UNIVERSITI TEKNOLOGI MARA INTERNATIONAL EDUCATION CENTRE (INTEC)

NAME

CLASS : DATE OF EXPERIMENT TITLE : : 5 AUGUST 2010.

THE EFFECT OF ANTIBIOTICS ON BACTERIA.

Abstract This experiment is to investigate the effect of different antibiotics on bacteria. The manipulated variable is the types of antibiotics used, the responding variable is the areas of the inhibition zones whereas the constant variables are the concentrations of antibiotic solutions, incubation temperature, the type of growth medium (agar) and the size of the antibiotic-impregnated paper discs. All the apparatus has been treated with aseptic methods and both the bacteria, Staphylococcus aureus and E.coli were grown on growth medium in separate Petri dishes with antibiotic-impregnated paper discs placed on different positions. Tetracycline is observed to give the biggest inhibition zone area on Staphylococcus aureus, and streptomycin gives the biggest inhibition zone area on E.coli. In conclusion, tetracycline works on both Staphylococcus aureus and E.coli, whereas streptomycin is much more effective against E.coli than Staphylococcus aureus.

Objective Hypothesis

To investigate the effect of different antibiotics on bacteria.

Tetracycline is effective against both Staphylococcus Aureus and E.coli, while streptomycin is only effective against E.coli.

Variables :
Manipulated variable Responding variable Fixed variable : : : Types of antibiotics. Areas of inhibition zones. Concentrations of antibiotic solutions, incubation temperature, the type of growth medium (agar), the size of the antibiotic-impregnated paper discs.

Introduction :
Bacteria
Bacteria are sorted into two groups: helpful bacteria and harmful bacteria. Some helpful bacteria live in human intestines and warm-blooded animals. These bacteria help the digestion and destroy harmful organisms. Intestinal bacteria, including the bacteria Escherichia coli, also produce vitamins needed by the human body, and help decompose (break down) dead organisms and animal wastes into chemical elements. Other bacteria help change chemical elements into forms that can be used by plants and animals. Harmful bacteria prevent the body form functioning properly, destroying healthy cells. These harmful bacteria enter the body through natural openings such as the nasal passage, mouth, and openings in the skin. Diseases that bacteria have introduced to humans are cholera, gonorrhea, leprosy, pneumonia, syphilis, typhoid fever, and whooping cough. Bacteria are absolutely necessary for all life on the human ecosystem. Without bacteria, there would be no life, on earth. Although it is a good thing that most bacteria die-out. Bacteria are single-celled organisms. A single bacterium cell like E. coli for example, divides every twenty minutes. At this rate, in only 43 hours there would be enough E. coli to occupy the entire volume of the earth (1,090,000,000,000,000,000,000 cubic meters)! In 45 hours these bacteria would weigh as much as the earth 6,600,000,000,000,000,000,000 tons! But most bacterial cells die because of the enormous competition for food, and other tiny organisms, which produce substances (antibiotics) that kill them. Bacteria are often classified on the basis of their physical shapes. Bacteria can be spherical (cocci), rod-shaped (bacilli), or corkscrew (spirochetes). Another classification system divides bacteria into gram-negative or gram-positive according tot he composition of their sell walls, a distinction identified by a staining technique call the gram strain. Scientists also classify bacteria according to whether or not they require oxygen to survive. Bacteria that require oxygen are called aerobic bacteria, or aerobes. Bacteria that

live without oxygen are called anaerobic bacteria, or anaerobes. Like all cells, bacteria contain genetic material known as deoxyribonucleic acid (DNA). Bacterial DNA is not enclosed in a nucleus, as is the DNA of eukaryotic cells. Like eukaryotic cells, bacteria have ribosomes, structures active in protein synthesis, but they are smaller and have a slightly different molecular structure.

