BIOLOGY INVESTIGATORY

PROJECT
STUDY OF EFFECT OF ANTIBIOTICS
ON MICROBS

SESSION:-2019-2020
CLASS:-12th
SECTION:-PCB
ROLL NO. :-4
SUBMITTED BY:- YOGI ARYA
SUBMITTED TO:- REKHA NAIK
 CERTIFICATE
This is to certify that YOGI ARYA has satisfactorily
completed course of BIOLOGY INVESIGATORY PROJECT
prescribed by the CBSE under the CBSE course in the
laboratory of the school in the year of the school in the
year 2019-2020.

Signature of the Signature of the

Candidate Teacher In-Charge

Signature of the Signature of the

Principal External Examiner

 ACKNOWLEDGEMENT

The enduring pages of the work are the cumulative sequence of extensive
guidance and arduous work. I wish to acknowledge and express my personal
gratitude to all those without whom this project could not have been reality.

First and foremost, I would like to express my deep gratitude to our principal,
JUHEE MISHRA for providing us with state of the art laboratories and
infrastructure and also providing her valuable suggestions and feedback, which
were instrumental in shaping up the project work. Without her help, this project
would remain unaccomplished.

I would like to sincerely thank our chemistry faculty Mrs. REKHA NAIK for
spending their precious time with us enhancing our knowledge regarding project.
Their help is unforgettable as this project is built on the concepts that they have
taught us. They always motivated us and ensured that we were on the right track.

My heartfelt thanks to my parents and other family members who have

constantly motivated and supported me during the making of this project work.

This project would be incomplete without thanking my peers who always lent a
helping hand and showed true spirit of unity and friendship.

I would also like to extend my heartfelt gratitude to the authors and publishers of
the books and managements of the websites, we referred to (as in Bibliography),
for having provided us with us valuable information.
 STUDY OF EFFECT OF
ANTIBIOTICS ON MICROBS
INDEX:-

 INTRODUCTION
 MATERIAL REQUIRED
 PROCEDURE
 OBSERVATION
 CONCLUSION
 BIBLOGRAPHY
#INTRODUCTION:-

An antibiotic is an agent that either kills or inhibits the

growth of a microorganism. The term antibiotic was
first used in 1942 by Selman Waksman and his
collaborators in journal articles to describe any
substance produced by a microorganism that is
antagonistic to the growth of other microorganisms in
high dilution. This definition excluded substances that
kill bacteria but that are not produced by
microorganisms (such as gastric juices and hydrogen
peroxide). It also excluded synthetic antibacterial
compounds such as the sulfonamides. Many
antibacterial compounds are relatively small molecules
with a molecular weight of less than 2000 atomic mass
units.

With advances in medicinal chemistry, most modern

antibacterial are semi synthetic modifications of various
natural compounds. These include, for example, the
beta-lactam antibiotics, which include the penicillin
(produced by fungi in the genus Penicillium), the
cephalosporin, and the carbapenems. Compounds that
are still isolated from living organisms are the amino
glycosides, whereas other antibacterial—for example,
the sulfonamides, the quinolones, and the
oxazolidinones—are produced solely by chemical
synthesis.

In accordance with this, many antibacterial compounds

are classified on the basis of chemical/biosynthetic
origin into natural, semi synthetic, and synthetic.
Another classification system is based on biological
activity; in this classification, antibacterial are divided
into two broad groups according to their biological
effect on microorganisms: Bactericidal agents kill
bacteria, and bacteriostatic agents slow down or stall
bacterial growth.
What is Antibiotic Resistance?
Antibiotic resistance is a form of drug resistance
whereby some (or, less commonly, all) sub-populations
of a microorganism, usually a bacterial species, are able
to survive after exposure to one or more antibiotics;
pathogens resistant to multiple antibiotics are
considered multidrug resistant(MDR) or, more
colloquially, superbugs.

