BIOLOGY LAB REPORT

ANTIBIOTICS

SUPERVISOR: KAISA-HELENA LUHT LOORE LIFLANDER 22.05.12

The aim of this experiment was to investigate the effects of antimicrobials on E.coli. Every student harvested E.coli on two Petri dishes, which were previously prepared by the tutor. For investigating the effect of antimicrobials to E.coli: 5 different concentrations of 4 different antimicrobials were put on the medium with harvested E.coli. Each students inhibition zone diameters, on their plates were measured. For this experiment 4 trials were conducted. The hypothesis of this experiment is that if the concentration of the antimicrobials is higher then there will be a wider inhibition zone on the E.coli. This happens because of the strength of the antibiotic pin points how strongly the antibiotic inhibits the growth of bacteria. Background information: The four antibiotics used are kanamycin, ampicillin, tetracycline, streptomycin. Antiseptics, disinfectants and antibiotics are used in different ways to combat microbial growth. Antiseptics are used on living tissue to remove pathogens. Disinfectants are similar in use but are used on inanimate objects. Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria. Many antibiotics are chemically altered to reduce toxicity, increase solubility, or give them some other desirable characteristic that they lack in their natural form. Other substances have been developed from plants or dyes and are used like antibiotics. A better term for these substances is antimicrobials, but the term antibiotic is widely used to mean all types of antimicrobial chemotherapy. AMPICILLIN is a semisynthetic antibiotic, a member of the penicillin family of antibiotics. To extend the usefulness of the penicillins to the treatment of infections caused by gram-negative rods, the broad-spectrum penicillins (ampicillin, amoxicillin, carbenicillin, and ticarcillin) were developed. It is created by attaching acid radicals to the central structure of 6-aminopenicillanic acid. Chemically, it is D-(-)-6-(2-amino-2phenylacetamido)-3,3-dimethyl-7 -oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid trihydrate. Similar in action to penicillin G--the most widely used natural penicillin-ampicillin (or alpha-aminobenzylpenicillin) is more stable in stomach acids and therefore may be given orally; it is also more active against certain strains of bacteria. It is used regularly to treat common urinary-tract infections, some respiratory infections, and bacterial meningitis in children, although many strains of Hemophilus influenzae (the most common cause of bacterial meningitis) are now resistant to ampicillin, and it commonly is used along with other drugs.The potential side effects of ampicillin are similar to those of other penicillins--i.e., chiefly allergic reactions ranging from skin rashes and hives to lifethreatening anaphylactic shock (very rare). People who are allergic to other drugs in this family are also likely to react to ampicillin. The incidence of skin rashes is higher with ampicillin than with other penicillins, a factor that suggests a possible toxic reaction, as well as a truly allergic response.

Streptomycin is a Water-soluble antibiotic drug that is principally used for the treatment of tuberculosis in conjunction with other drugs like isonicotinic acid hydrazide (isoniazid). Aminoglycoside is any of a group of antibiotics derived from various species of Streptomyces or Micromonosporaceae, while some of which are produced synthetically. Aminoglycosides inhibit bacterial protein synthesis by binding with the 30S ribosomal subunit and in some cases the 50S subunit, causing miscoding and thus inhibiting initiation and elongation during protein synthesis. They include;

Amikacin Gentamicin Kanamycin Neomycin Netilmicin Paromomycin Streptomycin Tobramycin

