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Antibiotics on Organisms
Materials Technology (Velammal Engineering College)
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BY:
KRISHA PRINCY N.S.
XII A
ACADEMIC YEAR 2023-2024
BIOLOGY
INVESTIGATORY
PROJECT
VELAMMAL VIDYALAYA
MUGAPPAIR WEST - 37
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VELAMMAL VIDYALAYA
MOGAPPAIR WEST, CHENNAI – 37.
BIOLOGY PROJECT
ACADEMIC YEAR: 2023 – 2024
To study the effects of
Antibiotics on microorganisms
PREPARED BY
NAME: KRISHA PRINCY N.S.
CLASS: XII A
SUBJECT CODE:
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ACKNOWLEDGEMENT
In the accomplishment of this project successfully, many people have bestowed
their blessings and heart-pledged support up on me, I take this opportunity to
express my gratitude to all, who have been instrumental in the successful
completion of this project.
Primarily, I express my deep sense of gratitude to the luminary, The Senior
Principal, The Vice Principal and The Head Master for providing the
best of facilities and environment to bring out innovation and spirit of inquiry
through this venture.
I am deeply indebted to my teacher ____________________, without whose
constructive feedback, this project would not have been successful.
The
valuable advice and suggestions for correction, modifications and improvement
did enhance the quality of the task.
I would also like to thank my parents, friends and all the members who
contributed to this project was vital for the success of the project.
I am grateful for their constant support and help.
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VELAMMAL VIDYALAYA
MOGAPPAIR WEST, CHENNAI – 37.
NAME:
BATCH NO:
CLASS:
REGD.NO.:
CERTIFICATE
Certified that this is a bonafide Record of Practical work done by
Mr. / Miss. _____________________________________________ in
the __________________________ Laboratory during the year 2023.
Teacher-In-Charge
Submitted
for
the
Practical
Examination
in
________________________
______________________________ held on ____________________
VICE PRINCIPAL
INTERNAL EXAMINERS
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EXTERNAL EXAMINERS
at
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INDEX
S. NO.
1.
TOPIC
ACKNOWLEDGEMENT
2.
BONAFIDE CERTIFICATE
3.
WHAT ARE ANTIBIOTICS?
4.
HOW ANTIBIOTICS WORKS?
5.
ANTIBIOTICS
6.
AIM & QUESTION
7.
MATERIALS
8.
INTRODUCTION
9.
PROCEDURE
PG. NO.
3
5
6
7
8-22
11
12
10.
RESULTS
11.
BIBLIOGRAPHY
13-22
23-29
30-41
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WHAT ARE ANTIBIOTICS?
Antibiotics are medications that destroy or slow down
the growth of bacteria. It includes a range of powerful
drugs used to treat diseases caused by bacteria. Doctors
prescribe them to treat bacterial infections. They do this
by killing bacteria or preventing them from multiplying.
Only substances that target bacteria are called
antibiotics. Antibiotics cannot treat viral infections, such
as colds, flu, and most coughs.
Antibiotics are produced in nature by soil bacteria and
fungi.
The first antibiotic was penicillin. Penicillin-based
antibiotics, such as ampicillin, amoxicillin, and penicillin
G, are still available to treat a variety of infections and
have been in use for many years.
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HOW ANTIBIOTICS WORKS?
Before bacteria can multiply and cause symptoms, the
immune system can typically kill them. White blood cells
(WBCs) attack harmful bacteria — even if symptoms
occur, the immune system can usually cope and fend off
the infection.
However, sometimes the number of harmful bacteria is
excessive, and the immune system cannot clear them
all. Antibiotics are useful in this scenario.
There are different types of antibiotics, which work in
their unique way. Antibiotics are used to treat bacterial
infections. Antibiotics take advantage of the difference
between the structure of the bacterial cell and the host's
cell.
They either prevent the bacterial cells from multiplying
so that the bacterial population remains the same,
allowing the host's defence mechanism to fight the
infection, or kill the bacteria, for example stopping the
mechanism responsible for building their cell walls.