Escherichia Coli
E. coli, the most significant species in the genus Escherichia, is recognized as an important potential pathogen in humans. Being a gram-negative bacillus, it is a common isolate from the colon flora. On most occasions it may become visible as a non-lactosefermenter or as a mucoid colony, E. coli usually produces a dry, pink (lactose positive) colony with a surrounding pink area of precipitated bile salts on MacConkey agar. A large amount of E. coli strains are motile and generally have both sex pili and adhesive fimbriae. The presence of E. coli and other kinds of bacteria within our intestines is necessary for us to develop and operate properly, and for us to remain healthy. Our bodies are dependent on E. coli for the development of the vitamins K and B-complex. Although most strains are harmless, several are known to produce toxins that can cause diarrheal side effects. The E. coli organism also possesses O, H, and K antigens. E. coli, first described by Theodore Esherich in 1885 was considered a non-harmful member of the colon flora. Since then, E. coli has been associated with a wide variety of diseases and infections, including meningeal (predominantly in the newborn), gastrointestinal, urinary tract, wound, and bacteremic infections in all age groups. E. coli may cause numerous types of diarrheal illnesses. There are five major categories of diarrheogenic E. coli. These include the following: Enteropathogenic (EPEC) Enterotoxigenic (ETEC) Enteroinvasive (EIEC) Enterohemorrhagic (EHEC serotype O157:H7 Enteroadherent (EAEC)

Figure 1 : Escherichia Coli [http://lacocinadebender.com/wp-content/uploads/2008/10/ecoli-0-157-3.jpg] Enteropathogenic E. Coli The enteropathogenic E. coli strain has been recognized to cause infantile diarrhea since the 1940s. Particular O serogroups of EPEC were acknowledged in the late 60s and 70s as a cause of diarrhea, but only particular groups of H types within each serogroup were connected to the intestinal infections. Studies in 1978 now show that EPEC strains cause distinct diarrhea. Diarrheal outbreaks due to EPEC have occurred in hospital nurseries and day care centers. Cases in adults are rarely seen. Case characterization is established by the presence of low-grade fever, malaise, vomiting, and diarrhea. Contaminated stools contain large amounts of mucus, but gross blood in not usually present. When severe diarrhea in children younger than one year, infection with EPEC should be assumed. Enterotoxigenic E. coli Diarrhea of infants and adults in tropical and subtropical climates, especially in developing countries, strains of enterotoxigenic (ETEC) should be the suspected cause. In the United States and other developed countries, ETEC diarrhea, sometimes referred to as travelers diarrhea, is the most common cause of diarrheal diseases. The ETEC infection is commonly acquired by consuming infected food or water. The major contributing factors in the spread and transmission of the disease include poor hygiene, inadequate sources of drinking water, and lack of proper sanitation. 106 to 1010

organisms are necessary to initiate disease in an immunocompetent host. Various protective mechanisms include stomach acidity have been described as inhibiting colonization and initiation of disease. Which means those who are suffering from achlorhydria are at greater risk of inhibiting the disease than those without achlorhydria. Colonization of ETEC begins near the small intestine. Once established enterotoxigenic strains of E. coli release into the small intestine. Usually the disease caused by ETEC is characterized by non-bloody, watery diarrhea, nausea, abdominal cramps, and low-grade fever. To date there is no evidence of mucosal penetration or invasion. The infirmity may last from one day up to five days. Enteroinvasive E. coli Strains of Enteroinvasive E. coli (EIEC) are very different from the strains of EPEC and ETEC. EIEC strains create dysentery, with direct penetration, invasion, and destruction of the intestinal mucosa. This diarrheal sickness is similar to that produced by Shigella. The EIEC infections are found in adults and children. Clinical infection is characterized by fever, severe abdominal cramps, malaise, and watery diarrhea (stools containing pus, mucus, and blood). While EIEC and Shigella have been discovered to be similar in morphology and in clinical presentation, the infective dose of EIEC necessary to produce disease in much higher than that of Shigella. Enterohemorrhagic E. coli In 1982, the O157:H7 strain of E. coli was first recognized during an outbreak of hemorrhagic diarrhea and colitis. Strain serotype O157:H7 of the enterohemorrhagic E. coli (EHEC) has since then been associated with hemorrhagic diarrhea, colitis, and hemolyticuremic syndrome (HUS). HUS is characterized by low platelet count, hemolytic anemia, and kidney failure. Illness brought on by EHEC is characterized by watery diarrhea that progresses to bloody diarrhea and cramping abdominal pain, with low-grade fever or no fever at all. The stool of a contaminated person contains no leukcytes, which differentiates it from Shigella dysentery or EIEC strain infection. The infection is potentially fatal to young children and the elderly. Meats, such as

undercooked hamburger, unpasteurized milk, and apple cider, have been known to spread the infection. Enteroadherent E. coli Enteroadherent E. coli, most recently spoken as of enteroaggregatice E. coli (EAggEC), causes diarrhea by adhering to the mucosal surface of the intestine. The following symptoms are the result to a EaggEC infection; watery diarrhea, vomiting dehydration, and occasionally abdominal pain.