Antibiotic resistance is a serious and growing

phenomenon in contemporary medicine and has
emerged as one of the pre-eminent public health
concerns of the 21st century, in particular as it pertains
to pathogenic organisms (the term is especially relevant
to organisms that cause disease in humans). A World
Health Organization report released April 30, 2014
states, "this serious threat is no longer a prediction for
the future, it is happening right now in every region of
the world and has the potential to affect anyone, of any
age, in any country. Antibiotic resistance–when bacteria
change so antibiotics no longer work in people who
need them to treat infections–is now a major threat to
public health."

In the simplest cases, drug-resistant organisms may

have acquired resistance to first-line antibiotics,
thereby necessitating the use of second-line agents.
Typically, a first-line agent is selected on the basis of
several factors including safety, availability, and cost; a
second-line agent is usually broader in spectrum, has a
less favorable risk-benefit profile, and is more
expensive or, in dire circumstances, may be locally
unavailable. In the case of some MDR pathogens,
resistance to second- and even third-line antibiotics is,
thus, sequentially acquired, a case quintessentially
illustrated by Staphylococcus aureus in some
nosocomial settings. Some pathogens, such as
Pseudomonas aeruginosa, also possess a high level of
intrinsic resistance.

It may take the form of a spontaneous or induced

genetic mutation, or the acquisition of resistance genes
from other bacterial species by horizontal gene transfer
via conjugation, transduction, or transformation. Many
antibiotic resistance genes reside on transmissible
plasmids, facilitating their transfer. Exposure to an
antibiotic naturally selects for the survival of the
organisms with the genes for resistance. In this way, a
gene for antibiotic resistance may readily spread
through an ecosystem of bacteria. Antibiotic-resistance
plasmids frequently contain genes conferring resistance
to several different antibiotics. This is not the case for
Mycobacterium tuberculosis, the bacteria that causes
Tuberculosis, since evidence is lacking for whether
these bacteria have plasmids. Also M. tuberculosis lack
the opportunity to interact with other bacteria in order
to share plasmids.

Genes for resistance to antibiotics, like the antibiotics

themselves, are ancient. However, the increasing
prevalence of antibiotic-resistant bacterial infections
seen in clinical practice stems from antibiotic use both
within human medicine and veterinary medicine. Any
use of antibiotics can increase selective pressure in a
population of bacteria to allow the resistant bacteria to
thrive and the susceptible bacteria to die off. As
resistance towards antibiotics becomes more common,
a greater need for alternative treatments arises.
However, despite a push for new antibiotic therapies,
there has been a continued decline in the number of
newly approved drugs. Antibiotic resistance therefore
poses a significant problem.

The growing prevalence and incidence of infections due

to MDR pathogens is epitomized by the increasing
number of familiar acronyms used to describe the
causative agent and sometimes the infection; of these,
MRSA is probably the most well-known, but others
including VISA (vancomycin-intermediate S. aureus),
VRSA (vancomycin-resistant S. aureus), ESBL (Extended
spectrum beta-lactamase), VRE (Vancomycin-resistant
Enterococcus) and MRAB (Multidrug-resistant A.
baumannii) are prominent examples. Nosocomial
infections overwhelmingly dominate cases where MDR
pathogens are implicated, but multidrug-resistant
infections are also becoming increasingly common in
the community.

Although there were low levels of preexisting antibiotic-

resistant bacteria before the widespread use of
antibiotics, evolutionary pressure from their use has
played a role in the development of multidrug-resistant
varieties and the spread of resistance between bacterial
species. In medicine, the major problem of the
emergence of resistant bacteria is due to misuse and
overuse of antibiotics. In some countries, antibiotics are
sold over the counter without a prescription, which also
leads to the creation of resistant strains. Other practices
contributing to resistance include antibiotic use in
livestock feed to promote faster growth.] Household
use of antibacterial in soaps and other products,
although not clearly contributing to resistance, is also
discouraged (as not being effective at infection control).
Unsound practices in the pharmaceutical manufacturing
industry can also contribute towards the likelihood of
creating antibiotic-resistant strains. The procedures and
clinical practice during the period of drug treatment are
frequently flawed — usually no steps are taken to
isolate the patient to prevent re-infection or infection
by a new pathogen, negating the goal of complete
destruction by the end of the course(see Healthcare-
associated infections and Infection control).