It may take a few hours or days after taking the first dose
before people feel better or their symptoms improve.
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Antibiotic classification:
An antibiotic can also be classified according to the
range of pathogens against which it is effective.
• Narrow-spectrum antibiotic: Penicillin G will destroy
only a few species of bacteria.
• Broad-spectrum antibiotic: Tetracycline is effective
against a wide range of organisms.
Fast facts on antibiotics
•
Alexander Fleming discovered penicillin, the first
natural antibiotic, in 1928.
•
Antibiotics cannot fight viral infections.
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Fleming predicted the rise of antibiotic resistance.
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Antibiotics either kill or slow the growth of bacteria.
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Side effects can include diarrhoea,
stomach, and nausea.
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7 types of antibiotics
Although there are well over 100 antibiotics, the majority
come from only a few types of drugs. These are the main
classes of antibiotics.
ANTIBIOTIC
EXAMPLE
Penicillin
amoxicillin
Cephalosporins
cephalexin
Macrolides
erythromycin
Fluoroquinolones
ofloxacin
Sulphonamides
Bactrim
Tetracyclines
tetracycline
Aminoglycosides
gentamicin
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The Impact of Antibiotics on Microorganisms:
Antibiotics have been widely used to treat bacterial
infections for over a century. However, they have also
had a profound impact on the microbial world.
Antibiotics can kill beneficial microorganisms along with
harmful ones, leading to an imbalance in the microbial
ecosystem. This can result in the growth of antibioticresistant bacteria, which can be difficult to treat and
pose a significant threat to public health.
Antibiotic Resistance:
Antibiotic resistance occurs when bacteria evolve to
resist the effects of antibiotics. This can happen naturally
or as a result of overuse or misuse of antibiotics.
The spread of antibiotic-resistant bacteria is a growing
concern, as it can lead to the failure of antibiotic
treatments and the development of new, more
dangerous infections.
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EXPERIMENT
AIM:
To study the effects of antibiotics on bacteria count
Question/ Purpose:
What do you want to find out? Write a statement that
describes what you want to do. Use your observations
and questions to write the statement.
The purpose of this investigation is to see the effect of
antibiotics on bacteria count.
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Materials and Equipment:
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10 test tubes of sterilized water
10 PCA plates
blender or mixer (optional)
Bunsen burner
Graduated cylinder
Ethanol (Used for sterilizing. Just flame is enough in
most cases)
Glass hockey stick
Pipettes
Refrigerator
Incubator (A warm cabinet for growing bacteria)
Microwave
Scale
Large beaker
Hot plate
Sample anti-biotic
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Introduction (Initial Observation):
An antibiotic is a substance, such as penicillin or
streptomycin, produced by or derived from certain fungi,
bacteria, and other organisms, that can destroy or inhibit
the growth of other microorganisms (specifically
bacteria).
The first antibiotic, penicillin, was discovered about
seven decades ago. Sir Alexander Fleming discovers the
drug penicillin, which counteracts harmful bacteria.
Fleming makes the discovery by accidentally
contaminating a bacteria culture with a "Penicillium
notatum" mould. He notices that the non-toxic mould
halts the bacteria's growth, and later conducts
experiments to show penicillin's effectiveness in
combating a wide spectrum of harmful bacteria.
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In this project, we will investigate the effect of antibiotics
on bacterial count.
The procedures and experiments that I am proposing
here are for actual bacteria count. If you just want to see
the effect of antibiotics on bacteria growth, you can do it
much easier.
Your test media can be a cup of chicken broth, mixed with
some sugar. When the bacteria grow, they will create
gases resulting in a very bad odour. They also convert
sugar to acid and drop the pH in a solution. So, you can
simply get some chicken broth (canned powder),
dissolve it in water, and add sugar and a few drops of
polluted water. Then you divide your sample into two
parts. Add some antibiotics to one part. Cover both with
filter paper or aluminium foil. Keep both samples in a
warm place such as an incubator for 24 hours. Check
both samples to see which one smells bad or has a lower
pH. That is the one with a higher level of bacteria.
