BIOLOGY LAB REPORT TITLE PREPARED BY I/C NUMBER STUDENT ID GROUP LAB PARTNER LECTURERS NAME PRACTICAL DATE

SUBMISSION DATE : THE EFFECTS OF DIFFERENT ANTIBIOTIC ON BACTERIA : : : : : : : :

Abstract Different antibiotics have different reaction towards different bacteria, thus exhibit different degree of effectiveness towards bacteria. In this experiment, different antibioics was left to soak up by the paper discs. These are then take using a pair of sterilize forceps and pressed onto an agar which has been prepared with bacteria (Bacillus subtilis and Staphylococcus aureus). The area of inhibition of the clear zone around the paper disc was measured and recorded.

Introduction 1. Bacillus subtilis (1) Bacillus subtilis is a Gram-positive, catalase-positive bacterium that commonly found in soil. It is rodshaped, and has the ability to form tough, protective endospore which enables this organism to tolerate extreme environmental condition. B. subtilis has proven highly amenable to genetic manipulation, and has become widely adopted as a model organism for laboratory studies, especially of sporulation, which is a simplified example of cellular differentiation. It is also heavily flagellated, enabling B. subtilis to move quickly in liquids. This organism also often used as the Gram-positive equivalent of Escherichia coli, an extensively studied Gram-negative bacterium.Enzymes produced by B. subtilis are widely used as additives in laundry detergents, commercial production of the Japanese food natto, as well as the similar Korean food cheonggukjang, as a natural fungicidal activity, as an immune stimulatory agent to aid treatment of gastrointestinal and urinary tract diseases and help in speeding corm growth and increasing stigma biomass yield.

Figure 1: Reproductive cycle of B.subtilis (2)

Figure 2 :B.subtilis (3)

2. Staphylococcus Aureus(4) S. aureus is a facultatively anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope, and has large, round, golden-yellow colonies, often with haemolysis, when grown on blood agar plates. S. aureus is catalase-positive, so is able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci. A small percentage of S. aureus can be differentiated from most other staphylococci by the coagulase test: S. aureus is primarily coagulasepositive that causes clot formation, whereas most other Staphylococcus species are coagulasenegative. However, while the majority of S. aureus are coagulase-positive, some may be atypical in that they do not produce coagulase. Strains are responsible for food poisoning through the production of an enterotoxin, and pathogenicity is also associated with coagulase positivity. S. aureus may occur as a commensal on skin; it also occurs in the nose frequently and the throat less commonly. S. aureus can infect other tissues when barriers have been breached (e.g., skin or mucosal lining). This leads to furuncles and carbuncles. In infants, S. aureus infection can cause a severe disease - staphylococcal scalded skin syndrome (SSSS).

Figure 3: Staphylococcus Aureus(5)

3. Antibiotics(6) i. Mechanism of Antibiotic Action

ii.

Mechanism of Resistance

iii.

How antibiotic resistance genes are transferred among bacterial cells

4. Tetracycline(7) Tetracyclines are medicines that kill certain infection-causing microorganisms. Tetracyclines are called "broad-spectrum" antibiotics, because they can be used to treat a wide variety of infections. Physicians may prescribe these drugs to treat eye infections, pneumonia, gonorrhea, Rocky Mountain spotted fever, urinary tract infections, certain bacteria that could be used in biological weapons, and other infections caused by bacteria. The medicine is also used to treat acne. The tetracycline will not work for colds, flu, and other infections caused by viruses. Tetracyclines are generally a lowcost alternative among antibiotics. Tetracycline medicine works best when taken on an empty stomach, with a full glass of water. The water will help prevent irritation of the stomach. If the medicine still causes stomach upset, it may be necessary to take it with food. Milk or dairy products should be avoided within one to two hours of taking tetracycline (except doxycycline and minocycline). The most common side effects using tetracycline are stomach cramps or a burning sensation in the stomach, mild diarrhea, nausea, or vomiting. These problems usually go away as the body adjusts to the drug and do not require medical treatment. Less common side effects, such as sore mouth or tongue and itching of the rectal or genital areas also may occur and do not need medical attention unless they do not go away or they are bothersome.