Staphylococcus
Staphylococcus is a genus of Gram-positive bacteria. Under the microscope they appear round (cocci), and form in grape-like clusters. The Staphylococcus genus includes thirty-two species and eight sub-species. Most are harmless and reside normally on the skin and mucous membranes of humans and other organisms. Found worldwide, they are a small component of soil microbial flora.

Figure 2 : Staphylococcus
[http://en.academic.ru/pictures/enwiki/83/Staphylococcus_aureus_01.jpg]

Staphylococcus aureus
Staphylococcus aureus is a facultatively anaerobic, gram-positive coccus and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. About 20% of the human population are long-term carriers of S.

aureus. The carotenoid pigment staphyloxanthin is responsible for S. aureus' characteristic golden colour, which may be seen in colonies of the organism. This pigment acts as a virulence factor with an antioxidant action that helps the microbe evade death byreactive oxygen species used by the host immune system. Staph organisms which lack the pigment are more easily killed by host defenses. S. aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis,endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections. Abbreviated to S. aureus or Staph aureus in medical literature, S. aureus should not be confused with the similarly named and similarly dangerous (and also medically relevant) species of the genus Streptococcus.

Antibiotics
Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics cure disease by killing or injuring bacteria. The first antibiotic was penicillin, discovered accidentally from a mold culture. Today, over 100 different antibiotics are available to doctors to cure minor discomforts as well as life-threatening infections. Although antibiotics are useful in a wide variety of infections, it is important to realize that antibiotics only treat bacterial infections. Antibiotics are useless against viral infections (for example, the common cold) and fungal infections (such as ringworm).

Ampicillin
Ampicillin , a penicillin -type antibiotic that is effective against both gramnegative microorganisms and gram-positive microorganisms such as Escherichia coli. It

is often used in the treatment of urinary tract infections, but resistant organisms are increasingly common (see drug resistance ). Like other penicillin antibiotics, ampicillin acts against bacteria by inhibiting the synthesis of bacterial cell wall components.

Tetracycline
Tetracycline is a broad-spectrum polyketide antibiotic produced by the Streptomyces genus ofActinobacteria, indicated for use against many bacterial infections. It is a protein synthesis inhibitor. It is commonly used to treat acnetoday, and, more recently, rosacea, and played a historical role in reducing the incidence of mortality because of cholera. It is sold under the brand names Sumycin, Terramycin, Tetracyn, and Panmycin, among others. Actisite is a thread-like fiber form, used in dental applications. It is also used to produce several semi-synthetic derivatives, which together are known as the tetracycline antibiotics. Tetracyclines work by binding the 30S ribosomal subunit and through an interaction with 16S rRNA, they prevent the docking of amino-acylated tRNA. Resistance to tetracyclines can arise through drug efflux, ribosomal protection proteins, 16S rRNA mutation, and drug inactivation through the action of a monooxygenase.

Streptomycin
Streptomycin, antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria, including species resistant to other antibiotics, e.g., some streptococci, penicillin-resistant staphylococci, and bacteria of the genera Proteus and Pseudomonas. Originally isolated by Selman A. Waksman and Albert Schatz in 1947, streptomycin is effective against tubercle bacilli and is a mainstay of tuberculosis therapy. Because streptomycin-resistant tubercle bacilli emerge during treatment, the antibiotic is usually used in combination with one or more of the drugs isoniazid, ethambutol, and aminosalicylic acid. Streptomycin acts by inhibiting protein synthesis and damaging cell membranes in susceptible microorganisms. Possible side effects include injury to the kidneys and nerve damage that can result in dizziness and deafness.

Autoclave
An autoclave is a device to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 C or more, typically for 15 to 20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879, although a precursor known as the steam digester was created by Denis Papin in 1679. The name comes from Greek auto, ultimately meaning self, and Latin clavis meaning key a self-locking device. A notable growing application of autoclaves is in the pre-disposal treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in this category largely operate under the same principles as the original autoclave in that they are able to neutralize potentially infectious agents by utilizing pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without any pressure vessels to sterilize culture media, rubber material, gowns, dressing, gloves etc. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven. For all-glass syringes, a hot air oven is a better sterilizing method.

Figure 3 : Autoclave

Material and apparatus

autoclaved forceps, paper discs.