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Basic information:
Growing bacteria is one of the most rewarding and
educational activities that a student can do. You will
learn a lot of things just by growing bacteria. We usually
grow bacteria as a way of identifying or counting
bacteria. Bacteria can grow anywhere as long as food,
moisture and proper temperature is available. The
optimum temperature for growing most bacteria is
about 37 to 40 degrees centigrade. This is the same as the
body temperature of warm-blooded animals. That does
not mean that bacteria do not grow in other
temperatures. If the temperature is not favourable,
bacteria will grow slower. If you have ever placed some
cut flowers into a clear jar, you may have noticed that in
a few days, the water becomes cloudy and smells bad.
Bacteria are the cause of cloudiness in water. Recently
this level of cloudiness is measured by special machines
and used as a method of counting or estimating the
number of bacteria (bacteria count). This method is not
accurate, but it is fast and does not need a 24 hours or
more waiting time.
To accurately count bacteria in a sample, first, a dilution
of the sample is made. Then the dilution is spread on a
nutrient agar petri-dish for bacteria growth. Each
bacterium will reproduce and become a bacteria colony.
So, we can simply count the colonies.
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I said nutrient agar because agar by itself is not food for
bacteria. You need to add some food to the agar to make
it nutrient agar. What food is good for bacteria? Think for
a moment. What foods spoil faster and create the worst
odour? They are most likely good for bacteria. I usually
use some fat-free chicken broth or beef broth as food. I
may also add a small amount of sugar. If you want to do
this, make sure you filter the broth so it will be clear.
Chicken broth powder can be purchased from
supermarkets, the only problem is that they also contain
some flavour and vegetables so they can be served as
soup. If you use them, you still need to filter them with a
coffee filter.
But why do we use agar? We use agar because agar can
form a gelatinous moist and clear medium for growing
bacteria. There are a few reasons that you cannot use
gelatin itself. The first reason is that gelatin melts in
warm temperatures, so you have to keep it cold and
bacteria don't grow fast in cold temperatures. The other
reason is that I think manufacturers of gelatin add some
preservatives that stop or slow down the bacteria
growth. So far agar is the best-known gelatinous
substance for growing bacteria.
Since agar is also used as a food additive, you may
purchase agar from health food stores or whole food
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stores. You may need to do some search on that. Some of
these stores don't know what the agar is.
So, start today. Prepare your broth, add some agar, let it
boil for a few minutes and fill up your Petri dishes or any
other thing that you want to use for bacteria growth, keep
it open for a few minutes so the bacteria from the air will
get to that. Cover it and keep it in a warm place for 24
hours. you should then be able to see the bacteria
colonies.
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Notes:
Agar concentration must be 1 to 3 % to get a good gel. I
think you better do it with 2% agar. The same amount of
chicken broth and half of that sugar must be sufficient. In
each petri dish add enough agar to cover the bottom of
the dish. Usually, you cover the petri-dish and keep it
upside down in warm storage such as an incubator.
When the petri dish is upside down, agar does not dry and
condensation does not form on the petri-dish cap.
If you are growing household bacteria, you can just dump
your used Petri dishes in the garbage and wash
everything else with warm water and liquid detergent.
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Determining which antibiotic is most effective:
This is how we determine which antibiotic will kill a
particular organism like E. coli in the laboratory. The
picture that you see is showing a culture plate with
bacteria and antibiotic disks. The bacteria were spread
evenly all over the culture plate. Next, little white disks
with different antibiotics in them were dropped on the
plate. Each white disk represents a different antibiotic
like ampicillin, tetracycline, gentamicin, and others. The
culture plate was then placed in an incubator for 18-24
hours to allow the bacteria to grow. If the bacteria is
sensitive to a particular antibiotic, it will not grow close to
the disk. If the bacteria is resistant to an antibiotic, it will
grow right up to the disk.