Figure 4: Tetracycline(7)

Figure 5: Action of Teracyclin(7)

5. Ampicillin(8) Ampicillin is a beta-lactam antibiotic that is part of the aminopenicillin family and is roughly equivalent to its successor, amoxicillin in terms of spectrum and level of activity. It can sometimes result in reactions that range in severity from a rash to potentially lethal allergic reactions such as anaphylaxis. However, as with other penicillin drugs, it is relatively non-toxic and adverse effects of a serious nature are encountered only rarely. Belonging to the penicillin group of beta-lactam antibiotics, ampicillin is able to penetrate Grampositive and some Gram-negative bacteria. It differs from penicillin only by the presence of an amino group. That amino group helps the drug penetrate the outer membrane of gram-negative bacteria. Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell lysis. Ampicillin has received FDA approval for its mechanism of action. Ampicillin are used to treat urinary tract infections, otitis media, Haemophilus influenzae, salmonellosis and Listeria meningitis. It is used with flucloxacillin in the combination antibiotic cofluampicil for empiric treatment of cellulitis; providing cover against Group A streptococcal infection whilst the flucloxacillin acts against the Staphylococcus aureus bacterium.

Figure 6: Ampicillin(8)

6. Gentamicin(9) Gentamicin is an amino glycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms.. It is synthesized by Micromonospora, a genus of Gram-positive bacteria widely present in the environment (water and soil). Gentamicin is a bactericidal antibiotic that works by binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis. Like all amino glycosides, when gentamicin is given orally, it is not systemically active. This is because it is not absorbed to any appreciable extent from the small intestine. It is administered intravenously, intramuscularly or topically to treat infections. It appears to be completely eliminated unchanged in the urine. Urine must be collected for many days to recover all of a given dose because the drug binds avidly to certain tissues. E. coli has shown some resistance to gentamicin, despite being Gram-negative. Reluctance to use gentamicin for empirical therapy has led to increased use of alternative broad-spectrum antibiotics, which some experts suggest has led to the prevalence of antibiotic-resistant bacterial infections by Golden Staph and other so-called "superbugs. Gentamicin is one of the few heat-stable antibiotics that remain active even after autoclaving, which makes it particularly useful in the preparation of some microbiological growth media. It is used during orthopaedic surgery when high temperatures are required for the setting of cements. These amino glycosides are toxic to the sensory cells of the ear, but they vary greatly in their relative effects on hearing versus balance. Gentamicin is a vestibulotoxin, and can cause permanent loss of equilibrioception, but there are well documented cases in which gentamicin completely destroyed the vestibular apparatus after three to five days. Gentamicin can also be highly nephrotoxic, particularly if multiple doses accumulate over a course of treatment. For this reason gentamicin is usually dosed by body weight.

Figure 7: Gentamicin(9)

7. Streptomysin(10) Streptomycin is an antibiotic drug, the first of a class of drugs called amino glycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It is derived from the actinobacterium Streptomyces griseus. Streptomycin is a bactericidal antibiotic. Streptomycin cannot be given orally, but must be administered by regular intramuscular injections. An adverse effect of this medicine is ototoxicity, nephrotoxicity, fetal auditory toxicity and neuromuscular paralysis. Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells through mechanisms that are still not understood. Speculation on this mechanism indicates that the binding of the molecule to the 30S subunit interferes with 50S subunit association with the mRNA strand. This results in an unstable ribosomal-mRNA complex, leading to a frameshift mutation and defective protein synthesis; leading to cell death. Humans have structurally different ribosomes from bacteria, thereby allowing the selectivity of this antibiotic for bacteria. However at low concentrations Streptomycin only inhibits growth of the bacteria by inducing prokaryotic ribosomes to misread mRNA. Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-negative bacteria, and is therefore a useful broad-spectrum antibiotic.

Figure 8: Streptomycin(10)

Objective To investigate the effect of different antibiotics on bacteria. Problem Statement Which antibiotic is most effective against bacteria? Hypothesis Different antibiotic has different effectiveness towards bacteria. The ingredient disc that has the biggest clear zone around the disc shows that it is able to inhibit bacterial growth near it thus, has the highest effectiveness towards bacteria. It is predicted that tetracycline has the greatest inhibition zone ampicillin, gentamicin and streptomysin..

Apparatus Sterile paper discs, micropipette with sterile tips, sterile Petri dish, sterile forceps, tape, marker pen, Bunsen burner, vortex machine, laminar flow chamber, small beakers, cellophane tape, sanitary hand wash, paper towels.

