INTERNATIONAL EDUCATION COLLEGE BIOLOGY LABARATORY REPORT

NAME : YUVANESHWARY CLASS : 12M8 TITLE : THE EFFECTIVITY OF DIFFERENT ANTIBIOTICS ON BACTERIA DATE : 9.8.2012

Bacteria play an important role in recycling nutrients. ranging from spheres to rods and spirals. with many steps in nutrient cycles. . for instance. as well as in organic matter. endoplasmic reticulum and Golgi apparatus.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. Typically a few micrometres in length. The bacteria will retain the crystal violet stain. bacteria can be divided into two groups. nitrogen fixation and putrefaction. radioactive waste. Teichoic acids and lipoids serve to act as chelating agents and also for certain types of adherence by forming lipoteichoic acids. bacteria can also have many shapes. teichoic acid and polysaccharides as part of their cell wall structure. They are ubiquitous in every habitat on Earth. aures) used in this experiment is an example of gram-positive bacteria. Gram-positive bacteria can be differentiated from gram-negative bacteria by gram-staining techniques. The bacterium Staphylococcus aures (S. Gram-positive bacteria are those that are characterised by having thick peptidoglycan. Basically. acidic hot springs. water and deep in the Earth‟s crust. 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 peptidoglycans are heteropolymers of glycan strands. growing in soil. which are cross-linked through short peptides. which are gram-positive bacteria and gram-negative bacteria.

such as inflammation. It was not until 1935 that a strain of E. This is because the LPS will trigger an innate immune response characterised by cytokine production and immune system activation. the enteric bacteria. as well.g. Enterobacter. but these bacteria. Escherichia. 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. The Enterobacteriaceae are among the most important bacteria medically. safranin is added after crystal violet. may occasionally be associated with diseases of humans. coli). The pathogenic capability of these bacteria is often associated with the lipopolysaccharide (also known as LPS or endotoxin) layer. Green Sulphur and Green Non-Sulphur. There is no teichoic acid in the cell wall of gram-negative bacteria. Some examples of gramnegative bacteria include proteobacteria Escherichia coli (E. Figure 2 : Escherichia coli Theodor Escherich first described E. As both gram-positive and gram-negative bacteria are used in this example. cyanobacteria. Several others are normal colonists of the human gastrointestinal tract (e. coli was shown to be the cause of an outbreak of diarrhoea among infants. coli is the head of the large bacterial family. Enterobacteriaceae. The cell wall of this group of bacteria is thinner with distinct layers as compare to gram-positive bacteria. Yersinia).On the other hand. .which he isolated from the faeces of newborns. spirochaetes. thus the effect of antibiotics on these bacteria can be determined.Shigella. coli in 1885. In this test. colouring all the gram-negative bacteria with red or pink colour. Klebsiella). A number of genera within the family are human intestinal pathogens (e. Salmonella.g. which are facultatively anaerobic Gram-negative rods that live in the intestinal tracts of animals in health and disease. as Bacterium coli commune. E. It was later renamed Escherichia coli. and for many years the bacterium was simply considered to be a commensal organism of the large intestine.

treatment of these infections has become more difficult because this bacteria has become resistant to penicillin and similar drugs. often referred to simply as “Staph”. Most of these infections are minor (such as pimples. aureus bacteria can also cause serious. These infections can be spread through contact with pus from an infected would. boils. and pneumonia. are bacteria commonly found on the skin and in the noses of healthy people. S. and sometimes. Firmicutes. These are all classified in the Prokaryote Domain. most serious S. surgical wound infections. aureus bacterial infections were treated with several forms penicillin. The S. Over the past 50 years. Sometimes. and other skin conditions). aureus can cause infection and is one of the most common causes of skin infections. . towels or exercise equipment. Staphylococcus aureus. skin to skin contact with an infected person.Figure 3 : Staphylococcus aureus Staphylococcus aureus is classified within a phylum. fatal infections such as bloodstream infections. In the past. or taxanomical grouping of similar classes of organisms. and contact with shared objects such as clothing.

Ampicillin is a broad-spectrum antibiotic which kills a wide variety of bacteria that cause a wide variety of commonly-occuring infections. Antibiotics are chemicals produced by or derived from microorganisms (i. It may also be used to treat urine infections. Gram positive and gram negative bacteria are controlled with this type of medication. Antibiotics are a specific type of antimicrobial drug. and certain infections affecting the blood or internal organs. and anti-parasitic drugs. and therefore can be used to treat infection. a larger group which also includes anti-viral. nose and throat. 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. This allows holes to appear in the cell walls and so kills the bacteria. However. Antibiotics are one class of "antimicrobials". They keep unwanted substances from entering their cells and stop the contents of their cells from leaking out. The cell walls of bacteria are vital for their survival. Ampicillin impairs the bonds that hold the bacterial cell wall together. Ampicillin works by interfering with the ability of bacteria to form cell walls. They are relatively harmless to the host.e. ears. certain sexuallytransmitted infections.Antibiotics An antibiotic is a drug that kills or slows the growth of bacteria. antifungal. . Ampicillin may be used to treat infections of the airways. Bugs or germs such as bacteria and fungi).