In the picture above, the bacteria is sensitive to all the
antibiotics. Without laboratory tests such as this, the
physician would be guessing which antibiotic to
use. Only medical laboratory technologists are licensed
to perform and interpret such tests.
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Identify Variables:
When you think you know what variables may be
involved, think about ways to change one at a time. If you
change more than one at a time, you will not know what
variable is causing your observation. Sometimes
variables are linked and work together to cause
something. At first, try to choose variables that you think
act independently of each other.
If you are going to do this experiment in an advanced
biology lab with all possible equipment, your
independent variable is the amount of antibiotic that you
use. But for now, the variable is antibiotic (presence,
absence).
The dependent variable is the bacteria count.
The constant is the type of antibiotic.
Controlled variables are temperature, light and any
other factor that may affect the bacteria growth in our
different experiment runs.
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Hypothesis:
Based on your gathered information, make an educated
guess about what types of things affect the system you
are working with. Identifying variables is necessary
before you can make a hypothesis.
I think antibiotics can reduce the bacteria count to zero
unless there are some antibiotic-resistant bacteria in
our test sample. I also think that the amount of
antibiotics or the ratio of antibiotics to bacteria is
important because if an antibiotic is not enough to
disable all bacteria, then bacteria may get a chance to
mutate and become resistant to antibiotics.
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Experiment Design:
Design an experiment to test each hypothesis. Make a
step-by-step list of what you will do to answer each
question. This list is called an experimental procedure.
For an experiment to give answers you can trust, it must
have "control." A control is an additional experimental
trial or run. It is a separate experiment, done exactly like
the others. The only difference is that no experimental
variables are changed. A control is a neutral "reference
point" for comparison that allows you to see what
changing a variable does by comparing it to not
changing anything. Dependable controls are sometimes
very hard to develop. They can be the hardest part of a
project. Without a control, you cannot be sure that
changing the variable causes your observations. A series
of experiments that includes a control is called a
"controlled experiment."
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If an antibiotic is able to kill bacteria or disrupt the growth
or reproduction of bacteria, we will have a lower bacteria
count on samples exposed to antibiotics.
PROCEDURE:
For your experiment, you will get a sample containing
bacteria and divide it into two parts. Expose one part to
antibiotic and then test bacteria count in both parts. To
count the bacteria, grow the bacteria on a nutrient agar
plate. Each bacterium will grow to a colony in about 24
hours. That's when it is visible and you can count them
Each colony represents one bacterium in the test
sample. Note that sometimes there are millions of
bacteria in a very small sample. If so many new colonies
grow on a petri-dish, we will not be able to count
anything. That is why we dilute our sample using distilled
water. We make many different dilutions such as 1:1000
and 1:10,000 and 1:100,000 and 1:1000,000. Then we do
bacteria count test on all of them, hoping that in one of
them, bacteria colonies will be in a countable quantity.
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Step 1:
Prepare 24 culture media
plates for growing bacteria.
You may purchase a bacteria
culture kit and prepare your
plates using the agar that
comes in the kit.
Make your own nutrient agar using the following formula
and then use that to prepare your plates.
TGY Tryptone Glucose Yeast
• Tryptone- 5.0g
• Agar- 10.0g
• Yeast extract- 5.0g
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• Glucose- 1.0g
• K2HPO4- 1.0g
• Spring water-1000 mL
This is also called Plate Count Agar. It supports more
species of bacteria than any other medium. 1 gram of
powdered CaCO3 can be added to counteract the acid
generated by many bacteria from the glucose, helping
stock cultures for years. Substitutes: Limestones or
chalk.
Step 2:
Get a sample of polluted water for the test. Mix 2 ml of
polluted water with 10 ml chicken broth in a test tube and
incubate it for 24 hours so the bacteria will reproduce and
increase. Usually, this is done on a device that constantly
moves, so the bacteria can freely move in the liquid. Most
likely you will not have a vibrator, so it is good if you
shake the test tube a few times during this incubation
period.