Bench spray of disinfectant (ethanol), Petri dishes, handwash, paper towels, marker pen,

Procedure

1. Hands were washed thoroughly with handwash. 2. The bench was sprayed thoroughly with ethanol prepared by the lab assistant. It was left for a few minutes, then wiped with a paper towel. 3. Three 50ml beakers containing 3 different types of antibiotics, namely ampicillin, tetracycline and streptomycin were obtained. Another 50ml beaker filled with distilled water (as control) was also obtained. 4. A autoclaved and sterilized Petri dish was obtained. The lid was kept close to prevent contamination. 5. The Petri dish was then labeled on the base at the edge by using a marker pen with initials, the date and the type of bacterium it was inoculated with, which in this case, was E.coli. 6. The base of the Petri dish was then labeled at 4 different areas with T (tetracycline), S (streptomycin), A (ampicillin), C (control, distilled water). 7. A micropipette was calibrated to 200 microlitres. 8. Teat of micropipette was placed at the edge of the micropipette. The teat was not touched to avoid contamination. 9. By using the micropipette, 200ml of E.coli was transferred from a small glass bottle to the Petri dish. 10. The teat was then expelled into a bio-hazard waste bin, which the used teats can be recycled. 11. Molten agar solution in a conical flask was taken out from the oven. 12. The mouth of the conical flask was heated by using Bunsen burner to sterilise it.

Figure 4 : The mouth of the conical flask was flamed to sterilize it. 13. The molten agar was poured into the Petri dish until it was one-third full. 14. The Petri dish was then swirled very gently in a motion shaped 8 to mix the solution and E.coli evenly. 15. The Petri dish was then left for 10 minutes for the molten agar to solidify. Meanwhile, the lid was left opened slightly to avoid the water vapour from dripping back into the Petri dish. 16. Steps 4 to 15 were repeated by replacing E.coli with Staphylococcus aureus. 17. The forceps were flamed and it was used to pick up a paper disc and the disc was dipped into the solution of antibiotic containing Tetracycline. 18. The paper disc was placed on the marked T region in both of the Petri dishes. 19. Steps 17 and 18 were repeated by replacing tetracycline with distilled water, ampicillin and streptomycin at different marked regions. 20. Both the Petri dishes were placed upside down inside an incubator for the growth of bacteria. They were left inside for a day. 21. The next day, the Petri dishes were taken out from the incubator and the diameter of each inhibition zone was measured using a ruler. Three measurements are taken and the average diameter were then calculated. 22. Hands were washed with handwash again before leaving the laboratory. 23. The readings were tabulated and two bar charts were plotted.

Results

Tabulation of data

Type of bacteria Staphylococc us aureus

Diameter of Inhibition Zones in Distilled Water / cm 0.0 (contamination observed) 0.0 0.0 0.0

Average Diamete r / cm 0.0

Area / cm2 0.0

Escherichia coli

0.0

0.0

0.0

0.0

Table 1 : Area of the inhibition zones in distilled water. Type of bacteria Staphylococc us aureus Escherichia coli Diameter of Inhibition Zones in Ampicillin / cm 4.0 0.8 3.8 0.9 4.1 1.0 Average Diamete r / cm 4.1 1.0 Area / cm2 52.8 3.1

Table 2 : Area of the inhibition zones in ampicillin. Type of bacteria Staphylococc us aureus Escherichia coli Diameter of Inhibition Zones in Streptomycin / cm 2.3 1.9 2.3 4.1 2.4 3.9 Average Diamete r / cm 2.3 4.0 Area / cm2 16.6 50.3

Table 3 : Area of the inhibition zones in streptomycin.

Type of bacteria Staphylococc us aureus Escherichia coli

Diameter of Inhibition Zones in Tetracycline / cm 4.2 3.2 4.1 3.2 4.3 3.2

Average Diamete r / cm 4.2 3.2

Area / cm2 55.4 32.2

Table 4 : Area of the inhibition zones in tetracycline.

Interpretation of data

Areas of Inhibition Zones Against Types of Antibiotics For Bacterium Staphylococcus Aureus

60 50 40
Areas of Inhibition Zones / cm

30 20 10 0

Control

Am pilicillin

Tetracycline

Streptom ycin

Types of Antibiotics

Chart 1 : Areas of inhibition zones against types of antibiotics (Staphylococcus aureus).

Areas of Inhibition Zones Against Types of Antibiotics For Bacterium Escherichia Coli

60 50 40
Areas of Inhibition Zones / cm

30 20 10 0

Control

Am pilicillin

Tetracycline

Streptom ycin

Types of Antibiotics

Chart 2 : Areas of inhibition zones against types of antibiotics (Escherichia coli).

