1. One antibiotic is taken at a time, say first Terramycin- one
250mg tablet is grinded into a powder by a pestle and
mortar.
2. This powder is carefully added to an eppendorf. Now, 1ml of
distilled water is added to the eppendorf by a pipette.
3. The solution formed is naturally shaken vigorously to make
the solute dissolve in the solvent; if it doesn’t dissolve, use a
cyclo-mixer.
4. Once the solute dissolves, we get a solution of conc. 250mg.
This is solution A.
5. Now, 500ul of the solution A is pipette out, and added to an
eppendorf. To this 500ul of distilled water is added.
6. The solute is made to completely dissolve in the solvent, and
the solution formed is of concentration 125mg. this is
solution B.
7. Now, 500ul of the solution B is pipette out and added to an
eppendorf to this 500ul of distilled water is added.
8. The solute is made to completely dissolve in the solvent, and
the solution formed is of the concentration 62.5mg. This is
solution C.
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9. The same method is used to prepare solutions of the rest of

the antibiotic i.e., Amoxycillin.
C. Spreading of soil solution
The cover of the Petri dish is lifted, and 50ul of the soil sample
solution is pipette in the solid medium and spread over the LB agar
plate.

D. Formation of Wells
(Apparatus: Cork borer, spirit, Laminar flow, Petri plate prepared in
the previous step)
1. The cork borer is made sterilized by dipping in spirit and
heating over the burner for a few minutes.
2. The cover of the Petri plate is marked at different positions
with a marker to ascertain the positions of the wells along with
the concentration of antibiotic solution to be added later.
3. The cover of the Petri plate is removed and using the cork borer
and the needle, wells are made into the solid medium at the
inclined spots.
4. The spreader is sterilized using spirit and flame.
5. This spreader is used to spread Soil sample evenly over the
surface of the medium.
6. The cover of the Petri plate is placed on top of it again.
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E. Addition of Antibiotic Solutions

(Apparatus: 10ul pipette, Spirit, Laminar Flow, and Marker)
1. The three plates prepared are marked as control, Amoxycillin,
and Terramycin.
2. A 10ul pipette is used to pipette put 10ul of one concentration
of the antibiotic into its respective spot in the particular
antibiotic Petri plate.
3. The same is done with different concentrations of the same
antibiotic till its plate has 3 wells filled.
4. The plates are now ready, and he process is repeated for other
antibiotic.
5. To prepare the control instead of the antibiotic solutions, 10ul
of distilled water is added to the wells.
6. Once the Petri plates are ready, they are put into the incubator
set at 37˚C for 24 hours.

F. Recording of Observations
(Apparatus: Camera, scale, screw gauge, and autoclave)
1. After the incubation period is over, the Petri plate are taken out
of the incubator, and the diameter of the inhibiton zones is
noted with the help of a scale. Diameter is taken at 3 different
points to ensure accurate results.
2. The diameter of the cork is noted with the help of a screw
gauge.
3. Photograph of the petri plates are taken.
4. After the experiment is over, the Petri plates are autoclaved.
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Agar

Photo of performing experiment

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Observations
A. Once the antibiotic solutions have been added to the wells, and
incubation has been done, a zone of inhibition is seen very
close to the wells, this inhibition zone is circular in shape, and
has no whitish cream colour indicating the absence of the
bacterial growth.
B. From this, it can be inferred that the antibiotic solutions have
diffused out form the wells into the medium, and have stopped
the bacteria from growing.
C. The diameter of the inhibition zone is more in the case of
higher concentration of the same antibiotics from which it can
be concluded that higher concentrations of antibiotics are
more effective in killing bacteria.
D. In the control plate, no inhibition zone is found, and the whole
plate is covered with the whitish cream layer of bacterial
growth. This can be explained as in the control plate only
distilled water is added to the wells and since no substance that
restricts the growth of bacteria is present in this control plate,
bacteria grow unchecked.

Lawn of Bacteria in Control

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Antibiotic activity of Amoxycillin on soil bacteria

Antibiotic activity of Terramycin on soil bacteria

Observation Table
Name Concentration Zone of Inhibition
250 mg 1.8 cm
Amoxycillin 125 mg 1.5 cm
62.5 mg 1.5 cm
250 mg 1.3 cm
Terramycin 125 mg 1.25 cm
62.5 mg 1.15 cm
Control water - -
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Sample loaded in each well=50µL

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Conclusion
The results are seen as the bacteria do not grow in the regions
around the wells; the area far away from the wells is observed to
have the whitish cream of bacterial growth. In contrast to this, in the
control plate, a lawn of bacteria is observed- TNTC.
Amoxycillin is more effective than Terramycin as the zone of
inhibition in the case of Amoxycillin is wider as compared to
Terramycin.
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Bibliography
 NCERT Biology Class XII
 www.google.com
 www.wikipedia.com
 www.wikihow.com