INTERNATIONAL EDUCATION COLLEGE

BIOLOGY LABARATORY REPORT

NAME : YUVANESHWARY

CLASS : 12M8

TITLE : THE EFFECTIVITY OF DIFFERENT ANTIBIOTICS ON BACTERIA

DATE : 9.8.2012

INTRODUCTION

Figure 1 : Gram-positive bacteria and gram-negative bacteria

Bacteria are a large group of unicellular microscopic organisms whose cells have

neither a membrane-bound nucleus nor other membrane-bound organelles such as mitochondria,

endoplasmic reticulum and Golgi apparatus. Typically a few micrometres in length, bacteria can

also have many shapes, ranging from spheres to rods and spirals. They are ubiquitous in every

habitat on Earth, growing in soil, acidic hot springs, radioactive waste, water and deep in the

Earth’s crust, as well as in organic matter. Bacteria play an important role in recycling nutrients,

with many steps in nutrient cycles, for instance, nitrogen fixation and putrefaction. Basically,

bacteria can be divided into two groups, which are gram-positive bacteria and gram-negative

bacteria.

Gram-positive bacteria are those that are characterised by having thick peptidoglycan,

teichoic acid and polysaccharides as part of their cell wall structure. Teichoic acids and lipoids

serve to act as chelating agents and also for certain types of adherence by forming lipoteichoic

acids. The peptidoglycans are heteropolymers of glycan strands, which are cross-linked through

short peptides. Gram-positive bacteria can be differentiated from gram-negative bacteria by

gram-staining techniques. The bacteria will retain the crystal violet stain, instead of taking up the

counterstain (safranin or fuchsine) and appearing red or pink due to the high amount of

peptidoglycan in the cell wall. The bacterium Staphylococcus aures (S. aures) used in this

experiment is an example of gram-positive bacteria.

On the other hand, gram negative bacteria are bacteria that do not retain the crystal violet

colour in their cell wall when carrying out the Gram’s stain test. In this test, safranin is added

after crystal violet, colouring all the gram-negative bacteria with red or pink colour. The cell wall

of this group of bacteria is thinner with distinct layers as compare to gram-positive bacteria.

There is no teichoic acid in the cell wall of gram-negative bacteria. The pathogenic capability of

these bacteria is often associated with the lipopolysaccharide (also known as LPS or endotoxin)

layer. This is because the LPS will trigger an innate immune response characterised by cytokine

production and immune system activation, such as inflammation. Some examples of gram-

negative bacteria include proteobacteria Escherichia coli (E. coli), cyanobacteria, spirochaetes,

Green Sulphur and Green Non-Sulphur. As both gram-positive and gram-negative bacteria are

used in this example, thus the effect of antibiotics on these bacteria can be determined.

Figure 2 : Escherichia coli

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune,which he

isolated from the faeces of newborns. It was later renamed Escherichia coli, and for many years

the bacterium was simply considered to be a commensal organism of the large intestine. It was

not until 1935 that a strain of E. coli was shown to be the cause of an outbreak of diarrhoea

among infants. E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric

bacteria, which are facultatively anaerobic Gram-negative rods that live in the intestinal tracts of

animals in health and disease. The Enterobacteriaceae are among the most important bacteria

medically. A number of genera within the family are human intestinal pathogens

(e.g. Salmonella,Shigella, Yersinia). Several others are normal colonists of the human

gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may

occasionally be associated with diseases of humans.

Figure 3 : Staphylococcus aureus

Staphylococcus aureus is classified within a phylum, or taxanomical grouping of similar

classes of organisms, Firmicutes. These are all classified in the Prokaryote Domain.

Staphylococcus aureus, often referred to simply as “Staph”, are bacteria commonly found on the

skin and in the noses of healthy people. Sometimes, S. aureus can cause infection and is one of

the most common causes of skin infections. Most of these infections are minor (such as pimples,

boils, and other skin conditions). The S. aureus bacteria can also cause serious, and sometimes,

fatal infections such as bloodstream infections, surgical wound infections, and pneumonia.

These infections can be spread through contact with pus from an infected would, skin to skin

contact with an infected person, and contact with shared objects such as clothing, towels or

exercise equipment. In the past, most serious S. aureus bacterial infections were treated with

several forms penicillin. Over the past 50 years, treatment of these infections has become more

difficult because this bacteria has become resistant to penicillin and similar drugs.