Certain antibiotic classes are highly associated with

colonization with "superbugs" compared to other
antibiotic classes. A superbug, also called multiresistant,
is a bacterium that carries several resistance genes. The
risk for colonization increases if there is a lack of
susceptibility (resistance) of the superbugs to the
antibiotic used and high tissue penetration, as well as
broad-spectrum activity against "good bacteria". In the
case of MRSA, increased rates of MRSA infections are
seen with glycopeptides, cephalosporins, and especially
quinolones.In the case of colonization with Clostridium
difficile, the high-risk antibiotics include cephalosporins
and in particular quinolones and clindamycin.
Of antibiotics used in the United States in 1997, half
were used in humans and half in animals; in 2013, 80%
were used in animals.

Need of this Experiment:-

Antibiotic resistance is becoming more and more
common. Antibiotics and antimicrobial agents are drugs
or chemicals that are used to kill or hinder the growth
of bacteria, viruses, and other microbes. Due to the
prevalent use of antibiotics, resistant strains of bacteria
are becoming much more difficult to treat. These "super
bugs" represent a threat to public health since they are
resistant to most commonly used antibiotics. Current
antibiotics work by disrupting so-called cell viability
processes. Disruption of cell membrane assembly or
DNA translation are common modes of operation for
current generation antibiotics. Bacteria are adapting to
these antibiotics making them ineffective means for
treating these types of infection. For example,
Staphylococcus aureus have developed a single DNA
mutation that alters the organism's cell wall. This gives
them the ability to withstand antibiotic cell disruption
processes. Antibiotic resistant Streptococcus
pneumoniae produce a protein called MurM, which
counteracts the effects of antibiotics by helping to
rebuild the bacterial cell wall.

Fighting Antibiotic Resistance:-

Researchers are attempting to develop new types of
antibiotics that will be effective against resistant
strains. These new antibiotics would target the
bacteria's ability to become virulent and infect the host
cell. Researchers at Brandeis University have discovered
that bacteria have protein "switches" that when
activated, turn "ordinary" bacteria into pathogenic
organisms. These switches are unique in bacteria and
are not present in humans. Since the switch is a short-
lived protein, elucidating its structure and function was
particularly difficult. Using nuclear magnetic resonance
(NMR) spectroscopy, the researchers were able to
regenerate the protein for one and one half days. By
extending the time frame that the protein was in its
"active state," the researchers were able to map out its
structure. The discovery of these "switches" has
provided a new target for the development of
antibiotics which focus on disrupting the activation of
the protein switches.

Monash University researchers have demonstrated that

bacteria contain a protein complex called Translocation
and Assembly Module (TAM). TAM is responsible for
exporting disease causing molecules from the inside of
the bacterial cell to the outer cell membrane surface.
TAM has been discovered in several antibiotic resistant
bacteria. The development of new drugs to target the
protein would inhibit infection without killing the
bacteria. The researchers contend that keeping the
bacteria alive, but harmless, would prevent the
development of antibiotic resistance to the new drugs.

Researchers from the NYU School of Medicine are

seeking to combat antibiotic resistance by making
resistant bacteria more vulnerable to current
antibiotics. They discovered that bacteria produce
hydrogen sulfide as a means to counter the effects of
antibiotics. Antibiotics cause bacteria to undergo
oxidative stress, which has toxic effects on the
microbes. The study revealed that bacteria produce
hydrogen sulfide as a way to protect themselves against
oxidative stress and antibiotics. The development of
new drugs to target bacterial gas defenses could lead to
the reversal of antibiotic resistance in pathogens such
asStaphylococcus and E.coli.