Streptomycin is the first isolated aminoglycoside antibiotic derived from Streptomyces griseus. It is effective against a wide variety of aerobic gram-negative bacilli and some gram-positive bacteria, including mycobacteria. But Its use is limited because of the emergence of resistant strains. Tetracycline is a member of the tetracycline family useful because of broad antimicrobial action. It is chiefly used in treating infections caused by streptococci, staphylococci, Gramnegative bacilli, rickettsiae, and certain protozoans and viruses. Tetracycline can be produced biosynthetically by fermentation with a strain of Streptomyces aureofaciens (or certain other species) or chemically by hydrogenolysis of chlortetracycline. Tetracyclines inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit. tetracyclines act as a inhibitor of a growth (bacteriostatic) rather than killer of the infectious agent (bacteriocidal) and is only effective against multiplying microorganisms. Kanamycin sulfate, or simply kanamycin, is one of several aminoglycoside antibioticsfrequently used in the treatment of infections caused by bacteria, especially the gram-negative bacteria. Examples of these gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, and Proteus organisms. Infection with Escherichia coli often results in stomach pain and diarrhea with blood in the stool. Klebsiella pneumonia infection usually leads to lung and urinary tract problems, and infection with Proteus organisms also causes diarrhea. This drug usually works by halting the production of essential proteins needed by bacteria for their growth, killing them. Patients with serious bacterial infections are frequently given kanamycin injections, either directly in the muscle or through the vein. Physicians usually consider the body weight of patients when determining the proper drug dosage. In some cases, specifically with infections caused by some strains of Staphylococcus organisms, a kanamycin ointment may also be used. It is often important for patients to follow their physician's instructions on how and when to take these medications, and when to stop using them in order for treatment to be effective.

Similar to most drugs, kanamycin also has several side effects. These include hearing loss,dizziness, breathing difficulty, and allergy symptoms, such as itching and swelling of the face. When these are experienced, patients are generally advised to get medical attention immediately. Other side effects include vomiting, diarrhea, headache, and pain at the injection site. Some women may also observe vaginal irritation or growth of fungi in the vaginal area when using this drug for prolonged periods. Kanamycin also has some harmful effects on the nerves and kidneys. This is why tests for nerve and kidney functions are regularly being done to monitor these patients. Signs of nerve problems include tingling of the skin, twitching of the muscles, and numbness in some parts of the body. Side effects in the kidneys can include low urine volume. In pregnant women and breastfeeding mother, the use kanamycin is usually not recommended. Before using this drug, patients are often urged to inform their physicians about other medications they may be taking. The mixture of kanamycin with certain drugs can sometimes result in dangerous drug interactions inside the body. Examples are diuretics, nonsteroidal anti-inflammatory drugs (NSAIDs), and some muscle relaxants, among many others. The experiment is conducted on E.coli otherwise known as Escherichia coli are bacteria that are commonly found in the intestines of humans and animals. There are different types of E. coli some are not harmful to people and some cause serious illness. The E. coli is placed upon agar which is the medium for E. coli growth. Agar is used throughout the world to provide a solid surface containing medium for the growth of bacteria and fungi. Microbial growth does not destroy the gel structure because most microorganisms are unable to digest agar. Agar is typically sold commercially as a powder that can be mixed with water and prepared similarly to gelatin before use as a growth medium. Other ingredients are added to the agar to meet the nutritional needs of the microbes. Many specific formulations are available, because some microbes prefer certain environmental conditions over others. As a gel, an agarose medium is porous and therefore can be used to measure microorganism motility and mobility. The gel's porosity is directly related to the concentration of agarose in the medium, so various levels of effective viscosity (from the cell's "point of view") can be selected, depending on the experimental objectives. A common identification assay involves culturing a sample of the organism deep within a block of nutrient agar. Cells will attempt to grow within the gel structure. Motile species will be able to migrate, albeit slowly, throughout the gel and infiltration rates can then be visualized, whereas non-motile species will show growth only along the now-empty path introduced by the invasive initial sample deposition. Another setup commonly used for measuring chemotaxis and chemokinesis utilizes the under-agarose cell migration assay, whereby a layer of agarose gel is placed between a cell population and a chemoattractant. As a concentration gradient develops from the diffusion of the chemoattractant into the gel, various cell populations requiring different stimulation levels to migrate can then be visualized over time using microphotography as they tunnel upward through the gel against gravity along the gradient. VARIABLES:

The independent variable: The concentration of the antibiotics (100, 150, 200, 500, 1000) The dependent variable: The inhibition zone of the E.coli (in mm) The controlled variables: Temperature 37 degrees Celsius The amount of E.coli The amount of agar The time of the experiment (24 hours) The units of measurement (mm) Equipment: Petri dish, Bunsen burner, Tweezers, E.coli, agar, 4 antibiotics, Cotton stick
Procedure Organism to be tested is Escherichia coli. 1. Students will work independently in the laboratory exercise. Each student gets 2 LB agar plates and marks on the bottoms of those the name of the antimicrobial and 5 dots and concentrations. 2. Obtain an overnight culture of Escherichia coli. 3. Make a dilution 1:1 in the sterile saline solution. Mix the solution. 4. Dip the sterile cotton stick into the broth culture of the organism. Gently squeeze the cotton against the inside of the tube to remove excess fluid. Use it to streak a Luria-Bertani agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. End by streaking the outside diameter of the agar. Make two plates. 6. Allow the plates to dry for about 5 minutes. 7. While the plates are drying, take 4 filter paper discs from 2 different antimicrobials solutions with different concentrations and place them on the paper towel to dry for ~5 minutes. 8. Using flame sterilize forceps gently press the disks onto the surface of the agar. 9. Invert the plates and incubate for 24 hours at 37 C. 10. Using a metric ruler measure the diameter of the zone of inhibition (if present) for each antimicrobial used of each student. 11. Compare the measurement obtained from the individual antimicrobials to the results of other students.

DATA:
concentration/inhibition zone (mm) uncertainty of 0.5 mm 100 150 200 500 1000 trial 1 2 antibiotics ampicillin ampicillin 14 20 17 23 26 34 27 27 32 31

3 4 1 2 3 4 1 2 3 4 1 2 3 4

ampicillin ampicillin kanamycin kanamycin kanamycin kanamycin tetracycline tetracycline tetracycline tetracycline streptomycin streptomycin streptomycin streptomycin

12 17 12 10 10 11 24 26 16 21 19 17 16 19

19 24 16 12 11 15 28 30 24 26 11 10 11 11

23 25 11 11 15 12 30 27 30 28 9 9 11 10

29 28 13 15 14 15 36 26 35 32 12 12 9 12

32 31 12 14 13 16 28 35 36 36 10 10 11 13

Antibiotic ampicillin

concentration 100 150 200 500 100 100 150 200 500 1000 100 150 200 500 1000 100 150 200 500 1000

Average diameter (in mm) of inhibition in different concentration 15.75 20.8 27 27.8 31.5 10.75 13.5 12.3 14.3 13.8 21.8 27 28.8 32.3 33.8 17.8 10.8 9.8 11.3 11

kanamycin

tetracycline

streptomycin

Change in inhibition zone diameter according to antibiotic concentration

40 35 30 25 20 15 10 5 0 0 200 400 600 800 1000 1200 concentration of antibiotic ampicillin kanamycin tetracycline streptomycin

The results of this experiment show the effects of antibiotics on E. coli. There are a few anomalies in the data gathered and that is why an average area of inhibition had to be calculated. Results show that the inhibition levels are higher at 1000 concentration in ampicillin and tetracycline. The inhibition is higher at around 100-150 in streptomycin and kanamycin. The most inhibition occurred in a 1000 concentration of tetracycline, and the least in streptomycin. The graph is based on the average of the four trials taken; therefore the results are only an interpretation and may have errors. The uncertainty of this experiment varies in about -0.5/+0.5 mm. My results support my hypothesis in some respect, but the kanamycin and streptomycin prove my hypothesis to be incorrect. Therefore it can be concluded that my hypothesis supports some antibiotic behaviour but not all. The evaluation of this experiment: The experiment could have been improved if the trials had all been done by the same person. Also the amount of E.coli placed on each medium varies due to the method of using the cotton stick, which cannot provide enough ability to control the exact amount of E. coli placed. Time management was also an issue when conducting the data; this may have caused some miscalculations. Some of the confounding variables were the motivation and attention levels of the experimenter. This experiment has high internal validity because it is a laboratory experiment where all the variables are appropriately controlled and manipulated. The reliability of this experiment is high due to the test-retest reliability, where many trials were taken to measure the inhibition zone. The inter-rater reliability is decreased because of one person measuring the inhibition zones, but it is increased by many experimenters conducting the same experiment with reasonably similar results.

the area of inhibition of E.coli in mm