Antibiotics

An antibiotic is a drug that kills or slows the growth of bacteria. Antibiotics are chemicals

produced by or derived from microorganisms (i.e. Bugs or germs such as bacteria and fungi).

Antibiotics are one class of "antimicrobials", a larger group which also includes anti-viral, anti-

fungal, and anti-parasitic drugs. They are relatively harmless to the host, and therefore can be

used to treat infection. Antibiotics are a specific type of antimicrobial drug. However, the term

antibiotic is now widely used to refer to all drugs that selectively target bacteria.

Figure 4 : structure of ampicillin

Ampicillin is another type of penicillin that is used to treat certain types of bacterial infections.

Gram positive and gram negative bacteria are controlled with this type of medication. Ampicillin

works by interfering with the ability of bacteria to form cell walls. The cell walls of bacteria are

vital for their survival. They keep unwanted substances from entering their cells and stop the

contents of their cells from leaking out. Ampicillin impairs the bonds that hold the bacterial cell

wall together. This allows holes to appear in the cell walls and so kills the bacteria.

Ampicillin is a broad-spectrum antibiotic which kills a wide variety of bacteria that cause a wide

variety of commonly-occuring infections. Ampicillin may be used to treat infections of the

airways, ears, nose and throat. It may also be used to treat urine infections, certain sexually-

transmitted infections, and certain infections affecting the blood or internal organs.

Figure 5 : structure of carbenicillin

Carbenicillin is a bacteriolytic antibiotic belonging to the carboxypenicillin subgroup of the

penicillins. It was discovered by scientists at Beecham and marketed as Pyopen. It works by

blocking the bacteria's cell wall growth, which kills the bacteria. It has Gram-negative coverage

which includes Pseudomonas aeruginosa but limited Gram-positive coverage. The

carboxypenicillins are susceptible to degradation by beta-lactamase enzymes, although they are

more resistant than ampicillin to degradation. Carbenicillin is also more stable at lower pH than

ampicillin.

Figure 5 : structure of tretracycline

Tetracycline is a broad-spectrum polyketide antibiotic produced by the Streptomyces genus of

Actinobacteria, indicated for use against many bacterial infections. The term "tetracycline" is

also used to denote the four-ring system of this compound. Tetracyclines bind to the 30S subunit

of microbial ribosomes. They inhibit protein synthesis by blocking the attachment of tRNA to

the A site on the ribosome. Thus, they prevent introduction of new amino acids to the nascent

peptide chain. Resistance to the tetracyclines results from changes in permeability of the

microbial cell envelope. It is commonly used to treat acne today, and, more recently, rosacea,

and is historically important in reducing the number of deaths from cholera.

OBJECTIVE

To investigate the effect of different antibiotics on bacteria. To dtermine which type of antibiotics among Ampicillin, Carbenicillin and Tetracycline

is most effective in inhibiting the growth of Escherichia coli, Bacillus and Staphylococcus aures).

To consider the reliability and validity of the results, thus find the ways to improve the result

HYPOTHESIS

Different antibiotics will affect the growth of bacteri differently by either inhibit the synthesise

of cell wall, protein synthesise or nucleic acid synthesis and produce different inhibition zone

size. The larger the inhibition zone, the more effective the antibiotic against bacteria.

NULL HYPOTHESIS

Antibiotics will not affect the growth of bacteri differently or produce different inhibition zone

size. The larger the inhibition zone, the less effective the antibiotic against bacteria.

VARIABLES

Manipulated variable : Types of antibiotics

Responding variable : Diameter of the inhibition zone (mm)

Fixed variable : Amount of bacteria used, incubation temperature, duration of incubation, concentration of antibiotics

APPARATUS AND MATERIALS

Bunsen burner, sterile forceps, sterile petri dish with cover, micropipette, pipette teats. Bacteria

such as Staphylococcus aures, Bacillus and Escherichia coli (E. coli) in bottle, antibiotics

solution such as Ampicillin, Carbenicillin and Tetracycline, sterilized distilled water, agar

solution, label stickers, marker pen, ruler, sterile filter paper discs, hand soap, disinfectant spray

PROCEDURES

1. The working place was sprayed thoroughly with the disinfectant spray and left a while

before wiping it with a clean paper towel.

2. Hands and fingers were washed thoroughly with hand soap before the experiments.

3. The base of the petri dish was labeled with respective antibiotics location where they will

be placed later and control (sterilized distilled water) was also labeled as well.