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Step 3:
While the bacteria are being incubated, prepare some
antibiotic disks as described here. (Antibiotic disks can
also be purchased from biology suppliers).
Break an antibiotic capsule (I used Ampicillin) and
empty the contents in a clean petri-dish. One capsule will
be enough for hundreds of disks.
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Dispose of the plastic shell and add a few drops of water
to the remaining powder. Cut some filter papers into
small pieces and soak them in the antibiotic solution. Let
the disks dry in a clean space. You may cover them with
filter paper to protect them from dust.
Although they are known as antibiotic disks, you can cut
them into small squares.
The reason that we use filter paper, is that other papers
often have starch and other polymers that may affect the
results of our experiments. Filter paper is pure cellulose
fibre.
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Step 4:
Use the bacteria that you grew in Step 2 and prepare
different dilutions of bacteria.
1. Prepare a 1:10 dilution of the sample. To do this, take 2
mL of the sample and blend it with 18 mL of distilled
water.
2. Prepare a 1:100 dilution of the sample by taking 1 mL
from the previous solution and adding 9 mL of water
to it in a test tube.
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3. Prepare 1:1000, 1:10,000 and 1:100,000 dilutions by
taking 1 mL of solution from the previous dilution and
adding 9 mL of distilled water
4. Pipette 0.1ml of each dilution onto a Plates Count
Agar (PCA) plate
5. Take a glass hockey stick submersed in ethanol and
run it through a flame to sterilize it. (Glass hockey
stick is a glass rod bent on one end like a hockey stick.
It is used to spread bacteria on the surface of the agar
plate. You may use a steel spoon instead.)
6. Let it cool and use it to spread dilution around the
plate
7. Do this on two plates for each of the five different
dilutions.
8. Place an antibiotic disk on one of the plates of each
dilution. Label the plates with did dilution level.
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9. incubate all plates at 35 degrees Celsius for 24 hours
and then count the bacterial colonies.
Record the results in a table like this:
Dilution
Bacteria count for a Bacteria count for
plate with antibiotic plate
without
disk
antibiotic disk
1:10 plates
8
120
1:100 plates
6
55
1:1000 plates
6
71
1:10,000 plates
7
97
1:100,000 plates 8
112
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How do you determine the actual bacteria count?
If a 0.1ml of 1:100 dilution shows 7 bacteria colonies, then:
•
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There have been 7 bacteria in 0.1ml of 1:100 solution.
There have been 700 bacteria in 0.1ml of the original
solution (before diluting).
There have been 7000 bacteria in 1ml of the original
solution.
Analyse your results:
The above table contains a set of sample results in grey.
This sample of results shows that the bacteria counted
are not from the samples. Instead, they are from the
environment or from the nutrient agar plates. Where the
bacteria are from samples, they must match the dilution
pattern. For example, if you have 7 bacteria in your 1:1000
sample, you must have about 70 bacteria in your 1:100
sample and about 700 in your 1:10 sample. (You see that
sanitation and a clean environment are crucial to the
bacteria growth tests.)
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Disregarding the source of bacteria, the effect of
antibiotics is clear. Take the average bacteria count with
antibiotics and without antibiotics and use them to make
a graph.
Use a bar graph:
You can use a bar graph to visually present your results.
Make two vertical bars. Label one bar "With Antibiotic".
Label the other bar "Without Antibiotic". The height of
each bar must show the average bacteria count in that
group.
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Note: The list of materials depends on your final
procedure design. You may modify these procedures
based on what is available to you.
Results of Experiment (Observation):
Experiments are often done in series. A series of
experiments can be done by changing one variable a
different amount each time. A series of experiments is
made up of separate experimental "runs." During each
run, you make a measurement of how much the variable
affected the system under study. For each run, a different
amount of change in the variable is used. This produces
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a different amount of response in the system. You
measure this response, or record data, in a table for this
purpose. This is considered "raw data" since it has not
been processed or interpreted yet. When raw data gets
processed mathematically, for example, it becomes
results.
