TITLE: EFFECTIVENESS OF ANTIOBIOTIC NAME: SITI NASHUHA BT OSMAN ID NO: 2010843638 CLASS: 11M2 DATE OF EXPERIMENT: 9 th August 2011

NAME OF LECTURER: Mdm. PUSPARANEE HAKIM

Objective
1. To investigate the effect of different antibiotics on bacteria 2. To compare the effectiveness of different antibiotics. 3. To increase experimental skills, such as working in a group, producing valid result and state valid conclusion based on results.

Introduction 1. ANTIBIOTICS

Figure 1: example of antibiotics( source2) Antibiotics are chemical that restrict the growth of bacteria by interfering with their metabolic pathway. It is grouped based on the mechanisms used to target the microorganisms. Antibiotics occur naturally in living organisms such as plants, fungi, and bacteria. The medicinal value of natural antibiotics are known long time ago and have been used as folk remedy. In pharmaceutical industry, the active ingredients are derived and chemically developed to form semi-synthetic antibiotics. These pharmaceutical antibiotics are administered by physicians in man or animal to treat bacterial infection. Examples of pharmaceutical antibiotics are ampicillin, tetracycline, streptomycin and kanamycin.

AMPICILLIN Ampicillin is only effective against gram- positive organism such as Proteus spp. However, sometimes it is said that this antibiotic can result in reactions that range in severity from a rash to people with allergic reactions such as anaphylaxis.

TETRACYCLIN Tetracycline is a protein synthesis inhibitor. It is said to be used as medicine to treat acne and also reducing the incidence of mortality because of cholera.

STREPTOMYCIN Streptomycin is a bactericidal antibiotic. It is said to be the first antibiotic remedy for tuberculosis. Besides that, this antibiotic also cannot be consumed orally but must be given via injection. However, this antibiotic has adverse effect which is hearing loss.

KANAMYCIN Kanamycin is a water-soluble broad-spectrum antibiotic obtained from the soil bacterium Streptomyces kanamyceticus. It is said to be available in oral, intravenous, and intramuscular forms, and used to treat a wide variety of infections.

2. ESCHERICHIA COLI ( E.Coli)

Figure 2: E.Coli (source 1)

E.Coli is a gram negative bacterium. This bacterium is commonly found in the lower intestine and it is said that this bacterium is harmless but some of it can cause serious food poisoning in humans. The harmless E.Coli strain is part of the natural gut flora which is beneficial to the host. This natural gut flora prevents the establishment of pathogenic bacteria in the intestine.

Rationale
This experiment is conducted to investigate the effectiveness of antibiotics on bacteria. In this experiment Escherichia coli which is a gram negative bacteria is used. This is because this bacterium is one of the infectious disease-causing bacterium. For example, this bacterium can cause intestinal diseases. Therefore, this study can show which antibiotics is the most effective against bacterial infection especially gram negative bacteria.

Problem Statement
What is the most effective antibiotics?

Hypothesis
Different antibiotics have different effectiveness on bacteria.

Null Hypothesis
All antibiotics have the same effectiveness on bacteria.

Apparatus
Bunsen burner, sterile forceps, sterile Petri dish, incubator set at 30C, oven to warm agar solution,

Material
Agar plate with one type of bacteria which is E. Coli, bench spray of disinfectant, soap or handwash, paper towels, marker pen, adhesive tape, impregnated paper discs.

Variables
Manipulated variable: Type of antibiotic Responding variable: diameter of clear zone Constant variable: Temperature, humidity, amount of bacteria, concentration of antibiotic, time of incubation

Procedure
1. Hands are washed with soap or handwash. The working area is sprayed thoroughly with the disinfectant spray. Then, the it is wipe with a paper towel after at least 10 minutes. 2. Work is done very close to a lit Bunsen burner. An agar plate seeded with bacteria is prepared. The Petri dish is labeled on the base at the edge with name, date and type of bacterium it is inoculated with. 3. 200ml of bacteria solution is taken by using pipette and withdrawn into Petri dish. The cover is opened as little as possible to avoid contamination by surrounding atmosphere. 4. Agar is taken from incubator and poured into the Petri dish until it reaches a quarter of the Petri dish. 5. The Petri dish containing agar and bacteria is swirled to mix up the combination. The dish is left for a while for the agar to solidify. 6. The forceps is flamed and it is used to pick up a sterile paper disc which is then immersed into antibiotic solution. The lid of the Petri dish is raised and the disc is placed firmly in the center of the agar. Disc of each type of antibiotic: and distilled water as a control are spaced evenly around the dish. 7. The dish is taped securely with two pieces of adhesive tape. The lid of the Petri dish is opened as little as possible. 8. The dish is placed inverted in the incubator in the incubator at the temperature of 30C for 24 hours. 9. At the end of 24 hours, the dish is removed from the incubator. 10. The clear zone around dish is observed without opening the cover. The diameter of the inhibition zones is measured in millimeters.

11. The data is tabulated in Table 1.

Results
Type of antibiotic Diameter (mm) Table 1: table above shows the diameter of clear regions on agar plate for different types of bacteria and a control. 35 22 26 21 Ampicillin Streptomycin Tetracyclin Kanamycin Distilled water 0

40 35 30 25 20 15 10 5 0 ampicillin streptomycin tetracyclin kanamycin diameter of clear zone(mm)

Graph 1: graph above shows the diameter of clear regions on agar plate for different types of bacteria and a control.

