BIOLOGY LAB REPORT TITLE: THE EFFECT OF DIFFERENT ANTIBIOTICS ON BACTERIA

NAME CLASS SID

: NUR IZZATI NADHIRAH BT MOKHTAR : 12M10 : 2011214146

LECTURERS NAME: MDM RITA ROHAIZAH SOHARI

OBJECTIVE: To determine which antibiotics are most efficient in combating Gram positive bacteria INTRODUCTION: Antibiotic is a chemical substance produced by microorganism, which has the capacity to inhibit the growth and even destroy bacteria, in dilute solution. Generally, antibiotics are used to kill or inhibit the growth of bacteria. It can be classified as bactericidal and bacteriostatic. Bactericidal antibiotics destroys bacteria whilst it counterparts; the bacteriostatic prevent multiplication of bacteria from undergoing cell division, then the host immune system can destroy the pathogens. Mechanism of action of the antibiotics includes the act of restraining and disturbing the cell growth and division in several ways. The machinery involve the inhibition of bacterial cell wall synthesis, disruption of the cell membrane that will alter permeability leading to cell lysis, hindering of nucleic acid synthesis and replication that prevent cell division. Apart from that, they also restrain the process of protein synthesis, that cause enzymes and other essential proteins are not manufactured. Lastly, antibiotics prevent specific enzyme activities from taking place in the bacterial cell. Tetracycline is so named for their (tetra) hydrocarbon ring. They are known as derivatives of polycyclic napthacene carboxamide. It is widely used in the treatment of infection of the urinary tract and intestine. Besides, it also remain as the preferable choice of treating infection caused by Chlamydia, Rickettsia ( Rocky mountain spotted fever ), Spirochete infection ( syphilis ). There has been uncommon side effect that carried along with the used of this drug that comprises stomach or bowel upset, allergic reactions and very rarely severe headache with vision problem. Mode of action of tetracycline antibiotic is mainly influenced by its role as protein synthesis inhibitors that hold the binding of aminoacyl-tRNA on the A-site of ribosomal RNA. Hence, no new amino acid is joined to the previous polypeptide as the vacant aminoacyl-site has been occupied by the antibiotics. This interrupt the translation of mRNA into protein, as the process is incomplete. Resulting in no enzymes and protein are synthesised and available for cell growth. Ampicilin is a beta-lactam bacteriostatic antibiotic that is part of the amino penicillin family. It can acts as bactericidal in the presence of E.coli bacteria. This drug is used to treat urinary tract infection, Haemophilus influenza, Salmonellosis and Listeria Meningitis. Consuming this medicine can occasionally consequence in reaction that range in severity from a rash to potentially lethal allergic reaction such as anaphylaxis (an extreme allergic reaction to an antigen to which body has become hypersensitive following an earlier exposure). However, compared with other penicillin drugs, it is relatively non-toxic.

The operative mode of ampicilin is the ability to penetrate Gram positive bacteria and some Gram negative bacteria. The presence of amino group helps the drug to penetrate the outer membrane of bacteria. Other than that, it also acts as a competitive inhibitor of the enzyme transpeptidase found on the inner surface of cell surface membrane, which is needed by the bacteria to make their cell wall. This directs the inhibition of cell wall synthesis during asexual reproduction (binary fission) which ultimately leads to cell disintegration. Carbenicillin or carboxybenzylpenicillin, is a bacteriolytic (bacteriostatic) antibiotic belonging to subgroup of the penicillins. The antibiotic is very soluble in water and is acid labile (easily dissociated). It fights against infection in urinary tract from bacteria E.coli, Pseudomonas, Proteus Vulgara and Enterococci. Consuming this drug at high dose can cause bleeding and as applicable in pharmaceutical industry, carbenicillin can cause hypokalemia by promoting potassium loss at the distal convoluted tubule of kidney. The process of combating the bacteria by carbenicillin involve the interference with the final stage of bacterial cell wall synthesis by acylating(introducing an acyl group into a molecule) the penicillin-sensitive transpeptidase emzymes C- terminal domain by opening the lactam ring(an organic compound containg an amide group as part of ring). This prevents the cross-linking of peptidoglycan strands that lead to autolysis of bacteria cells by autolysin enzymes. Bacteria can be classified into gram positive and gram negative, differentiate by the way their cell wall take up gram staining. As for gram positive bacteria that consist of thick layer peptidoglycan containing chemicals such as techoic acid within their net-like structure. The crystal violet stain bind to techoic acid and resist decolouration and leaving the purple blue colour. While, gram negative bacteria have a thinner layer of peptidoglycan with no techoic acid within and the outer membrane made up of layer of lipopolysaccharides. Any crystal violets which does binds is readily decolurised and replaced with red sarfarine; so the cells appear red.