You may change the procedures suggested above in
order to adapt them to your equipment, supplies or
questions. For example, here initial sample of polluted
water was placed on a nutrient agar plate and incubated
for 48 hours. (The nutrient was chicken broth with a small
amount of sugar.) The bacteria colonies appeared to be
in two different colours. So, I decided to grow the bacteria
with a yellow colony. I used a sterile spatula to remove
one colony and transfer it to 2 ml of distilled water in a test
tube.
Then I took 3 nutrient agar plate and added 0.5 ml of the
solution on each of the plates. I left one plate without any
antibiotics, and placed one antibiotic disk on the second
plate and two antibiotic disks on the third plate. All plates
were incubated for 48 hours. Images show that no
bacteria is grown close to antibiotic disks.
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What Nutrient Agar did I use?
The nutrient agar in these plates was made using 9
grams of chicken broth (powder), 5 grams of Agar and
500 ml of water. I also used a few drops of food colouring
hoping that it will make the bacteria colonies more
visible, but I did not feel any difference. That made me 15
nutrient agar plates that I kept in the refrigerator for later
use.
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How to dispose the bacteria-infected plates?
One way is placing all the plates in an autoclave in a
temperature of 130º for one hour. Note that this
temperature is under pressure and hot steam will kill
bacteria. Dry hot air does not kill the bacteria at this
temperature.
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Another way is using antibacterial disinfectants. Soak
the plates in a strong antibacterial solution for a few days
and then dispose them.
Calculations:
Depending on your method you may or may not need to
do any calculations. If instead of a bacteria disk, you use
a bacteria solution, then you may also want to do some
calculations to find out how many bacteria will be killed
by the action of a certain amount of antibiotics.
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Related Questions & Answers:
What you have learned may allow you to answer other
questions. Many questions are related. Several new
questions may have occurred to you while doing
experiments. You may now be able to understand or
verify things that you discovered when gathering
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information for the project. Questions lead to more
questions, which lead to additional hypotheses that need
to be tested.
Possible Errors:
If you did not observe anything different than what
happened with your control, the variable you changed
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may not affect the system you are investigating. If you
did not observe a consistent, reproducible trend in your
series of experimental runs there may be experimental
errors affecting your results. The first thing to check is
how you are making your measurements. Is the
measurement method questionable or unreliable?
Maybe you are reading a scale incorrectly, or maybe the
measuring instrument is working erratically.
If you determine that experimental errors are influencing
your results, carefully rethink the design of your
experiments. Review each step of the procedure to find
sources of potential errors. If possible, have a scientist
review the procedure with you. Sometimes the designer
of an experiment can miss the obvious.
Summary of Results:
Summarize what happened. This can be in the form of a
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table of processed numerical data, or graphs. It could
also be a written statement of what occurred during
experiments.
It is from calculations using recorded data that tables
and graphs are made. Studying tables and graphs, we
can see trends that tell us how different variables cause
our observations. Based on these trends, we can
conclude the system under study. These conclusions
help us confirm or deny our original hypothesis. Often,
mathematical equations can be made from graphs.
These equations allow us to predict how a change will
affect the system without the need to do additional
experiments. Advanced levels of experimental science
rely heavily on the graphical and mathematical analysis
of data. At this level, science becomes even more
interesting and powerful.
Conclusion:
Using the trends in your experimental data and your
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experimental observations, try to answer your original
questions. Is your hypothesis correct? Now is the time to
pull together what happened, and assess the
experiments you did.
BIBLIOGRAPHY
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1.
2.
https://www.studocu.com/in/document/universit
y-of-kerala/reading-poetry/bio-projectestse/44582353
www.emedicinehealth.com
4.
www.scienceproject.com
3.
www.microbiologysociety.org
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