Materials Agar warmed at 60C, distilled water, Staphylococcus Aureus and Bacillus subtilis bacterial broth in sterile bijou bottles, tetracycline, ampicillin, gentamycin and streptomysin antibiotics in sterile bijou bottle.

Variable : Types of Variables Manipulated Variable: Type of bacteria and antibiotics used Use different types of bacterial cultures Ways to control the variables

(Staphylococcus Aureus and Bacillus subtilis) on different type of antibiotics namely tetracycline, ampicillin, gentamycin and streptomysin.

Responding Variables:

Area of the white zone

The radius clear zone calculated was measured using a ruler after 24 hours and by using formula area = r2, area of inhibition zone was calculated.

Fixed Variables: Volume of bacterial broth used About 200L of bacterial broth was used in all samples. Temperature at which agar was kept Liquidified agar was kept at 60C and kept back into incubator after used.

Procedure a) Preparing the agar plates 1. Hands were washed using disinfectant before the experiment carried out. 2. 2 sterile Petri dishes were labeled with respective name of bacteria and numbers were written according to the paper discs (immersed with antibiotics) on the back of Petri dish. 3. Using aseptic technique, 200L of the bacterial broth ( Staphylococcus Aureus and Bacillus subtilis) was transferred in sterile condition inside a laminar flow chamber (condition inside laminar flow chamber are very much sterile, clean and free from bacteria). 4. Using last finger, the bijou bottle containing bacterial broth was opened. The mouth of bijou bottle was then flamed with Bunsen burner to kill any pathogens that may present. 5. The bacterial broth was then pipette using micropipette that has been calibr ated to 200L. The knob of the micropipette was gently pressed until the first pressure exerted felt and released inside the bijou bottle (in the middle of the bacterial broth solution) so that no air bubble will be formed. 6. The mouth of bijou bottle flamed again before closed to avoid contamination. 7. The bacterial broth was dispensed into a Petri dish (lid of Petri dish only lifted enough to allow entry of the pipette tip) according to their label and closed quickly by putting the lid onto the Petri dish to avoid contact with air too long. 8. The tip of micropipette was ejected into the beaker of disinfectant and a new sterile one was used for another time. 9. Step 2 to 7 was repeated for each bacterium (Staphylococcus Aureus and Bacillus subtilis). 10. The agar solution that was kept at 60C to maintain its liquid form was also prepared using aseptic technique. 11. Using hand glove, the bottle containing the agar was taken out from vortex machine, opened and the agar was poured into the Petri dishes until enough to cover the bacteria (around 18-20 ml). 12. Both bacterial broth and agar was then gently mixed together by swirling the Petri dish in all direction. This has to be done to ensure thorough mixing of both components so that the bacteria will grow at a similar rate at all areas of the agar.

b) Applying the antibiotics 1. Four types of antibiotics were chosen namely tetracycline, ampicillin, gentamycin and streptomysin. 2. Two sterile paper discs were placed in each small beaker containing antibiotics using a pair of sterilized forceps. Then, they were taken out and left to dry on another Petri dish inside the laminar flow chamber. 3. After all the paper discs have been prepared, they were then placed onto the prepared agar plates according to their respective letters (table below) using a pair of sterilized forceps (different forceps used for each solution to prevent mixture of antibiotics).

Labels A B C D E

Staphylococcus Aureus Tetracycline Ampicillin Distilled water Gentamycin Streptomysin

Bacillus subtilis Tetracycline Ampicillin Distilled water Gentamycin Streptomysin

4.

The lid of Petri dishes were only lifted enough to place the paper discs.

5. The paper discs were pressed gently on the solidified agar to prevent them from fall of when the Petri dish is inverted. Paper discs that were soaked in only distilled water were also placed on the agar (act as control). 6. The Petri dishes were closed and cellophane tape was used to tape it in side ways to prevent growth of anaerobic bacteria and enable entering of oxygen for the bacteria to respire. 7. The Petri dish containing agar and paper discs with antibiotics were then inverted and placed into incubator and left for 24 hours to enable bacterial growth. Observations were done on the next day and the area of clear zone (area of inhibition of bacterial growth) was calculated.

Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any chemical substance or bacteria culture. Hands need to be thoroughly washed before and after performing the experiment. This is to avoid ourselves from getting infected from any of the microorganism. Since only bare hands were used in handling the bacteria, thus it is important to maintain a minimal contact, such as using only the last finger in opening the bijou bottle. Furthermore, the glassware such as beaker should be handled with full care because they are fragile. The apparatus such as forceps is also sterilized to prevent infection of microorganism. After using all samples and apparatus at the end of experiment, they should be discarded properly and returned back to their places to avoid injuries and unnecessary accidents that may result fatal results.

Risk Assessment The tip of the micropipette has to be changed each time different bacteria used to avoid contamination. All sterile apparatus such as sterile forceps were used to avoid contaminations. The preparation of agar and placing the paper discs that were soaked in antibiotics were carried out in laminar flow chamber to avoid contamination too. Hot agar is used while it is still in its liquid form and it is advisable to use it immediately after it has been taken out from the vortex machine. This is to avoid the agar from solidifying too fast even before the bacteria were mixed which may affect the result later. The agar is put inside the vortex machine as soon as it is finish used to prevent agar solidification that may contribute to wastage. After placing the bacteria in the Petri dishes (done at fast pace) to avoid long exposure to open air, the agar solution and bacteria needs to be mixed thoroughly as this will affect bacterial growth across the agar.

Results Type of antibiotics used Type of Bacteria Staphylococcus Aureus Radius r 1.20 3.142 X (1.20) Area of inhibition of bacterial growth (cm) Bacillus subtilis Radius r 1.00 3.142 X (1.00) Area of inhibition of bacterial growth (cm) 3.1420 No change No change 1.20 3.142 X (1.20) 4.5245 No change 4.5245 No change No change Tetracycline (A) Ampicillin (B) Distilled water (control) (C) 1.10 3.142 X (1.10) 3.8018 1.00 3.142 X (1.00) 3.1420 Gentamicin (D) Streptomysin (E)

Table 1 : Area of bacterial growth respective to the antbiotics

Data Analysis

Table 1 above show the area of clear zones surrounding the paper discs that were soaked into respective antibiotics. This data will be analysed according to each type of bacteria namely Staphylococcus Aureus and Bacillus subtilis. Both bacteria have shown the same results with distilled water that act as a control. For antibiotics, different results were obtained. Both bacteria didnt react with ampicillin. This is may be due to some limitations that may occurred during usage and handling way of ampicillin. For Staphylococcus Aureus, area of clear zone is bigger in tetracycline (4.5245cm). Gentamicin has the second biggest clear zone at about 3.8018 cm and followed by streptomysin with 3.1420 cm. Overall it can be concluded that the antibiotic that exhibit highest antimicrobial property is tetracycline, gentamicin and lastly followed by streptomysin. Bacillus subtilis also has reacted with only two antibiotics. Inhibition area of gentamicin (4.5245cm) is the highest among the all antibiotics, followed by tetracycline (3.142cm). Thus, it can be concluded that gentamicin has the highest antibacterial property against Bacillus subtilis, followed by tetracycline. It can be seen that no area of clear zone is noted for ampicillin in both Petri dishes that contain different microbial broth. For Bacillus subtilis, it didnt react with streptomysin. This may arise due to some limitation in the handling style. Since Staphylococcus Aureus has reacted with three antibiotics than Bacillus subtilis that has only reacted with two antibiotics this shows that Staphylococcus Aureus has the greatest microbial properties towards antibiotics.

DISCUSSION From the result obtained, it can be seen that different antibiotic exhibit different antimicrobial properties. According to the hypothesis, highest antimicrobial activity is exhibited by tetracyclin, followed by ampicillin, gentamicin and streptomysin.. Both bacteria showed reaction towards both antibiotics named tetracycline and gentamicin and no reaction noted for ampicillin and distilled water. For Staphylococcus Aureus, area of clear zone is bigger in tetracycline (4.5245cm) followed by gentamicin has the second biggest clear zone at about 3.8018 cm and streptomysin with 3.1420 cm. Thus, it can be concluded that the antibiotic that exhibit highest antimicrobial property is tetracycline, gentamicin and lastly followed by streptomysin. For Bacillus subtilis ,it has reacted with only two antibiotics :Gentamicin (4.5245cm) has the highest inhibition zone among the all antibiotics, followed by tetracycline (3.142cm). So, it can be concluded that gentamicin has the highest antibacterial property against Bacillus subtilis, followed by tetracycline. Since Staphylococcus Aureus has reacted with three antibiotics than Bacillus subtilis that has only reacted with two antibiotics this shows that Staphylococcus Aureus has the greatest microbial properties towards antibiotics. The results were for sure having some limitations during the experiment. Some of the antibiotics are milky due to uncontrolled division and the same applied for the bacterial broths. The Staphylococcus Aureus and Bacillus subtilis are both positive-gram bacteria, thus they are easily treated with antibiotics. This leads to larger inhibition one in both Petri dishes.