PROBLEM STATEMENT: Which antibiotics are the most effective in combating the gram positive bacteria? NULL HYPOTHESIS: There is no correlation between mechanisms of action of antibiotics on the bacterial activity HYPOTHESIS: The wider the zone that remain clear due to no bacterial growth, the more effective the antibiotic is in preventing growth of bacteria. VARIABLES: Manipulated Type of antibiotics used Ways to control the variables Different types of antibiotics are used including; tetracycline, ampicilin and carbenicillin The radius are measured using metre ruler and the area calculated taking the formula:

Responding

The area of inhibition zone

Constant

The type of bacteria used,

Both gram positive bacteria are tested

PROCEDURE: 1. The working table was sprayed with a disinfectant spray, which is 70% ethanol and wiped with the paper towel. 2. A Bunsen burner was lighted. 3. A Petri dish was labelled with the three types of antibiotics used and the control. Petri dish A was labelled with C (Control), T (Tetracycline), A (Ampicilin) and G (Carbenicillin). Petri dish B was labelled with the same labels. 4. With a micropipette, 100 micro litre of Bacillus subtilis was added into one of the sterile Petri dishes using aseptic technique. The lid of the Petri dishes were only lifted enough to allow the entry of the micropipette. Before that, the mouth of the bottle containing the E.coli was heated with naked flame to ensure that the mouth was sterile. 5. The agar was poured into the Petri dishes until about 3mm of height. However, before pouring the agar into the Petri dishes, the mouth of the jar containing the agar was heated with naked flame from the Bunsen burner to ensure that the mouth was sterile. The Petri dish was covered at all times when nothing is being added into the agar solution.

Figure: The mouth of the jar was heated.

6. The used tip of the micropipette was discarded into the biohazard waste box. 7. The contents in the Petri dishes were shaken gently to ensure that the bacteria and the agar solution mix. 8. Steps 3 to 8 were repeated for a second Petri dish but this time Staphylococcus aureus was used. 9. The agar solutions which contained the bacteria were allowed to solidify with the lids on the Petri dishes. 10. 5% of each antibiotics solution (Tetracycline, Ampicilin and Carbenicillin) was prepared. 11. Using a pair of sterilised forceps, sterilised paper discs were dipped into each antibiotics solution and the paper disc was placed onto the solidified agar. 12. Distilled water was used as a control in the experiment. 13. The Petri dishes were labelled with name, date, bacteria used and the antibiotics. 14. The plates were incubated for 24 hours at 37C. 15. After 24 hours, the plates were observed with the lids of the Petri dishes still intact (without opening the Petri dishes). 16. The diameters of the clear zones around the paper discs (inhibition zones) were measured.

Figure: The diameter of the paper disc together with the clear zones around the paper discs was measured. 17. At the end of experiment, the Petri dishes were disposed into the biohazard waste box. 18. Hands were washed with Dettol disinfectant and water after completing the practical.

RESULTS: Bacteria Staphylococcus aureus Bacillus subtilis Area of inhibition zone( Antibiotics Tetracycline Ampicilin 106.8 94.2 125.7 62.8 ) Carbenicillin 62.8 44.0

Table 1: the area of inhibition zone on gram positive bacteria

Calculation Measuring the area of inhibition of bacterial growth by tetracycline = 2 x r (including paper disc and the inhibition zone) = 2 x (17mm) =106.8 mm2 x r (paper disc)

Graph of area of inhibition (mm2)