The term "tetracycline" is also used to denote the four-ring system of this compound. more recently. Carbenicillin is also more stable at lower pH than ampicillin. which kills the bacteria. rosacea. they prevent introduction of new amino acids to the nascent peptide chain. . indicated for use against many bacterial infections. It was discovered by scientists at Beecham and marketed as Pyopen. Tetracyclines bind to the 30S subunit of microbial ribosomes. It is commonly used to treat acne today. although they are more resistant than ampicillin to degradation. Resistance to the tetracyclines results from changes in permeability of the microbial cell envelope. and is historically important in reducing the number of deaths from cholera. and.Figure 5 : structure of carbenicillin Carbenicillin is a bacteriolytic antibiotic belonging to the carboxypenicillin subgroup of the penicillins. They inhibit protein synthesis by blocking the attachment of tRNA to the A site on the ribosome. Thus. Figure 5 : structure of tretracycline Tetracycline is a broad-spectrum polyketide antibiotic produced by the Streptomyces genus of Actinobacteria. It works by blocking the bacteria's cell wall growth. The carboxypenicillins are susceptible to degradation by beta-lactamase enzymes. It has Gram-negative coverage which includes Pseudomonas aeruginosa but limited Gram-positive coverage.

sterile filter paper discs. Carbenicillin and Tetracycline. To consider the reliability and validity of the results. sterilized distilled water. Bacteria such as Staphylococcus aures. micropipette. hand soap. sterile petri dish with cover. the less effective the antibiotic against bacteria. pipette teats. Bacillus and Staphylococcus aures).OBJECTIVE   To investigate the effect of different antibiotics on bacteria. antibiotics solution such as Ampicillin. marker pen. To dtermine which type of antibiotics among Ampicillin. Bacillus and Escherichia coli (E. ruler. protein synthesise or nucleic acid synthesis and produce different inhibition zone size. The larger the inhibition zone. 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. concentration of antibiotics APPARATUS AND MATERIALS Bunsen burner. coli) in bottle. sterile forceps. incubation temperature. VARIABLES Manipulated variable : Types of antibiotics Responding variable : Diameter of the inhibition zone (mm) Fixed variable : Amount of bacteria used. disinfectant spray . duration of incubation. the more effective the antibiotic against bacteria. label stickers. NULL HYPOTHESIS Antibiotics will not affect the growth of bacteri differently or produce different inhibition zone size. agar solution. The larger the inhibition zone. Carbenicillin and Tetracycline is most effective in inhibiting the growth of Escherichia coli.

5. then later place them carefully onto the surface of the nutrient agar at the location labeled with that particular antibiotic. 3. 6. The pipette teat used is then disposed. A micropipette fixed with pipette teat was used to inoculate 200 µl of bacteria E. The petri dish was swirled gently so that the bacteria were spread and mix evenly throughout the solution. 12. After the incubation was done. 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. 8. coli into the petri dish carefully. 4. The working place was sprayed thoroughly with the disinfectant spray and left a while before wiping it with a clean paper towel. 2. Agar solution was taken out from oven and was then poured into the petri dish until it was half full. . Hands and fingers were washed thoroughly with hand soap before the experiments. 11. The mouth of the bottle containing agar solution was flamed and let it to cool down before pouring into the petri dish. 7. The solution was then left to solidify. Label stickers were used to label at the bottom of the petri dish with the type of bacterium it is inoculated. Sterile filter paper discs were then dipped into the antibiotic solutions and sterile distilled water. 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. the diameter of the inhibition zones was measured by ruler and the data obtained was recorded and tabulated. 9. 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.PROCEDURES 1.

The hot agar should be handled carefully to avoid scorching of hands. 3. 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. wear eye protection. 7. 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. During the experiment. 2.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. Clean hands and fingers with soap and the working place with alcohol spray to prevent bacterial contamination. 8. Flame the mouth of the conical flask containing agar solution before pouring it into the petri dish to prevent bacteria contaminating the solution. 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. 5. 6. lab coat and handle all the glass apparatus carefully to prevent any accident from happening. 4. .