Discussion
i. Data analysis

The objective of this experiment is to investigate the effect of different antibiotics on E.Coli bacteria. The manipulated variable is the different type of antibiotic used. The constant variables are humidity, type of bacteria used, concentration of antibiotic and time of incubation. In this experiment, distilled water is used as control which is use to make comparisons with antibiotics. Diameter of clear zone is the responding variable. The diameter is measured using ruler in millimeter unit. Table 1 and graph 1 shows the diameter of inhibition zones on agar plate after incubation. It can be seen that ampicillin shows the largest diameter of inhibition zone. This is followed by tetracylin, which is 26mm, streptomycin, 22mm and kanamycin, 21mm. Disc immersed in distilled water shows zero inhibition zone which shows that there is no antibiotic activity. A clear inhibition zones indicate the absence of bacteria thus showing that the bacteria are killed. I believe that ampicillin is the most effective antibiotic against E.Coli. It is shown by the largest diameter of clear zone measured. This is because ampicillin is a broad spectrum antibiotic which acts as bactericidal. This antibiotic will prevent the formation of cell membrane. Therefore, metabolites leak out thus killing the bacteria. Tetracycline, streptomycin and kanamycin are also effective against E.Coli but not as effective as ampicillin. Tetracyclin acts as protein synthesis inhibitors. It interrupts or prevents the transcription or translation of microbial genes, so protein production is affected. Streptomycin works by attaching to the specific site of bacteriums ribosome and stop it from producing the

right protein. Kanamycin stops the production of essential proteins needed by the bacteria to survive. In conclusion, ampicillin shows the greatest inhibition zones, thus it shows it is the most effective against E.Coli followed by tetracycline, streptomycin and kanamycin.

Validity and reliability

I believe the result obtained for this investigation of the effectiveness of antibiotics are valid reliable. To ensure validity, this experiment which was planned and carried out meticulously had used different types of antibiotics to test the effectiveness of it. So, comparison can be made among the antibiotics as it is tested on one type of bacteria which is E. Coli. This can ensure precision of the results. Besides that, safety precautions and procedures are followed properly. Other than that, Comparison can be made as the experiment was carried out by a large number of students. This would ensure the reliability of the experiment conducted. Furthermore, with advice of lecturer, I can ensure that the result obtained is valid. So, the results obtained are valid and reliable.

Sources of error
One of the sources errors is parallax error while taking measurement of the diameter of the clear zone. It is difficult to determine the exact diameter because horizontal and vertical diameter of the clear zone is different. Besides that, the agar might be contaminated with lots of impurities. Besides that, the apparatus used such as forceps, scissors and beaker which had been sterilized earlier may be contaminated

with bacteria from the air. So, the molten agar which had been poured into the beaker might be contaminated. It is difficult to have a very sterile set up. Error also occurs when discs plate is placed too late into the agar. The extract of antibiotic on the disc would be too dried and its effectiveness on bacteria might lose. Moreover, error is also detected when agar has been solidified .The agar should be in molten state so that the disc can be placed into the agar. If the agar is not in molten state, it would be difficult to insert the disc into the agar. Plus, the antibiotic extract cannot diffuse through the solidified agar and it would be not effective on the bacteria.

Safety precautions
Safety measures are very important during conducting an experiment. Apparatus especially glassware are handled with care. This apparatus are fragile and can break if it not used with care. First aid kit should be placed around the laboratory for easy access to first aid. Laboratory coat is worn during the experiment. This is to prevent any splitting of chemical substance on the skin and cloth. Besides that, rules and regulation in the laboratory should be followed to ensure efficiency of experiment. Moreover, extra care must be taken while pouring the hot agar into the Petri dish. This is important to avoid any injury occur during the cutting process.

Limitations

The first limitation is the contamination of the materials and apparatus used. Bacteria may have entered the agar due to contaminated beaker or forceps used during inserted the disc into the agar. When there is a contact, bacteria from bare hand may contaminate the apparatus hence affected the result obtained. Another limitation is that the inhibition zones are not circular. Therefore, it is difficult to get accurate measurement for the diameter of inhibition zones.

Modification or further work

For modification, replication of experiment can be done. By this, more results can be collected hence comparisons can be made to obtain more reliable data. Plus, any anomalous result or observation can be left out as there are replicates to be observed and compared. Besides that, the inhibition zone can be calculated by measuring its area. This can be determined by using this formula: x diameter x diameter 4 Further investigation can be carried out by test the antibiotics on another gram negative bacteria or gram positive bacteria. This study can be used to show which antibiotic is effective against gram positive bacteria.

Conclusion
Different antibiotics have different effectiveness on bacteria. The most effective antibiotic will show the largest diameter of clear zone. Ampicillin is the most effective antibiotic against E.Coli. This was determined by the largest diameter of clear zone. It is followed by tetracycline, streptomycin and kanamycin.

References
1. http://1492andiblair.deviantart.com/art/e-coli-211581820 2. http://www.lulusoso.com/products/Dimethylamine-Hydrochloride.html 3. www.emedicinehealth.com/antibiotics/article_em.htm 4. www.nlm.nih.gov/medlineplus/antibiotics.html 5. www.absoluteastronomy.com/topics/Streptomycin 6. 2009. Edexcel A2 Biology. 106p. Ann Fullick : Pearson Education Publisher.