Discussion

In this experiment, we are to investigate the effect of different antibiotics on bacteria. An antibiotic is a substance or compound that kills bacteria or inhibits their growth. There are two types of antibiotics, namely bactericidal antibiotics and bacteriostatic antibiotics, whereby the former is antibiotics which kills bacteria, and the latter works by limiting and slowing the growth of bacteria. In this case, ampicillin and streptomycin are bactericidal antibiotics whereas tetracycline is a type of bacteriostatic antibiotics. From the results obtained, it is observed that there is no inhibition zone observed with the treatment of distilled water, which acts as control in this experiment. This is observed because there is no compound or substance in distilled water which can kill bacteria. On the other hand, from the first bar chart plotted, it is observed that the area of the inhibition zone around tetracycline in the Petri dish containing Staphylococcus aureus is the biggest, followed by ampicillin within a 5% range. This is because ampicillin and tetracycline are both effective against gram-positive bacteria. However, streptomycin shows relatively small inhibition area. This is probably because streptomycin is highly active against gram-negative aerobic pathogens but only some activity against grampositive cocci, which in this case, Staphylococcus aureus is a gram-positive bacteria. Other than that, from Chart 2, streptomycin shows the biggest inhibition area in the Petri dish containing E.coli. As streptomycin is highly active against gram-negative pathogens, this observation is reasonable as E.coli is a gram-negative bacteria. Not forgetting also, tetracycline still shows significant reading as it is one of the broad spectrum bacteria, which it is effective against both gram-negative and gram-positive bacteria. However, for ampicillin, it shows relatively small inhibition area. As penicillin was found to be most effective against Gram-positive bacteria, and ineffective against Gram-negative organisms and fungi, it is reasonable for ampicillin to have such low reading as ampicillin belongs to the group of penicillin. Besides, lipopolysaccharides

is the major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. Thus, it is harder for ampicillin to kill E.coli.

Evaluation

There are some precautions to be taken into account in this experiment. Firstly, as the microorganisms are a potential biological hazard, aseptic techniques have to be used when transferring the bacteria to the Petri dishes. Only sterilized apparatus can be used. Besides, the bench has to be cleaned with ethanol and hands have to be washed thoroughly to prevent bacteria infection. Other than that, the Petri dishes cannot be opened once they have been incubated. On the other hand, there are also some sources of errors which may affect the accuracy of the data obtained. For example, systemic error might occur if the pipette is calibrated wrongly. Besides that, random error, such as zero error might happen while taking the diameter of the inhibition zones. To minimize zero error, the eye position while taking readings must be directly proportional to the scale. Besides, while it is assumed that the inhibition zones are circle in shape, it might not be a complete circle. This might affect the results, but only a little, as the inhibition zones are generally circular. If it is not, square grids can be used to determine the total surface areas of the inhibition zones. Not forgetting also, there are some limitations in this experiment. As bacteria is everywhere, it is almost impossible for us to sterilize the workplace completely. Therefore, the Petri dishes might be exposed to contamination, which will in turn affect the results obtained. All in all, the results obtained are valid because the trend of the curve obtained is explainable and supported by theories which are stated in the discussion. Besides that, the results are also reliable as the data obtained is almost the same as other practical groups and there are no apparent anomalies in the results and none of the sources of errors or limitations is enough to deem the results unreliable. A set of data is obtained from another practical group and it is presented in the table below:

Another experiment can also be carried out to investigate the effect of narrowspectrum antibiotics on bacteria as broad-spectrum antibiotics are used in this experiment. For instance, penicillin G, gentamicin, clindamycin, and gentamicin. Some are effective only against gram-negative bacteria, some against gram-positive bacteria, and some only against a few species of bacteria. However, while in real life, the antibiotics that gives the biggest inhibition zones might not be the best remedy for all bacteria infections. Other factors must also be taken into consideration while giving prescription. For example, the side effects of the antibiotics and also the suitable strength of antibiotics for different levels of infections.

Conclusion

It is proven that tetracycline works on both Staphylococcus aureus and E.coli, on the other hand, streptomycin is much more effective against E.coli than Staphylococcus aureus. This is because, tetracycline is effective against both gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (E.coli), whereas streptomycin works better on gram-negative bacteria than gram-positive bacteiria.

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