4. Label stickers were used to label at the bottom of the petri dish with the type of bacterium

it is inoculated.

5. Agar solution was taken out from oven and was then poured into the petri dish until it

was half full. The mouth of the bottle containing agar solution was flamed and let it to

cool down before pouring into the petri dish.

6. A micropipette fixed with pipette teat was used to inoculate 200 µl of bacteria E. coli into

the petri dish carefully. The pipette teat used is then disposed.

7. The petri dish was swirled gently so that the bacteria were spread and mix evenly

throughout the solution.

8. The solution was then left to solidify.

9. Sterile filter paper discs were then dipped into the antibiotic solutions and sterile distilled

water, then later place them carefully onto the surface of the nutrient agar at the location

labeled with that particular antibiotic.

10. The petri dish was then closed properly and placed upside down in the incubator at a

constant temperature of 37°C for at least 24 hours.

11. The working area was once again sprayed with disinfectant and wiped with a clean paper

towel while the hands also need to be sanitised after the experiment.

12. After the incubation was done, the diameter of the inhibition zones was measured by

ruler and the data obtained was recorded and tabulated.

SAFETY PRECAUTIONS

1. All the apparatus such as the forceps and petri dish used should be sterilised by wrapping

them with aluminium foil before the experiment to prevent microbial contamination.

2. Clean hands and fingers with soap and the working place with alcohol spray to prevent

bacterial contamination.

3. All the steps in the procedure must be carried out close to the flame of a Bunsen burner to

prevent contamination from the surrounding bacteria.

4. Flame the mouth of the conical flask containing agar solution before pouring it into the

petri dish to prevent bacteria contaminating the solution.

5. The hot agar should be handled carefully to avoid scorching of hands.

6. The cover of the agar plate is only opened slightly when transferring the bacteria and the

agar solution into the agar solution to minimize the chance of contamination.

7. Label the sticker on the side of the petri dish rather than the top or the bottom to prevent

obstruction of view when measuring the diameter of the circular inhibition areas.

8. During the experiment, wear eye protection, lab coat and handle all the glass apparatus

carefully to prevent any accident from happening.

RESULT

No results were obtained as there is no inhibition zone.

Escherichia coli Staphylococcus aureus Bacillus

DISCUSSIONS

This experiment is carried out to investigate the effectiveness of different antibiotics

against Staphylococcus aures, Bacillus and Escherichia coli. This experiment is conducted by

following all the steps carefully from the lab manual. However, there are no results obtained in

this experiment. As the procedure is quite long and complicated, errors might have arisen from

any of the steps causing negative result.

There are several sources of errors that might have caused negative result. Firstly, even

though the experiment has been carried out with several steps to avoid contamination, these steps

only reduce the risk of contamination but not prevent contamination completely. Aseptic

techniques should be used more strictly to improve the result. For example, the lid of the petri

dish should only be opened for the minimum amount to allow the micropipette tip in and for a

minimum amount of time. This experiment result can be further improved by carrying out this

experiment in the laminar flow chamber to minimize the risk of contamination. A constant flow

of clean air is produced when air is passed through a series of filter.

Moreover, contamination of the agar solution when it is exposed to air to solidify is also

the sources of error in the experiment. Even though the mouth of the bottle is flamed using a

Bunsen burner before pouring the agar into the petri dish, other bacteria and fungi can still

contaminate the solution as they are present in the surrounding all the time. The bacteria have to

compete against microorganisms for space. Besides that, putting the paper discs onto the agar

which has not completely solidified is also one of the errors. This is because the antibiotics on

the filter paper disc might have spread around the disc which is still moving. This will contribute

to the unreliability of the result.

There are several variables being kept constant in the experiment which are volume of

bacteria, size of paper discs, concentration of antibiotics and incubation temperature. The volume

of the bacterial culture solution used is kept constant at 200µml for each bacterium using the

graduated micropipette with teat to inoculate them into the Petri dish. The nutrient agar solution

is controlled by approximation method so as to ensure that the relative measure of dispersion of

bacteria is equal in each Petri dish. The size of paper discs dunked into the antibiotic solutions is

controlled by using paper discs cut out by same puncher prior to the experiment. This is to ensure

the volume of antibiotics absorbed by each paper disc is equal. Besides, the Petri dishes were

kept in constant temperature by incubating the plates in the same incubator for 24 hours. This is

to prevent the effect of other factors that might affect the actions of the antibiotics. Petri dishes

were kept covered at all times to ensure constant environmental conditions and also to prevent

contamination by any other microorganism in surrounding air.