Escherichia coli Staphylococcus aureus Bacillus DISCUSSIONS This experiment is carried out to investigate the effectiveness of different antibiotics against Staphylococcus aures. For example. There are several sources of errors that might have caused negative result. A constant flow of clean air is produced when air is passed through a series of filter. As the procedure is quite long and complicated. these steps only reduce the risk of contamination but not prevent contamination completely. This experiment result can be further improved by carrying out this experiment in the laminar flow chamber to minimize the risk of contamination. Firstly. there are no results obtained in this experiment. . Bacillus and Escherichia coli. errors might have arisen from any of the steps causing negative result. Aseptic techniques should be used more strictly to improve the result. 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 is conducted by following all the steps carefully from the lab manual. However.RESULT No results were obtained as there is no inhibition zone. even though the experiment has been carried out with several steps to avoid contamination.

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. Besides that. contamination of the agar solution when it is exposed to air to solidify is also the sources of error in the experiment. It's different from penicillin by the presence of an amino group. concentration of antibiotics and incubation temperature. Ampicillin interferes with the ability to synthesize cell wall. putting the paper discs onto the agar which has not completely solidified is also one of the errors. the Petri dishes were kept in constant temperature by incubating the plates in the same incubator for 24 hours. Although no results is obtained. 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. other bacteria and fungi can still contaminate the solution as they are present in the surrounding all the time. This is obtained through the comparison with other groups. size of paper discs. conclusion is made that ampicillin is the most effective antibiotics among all the plant extracts used. 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. There are several variables being kept constant in the experiment which are volume of bacteria. This is to ensure the volume of antibiotics absorbed by each paper disc is equal. This will contribute to the unreliability of the result. . That amino group helps the drug penetrate the outermembrane of bacteria. The bacteria have to compete against microorganisms for space. Even though the mouth of the bottle is flamed using a Bunsen burner before pouring the agar into the 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. which ultimately leads to cell lysis.Moreover. This is because the antibiotics on the filter paper disc might have spread around the disc which is still moving. 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. Besides. This is to prevent the effect of other factors that might affect the actions of the antibiotics. Also.

aures for each corresponding antibiotic. Most gram-negative bacteria. S. Carbenicillin is broad spectrum antibiotics as they target on all the bacteria.Generally. The thick peptidoglycan of S. like Escherichia coli. all antibiotics are effective against all bacteria. aures acts as a protective barrier against the antibiotics and thus the smaller diameter of inhibition zones can be seen for S. . aures and Bacillus are examples of gram-positive bacteria that have thick peptidoglycan in the structure of cell wall. Ampicillin and Tetracycline are narrow spectrum antibiotic as it only targets specific bacteria which is at least two out of three bacteria. For instance. Meanwhile. 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. In contrary. This could be due to the type of bacteria which is categorized into gram-positive or gramnegative bacteria.

Furthermore. Contamination can occur in any part of the experiment. Although the inhibition zones are expected and assumed to be circular in shape. the result will not be reliable. . Foreign bacteria can contaminate the solution when the bacteria are transferred from its bottle to the petri dish. The bacteria again. the spreading and the growth of the bacteria may been limited in a single position and not scattered. Other than that. for irregularly shaped inhibition zones. the amount of agar solution poured into each agar plate is not constant. This may affect the growth of the bacteria as the varying amount of agar could cause some bacteria to grow better. 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. 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. Different microorganisms have varying resistance against the antibiotics. it takes longer time for the agar solution to solidify. This will cause the result to be inaccurate as the foreign bacteria or fungi may alter the diameter of the inhibition zone. but not simultaneously. may not have been correctly distributed as the bacteria poured were divided into three sides of the petri dish. Last but not least. although several steps are taken to avoid contamination.LIMITATIONS There are some unavoidable limitations in the experiment that can cause the results to be less accurate. When the amount of agar solution is more. there are a few errors that could affect the validity of the experiment. The first limitation is measuring the diameter of the inhibition zones. only an estimate of the diameter can be obtained. this is not always the case. The bacteria have to compete against microorganisms for space. Consequently. Moreover. As such. 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. contamination of the agar solution when it is exposed to air to solidify is also the limitation in the experiment. these steps only reduce the risk of contamination and not completely prevent contamination. This is because the amount of agar poured is just an approximation. Thus.

Hodder Education A2.htm 4. 2008. REFERENCE 1. http://www. „Bacteria.CONCLUSION As no results have obtained.J. Ampicillin is the most effective antibiotic compared to other antibiotics. 2008.medicalnewstoday.html 6.nlm. Null hypothesis is rejected. „Introducing micro-organisms‟. Antibiotics.gov/books .ncbi. Edexcel A2 Biology. London. http://www. Clegg C.com/the-bloody-beet.elements4health. Edexcel Biology.com/antibiotics/article_em..com/articles/10278. UK. Different antibiotic has different effect on the growth of bacteria. A Pearson Company. conclusion is made based on comparing my friend‟s result.php 5.emedicinehealth. pg 70-72 2. Antibiotics. http://www.http://www.nih. pg 86-87 3.

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