These studies indicate how highly adaptable bacteria

are in relation to the application of antimicrobial
treatments. Antibiotic-resistant bacteria have become a
problem not only in hospitals, but in the food industry
as well. Drug-resistant microbes in medical facilities
lead to patient infections that are more costly and
difficult to treat. Resistant bacteria in turkey and other
meat products have caused serious public health safety
issues. Some bacteria may develop resistance to a single
antibiotic agent or even multiple antibiotic agents.
Some have even become so resistant that they are
immune to all current antibiotics. Understanding how
bacteria gain this resistance is key to the development
of improved methods for treating antibiotic resistance.

#Material Required:-
1. Sterilized Petri dishes

2. Sterilized culture tubes with media

3. Transfer loops

4. Forceps

5. Flask
6. Beaker

7. Burner

8. Penicillin
9. Aureomycin

10. Hay

11. Alcohol
12. Agar

13. Starch

14. Distilled water

#Experimental Procedure:-
1. To 200ml of distilled water in a flask, I added 8 grams
of agar powder and 2 grams of starch. Then putting a
few pieces of dry hay into the medium I covered the
flask with an Inverted beaker. Boiling the medium for 5
minutes and then cooling the medium to room
temperature. After that placing the flask in a warm
place. Within 2-3 days, formation of scum of cloudy
suspension appeared on the medium indicating the
growth of Bacillus subtilis.

2. Taking culture tubes with agar medium and heating

the test tubes in warm water to melt agar. Cooling each
test tube so that I can hold it in my hand and the agar
remains liquid. After that removing the cotton plug and
I passed the mouth of the test tube through the burner
flame twice. Flaming the transfer loop after dipping it in
alcohol and I let it cooled. After that picking up a loop
full of bacterial culture from flask and then I transferred
it to the warm agar in the culture tube. Flaming the loop
and the mouth of the culture tube and then I replaced
the cotton plug. Rolling the culture tube of warm agar
between palms to I mixed the bacteria well with agar.

*Transferring the bacteria should be done as quickly as

possible.

3. After that I took sterilized petridishes. Removing the

cotton plug and flamed the mouth of the culture tube.
Then I lifted the cover of the Petridish at an angle 45
Degree and then quickly pouring the medium of the
culture tube into the bottom half the dish. Removing
the culture tube and replacing the cover tube into the
bottom half of the dish. Removing the culture tube, and
replace the cover of the Petridish. Moving the covered
Petridish along the table top to distribute the medium
evenly. Then I allowed the agar to cool. After that I
prepared two petridishes and marked them A & B.

4. I prepared Penicillin and Aureomycin solution by

dissolving the powdered drugs in distilled water. Then I
cut down a few discs of filter paper of 1 cm diameter.
Then I soaked a disc in each of the penicillin and
Aureomycin solutions. Dipping the forceps in alcohol
and the I passed the forceps’ tip quickly over the burner
flame. Using the sterilized forceps I put Penicillin and
Aureomycin soaked discs at two distant sites of
Petridish A. Considering Petridish B as control. Then I
kept both the Petridishes undistributed in warm place
to allow the bacteria to grow. Then I observed the
Petridishes for several days.

#Observation:-

The area around the antibiotic discs in the Petridishes

will be clear. In other areas, colonies of bacteria will be
observed. Then I examined the clear area in each
Petridishes for few more days. A few very colonies may
appear in the clear areas. These are the colonies of
resistant strains of the bacteria.
#CONCLUSIONS:-

Antibiotic drugs killed most of the bacterial strain,

hence the areas appeared clear. However, a few strains
which were resistant in the bacterial population
survived and produced colonies later. This proves the
resistant strain to antibiotics were present in the
bacterial population.
#BIBLOGRAPHY:-

1. Comprehensive Laboratory Manual In Biology-XII 2.

Biology Text For Class XII – NCERT

2. http://www.wikipedia.org/

3.http://www.sciencedaily.com/articles/a/antibiotic_re
sistance.htm

4.http://www.betterhealth.vic.gov.au/bhcv2/bhcarticle
s.nsf/pages/Antibiotic_resistant_bacteria

5. http://www.rxlist.com/antibiotic_resistance-
page3/drugs-condition.htm