Limitations There are several limitations that have been identified throughout this experiment. The whole preparation wasnt done inside the laminar flow chamber except for some crucial steps may cause contamination by bacteria in air thus, contributing towards reduction in result accuracy. Bacterial culture that was prepared left for more than 24 hours. This bacterial culture actually should sub cultured after 24 hours to prevent the concentration of bacterial culture become too concentrated. And since the bacteria left for too long, the bacteria may enter lag phace or stationary phase which will affect the growth of bacteria in agar. The paper discs that were used werent sterile properly and some of the apparatus such as forceps were not in sterile condition which later affects the result. Forceps should be sterilized by dipping them in alcohol and burn it in Bunsen burners flame. But due to safety of the students, this step wasnt carried out, thus act as limiting factor for the result. Sources of errors Several sources of error in this experiment were identified and steps were taken to minimize these errors to make the result more accurate. The Petri dish with bacterial broth and agar solution were kept inside laminar flow chamber instead of being left outside. This is to avoid contamination of bacteria that may present in air. When the paper discs were placed onto the agar, the lid of Petri dish was only lift up enough to let the forceps to place the discs. This is to, prevent contamination of other bacteria. The Petri dish may open when it is inverted before placed into incubator. This was prevented by taping the sides of Petri dish. The Petri dishes were not taped all round as it will encourage growth of anaerobic bacteria, some which may be harmful and also exclude oxygen needed for growth of bacteria in the agar.

Conclusion Theoretically, each and every antibiotics has its own strength of antimicrobial property in order to fight against bacterial infection. Same goes to the bacteria. Different bacteria may have different effect towards the antimicrobial actions .All of this depend on its resistance towards the chemical it encounter. From this experiment, it can be concluded that both tetracycline and gentamicin has the highest antimicrobial property toward both Staphylococcus Aureus and Bacillus subtilis. Thus, the hypothesis is accepted.

Further Investigation Another experiment can be carried out using negative gram bacteria. It is also possible to use other antibiotics. References 1. Wikipedia Foundation. Last modified on 2012. Bacillus subtilis. Available from http://en.wikipedia.org/wiki/Bacillus_subtilis. Accessed on 17th August 2012 2. http://www.devbio.biology.gatech.edu/?page_id=15. Accessed on 17th August 2012 3. http://www.sciencephoto.com/media/11211/enlarge. Accessed on 17th August 2012 4. Todars online Text book of Bacteriology. Last modified on 2012. Staphylococcus. Available from http://textbookofbacteriology.net/staph.html. Accessed on 17th August 2012 5. http://www.microbeworld.org/index.php?option=com_jlibrary&view=article&id=6433. Accessed on 17th August 2012 6. http://www.wiley.com/college/pratt/0471393878/student/activities/bacterial_drug_resistance/in dex.html. Accessed on 17th August 2012 7. The free dictionary. Last modified on 2012. Tetracyclines. Available from http://medicaldictionary.thefreedictionary.com/Tetracycline+antibiotics. Accessed on 17th August 2012 8. The free dictionary. Last modified on 2012. Ampicillin. Available from http://www.antibioticslist.com/ampicillin.html. Accessed on 17th August 2012 9. The free dictionary. Last modified on 2012. Gentamicin. Available from http://en.citizendium.org/wiki/gentamicin. Accessed on 17th August 2012 10. The free dictionary. Last modified on 2012. Sreptomysin. Available from http://www.rsc.org/Publishing/Journals/OB/article.asp?Type=Issue&Journalcode=OB&Issue= 19&SubYear=2003&Volume=1&Page=0&GA=on. Accessed on 7th April 2012