Carbenicillin

Ampicilin

bacillius subtilis staphylococcus aureus

Tetracycline

20

40

60

80

100

120

140

Graph 1: Area of Inhibition

DATA ANALYSIS AND EVALUATION: Based on table 1, they are the calculated area of inhibition zone observed in bacteria; Staphylococcus Aureus and Bacillus Subtilis. Both are gram positive bacteria that are treated with three different types of antibiotics including; Tetracycline, Ampicilin and Carbenicillin. The earlier conclusion made which is the wider the zone that remains clear due to no bacteria growth, the more effective the antibiotics are at preventing growth. The results depicted Tetracycline has been very excellent in combating the bacteria with widest inhibition area in both bacteria. As recorded, 106.8mm2 is the clear zone in Staphylococcus Aureus while for Bacillus Subtilis the area is 125.7mm2. Tetracycline is bacteriostatic that bind to the 30S subunit of microbial ribosomes. They inhibit protein synthesis by blocking the attachment of charged aminoacyl-tRNA to the A-site on the ribosomal RNA. Thus they prevent production of new amino acid to the peptide chain. Hence the polypeptide chain is not completed, resulting in no enzyme or protein is encoded. Then multiplication of bacteria is prevented. The trends observed are decline in the potency of antibiotics in ampicilin and carbenicillin. In both bacteria the mechanism of action of ampicilin is largely contributed by the presence of amino group that help drug to penetrate the outer membrane of gram positive bacteria. In fact the existence of ampicilin that acts as competitive inhibitors of enzyme transpeptidase which is required by the bacteria to make their cell wall and eventually will lead to cell lysis. Ampicilin action on gram positive bacteria results in area of inhibition zone of 94.2mm2 in Staphylococcus Aureus and 62.8mm2 in Bacillus subtilis. Carbenicillin antibiotics interferes with the last stage of bacterial cell wall synthesis by preventing the cross linkage of peptidoglycan strands eventually leading to autolysis of bacteria cell by autolysin enzymes. The antibiotic inhibits the division of both gram positive bacteria. Hence the measured area of inhibition is 62.8mm2 in staphylococcus aureus and 44.0mm2 in bacillus subtilis. Here, we can observed that Tetracycline are the most potent and efficient bacteriostatic antibiotics as it prevents the multiplication of bacteria by interrupting the translation of mRNA into protein. However, compared with the rest; ampicilin, carbenicillinthat only invade the membrane of bacteria and this result in slower rate of action in combating these bacteria.

SAFETY PRECAUTION: There are a few safety measures and deliberate acts that we should apply and implemented during this experiment involving bacteria which comprises: In handling bacteria, gloves and proper hand washing must be done before and after the experiment, to avoid infection. The Petri dishes must be disposed of into an appropriate biohazard box, not to ordinary dustbin to prevent spreading of bacteria to the environment. Working around burning flame is encouraged as to ensure the sterile condition is preserved, especially the equipment used, for example the forceps. Students should not open the lid of Petri dishes, to restrain the bacteria from escaping to the surrounding and can lead to infection on human. The apparatus used must be exposed to burning flame to ensure sterile condition secured. (Aseptic technique) LIMITATION AND MODIFICATION: There are a few limitations that cause the result to be inaccurate. The agar solution may be too hot that can kill and cause the enzyme to denature due to elevated temperature rise. To overcome this, the bottle consisting of agar solution must be of within the range of optimum temperature for the bacteria to live by placing it in fixed incubation temperate. The inhibition zone observed is not cleared and some are overlapping with each other since the antibiotic action is very rapid and effective. Making the accuracy of the measured area obtained is inaccurate. In order to overcome this situation, the bigger container should be used and we also can separately place each antibiotic in one Petri dish only.

RELIABITY AND VALIDITY: As to ensure that my result is coherence, concise and dependable, I have compared y results with my other classmates. Their results are as referred below:

Bacteria Staphylococcus aureus Bacillus subtilis

Area of inhibition zone( Antibiotics Tetracycline Ampicilin 106.8 94.2 125.7 62.8

) Carbenicillin 62.8 44.0

Based on the observation and contrasting, I found my result is trusted to be reliable and truth as to support the hypothesis made earlier; the wider the inhibition zone, the more effective the antibiotics in combating the bacteria. This experiment was conducted with reference to the best methods and techniques that are proven to be effective to test for the variables desired. The technique used in this investigation includes aseptic technique that gives protection through favourable sterile condition in the laboratory where we are working on the bacteria. This method provides a valid result as the bacteria is not exposed directly to contaminate with environment. Hence, the bacteria are at its original and pure state at the time of pippeting them onto the Petri dishes.

CONCLUSION: The wider the clear inhibition zone spotted surrounding the paper disc, the more effective the antibiotics used. Thus, hypothesis is accepted.