Although no results is obtained, conclusion is made that ampicillin is the most effective

antibiotics among all the plant extracts used. This is obtained through the comparison with other

groups. Ampicillin interferes with the ability to synthesize cell wall. It's different from penicillin

by the presence of an amino group. That amino group helps the drug penetrate the outer-

membrane of bacteria. Also, transpeptidase (needed by bacteria to make their cell walls) which

acts as a competitive inhibitor of Ampicillin inhibits the final stage of cell wall synthesis, which

ultimately leads to cell lysis.

Generally, all antibiotics are effective against all bacteria. Carbenicillin is broad spectrum

antibiotics as they target on all the bacteria. Meanwhile, Ampicillin and Tetracycline are narrow

spectrum antibiotic as it only targets specific bacteria which is at least two out of three bacteria.

This could be due to the type of bacteria which is categorized into gram-positive or gram-

negative bacteria. Most gram-negative bacteria, has a thinner layer of peptidoglycan with no

teichoic acid that enables the antibiotic to penetrate through the cell wall and inhibit the growth

of the bacteria. For instance, like Escherichia coli. In contrary, S. aures and Bacillus are

examples of gram-positive bacteria that have thick peptidoglycan in the structure of cell wall.

The thick peptidoglycan of S. aures acts as a protective barrier against the antibiotics and thus

the smaller diameter of inhibition zones can be seen for S. aures for each corresponding

antibiotic.

LIMITATIONS

There are some unavoidable limitations in the experiment that can cause the results to be

less accurate. The first limitation is measuring the diameter of the inhibition zones. Although the

inhibition zones are expected and assumed to be circular in shape, this is not always the case. As

such, for irregularly shaped inhibition zones, only an estimate of the diameter can be obtained.

Furthermore, although several steps are taken to avoid contamination, these steps only

reduce the risk of contamination and not completely prevent contamination. Contamination can

occur in any part of the experiment. Foreign bacteria can contaminate the solution when the

bacteria are transferred from its bottle to the petri dish. This can occur when withdrawing the

bacteria through the mouth of the bottle and inoculate it into the petri dish with its cover opened

slightly.

Moreover, contamination of the agar solution when it is exposed to air to solidify is also

the limitation in the experiment. Even though the mouth of the bottle is flamed using a Bunsen

burner before pouring the agar into the petri dish, other bacteria and fungi can still contaminate

the solution as they are present in the surrounding all the time. The bacteria have to compete

against microorganisms for space. Different microorganisms have varying resistance against the

antibiotics. This will cause the result to be inaccurate as the foreign bacteria or fungi may alter

the diameter of the inhibition zone.

Other than that, there are a few errors that could affect the validity of the experiment. The

bacteria again, may not have been correctly distributed as the bacteria poured were divided into

three sides of the petri dish. Consequently, the spreading and the growth of the bacteria may been

limited in a single position and not scattered.

Last but not least, the amount of agar solution poured into each agar plate is not constant.

This is because the amount of agar poured is just an approximation. When the amount of agar

solution is more, it takes longer time for the agar solution to solidify. This may affect the growth

of the bacteria as the varying amount of agar could cause some bacteria to grow better. The time

when the filter paper discs with antibiotics introduced into the agar solution will also be different

as there is a tendency that they will be introduced to the agar solution that solidifies first, but not

simultaneously. Thus, the result will not be reliable.

CONCLUSION

As no results have obtained, conclusion is made based on comparing my friend’s result.

Ampicillin is the most effective antibiotic compared to other antibiotics. Different antibiotic has

different effect on the growth of bacteria. Null hypothesis is rejected.

REFERENCE

1. Clegg C.J., ‘Introducing micro-organisms’, Edexcel Biology, Hodder Education A2, London, UK, 2008, pg 70-72

2. A Pearson Company, ‘Bacteria, Edexcel A2 Biology, 2008, pg 86-87

3. Antibiotics, http://www.emedicinehealth.com/antibiotics/article_em.htm

4. Antibiotics,http://www.medicalnewstoday.com/articles/10278.php

5. http://www.elements4health.com/the-bloody-beet.html

6. http://www.ncbi.nlm.nih.gov/books