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 Earths 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 Grams 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 gramnegative 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, antifungal, 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 sexuallytransmitted 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 l 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 37C 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 200ml 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 outermembrane 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 gramnegative 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 friends 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