Testing Antibotics On Bacteria Essay, Research Paper

Introduction

Problem

To determine the effect of ten different antibiotics on two different types of bacteria. I will test six antibiotics on Escherichia coli, and six

antibiotics on Bacillus subtilis. On Escherichia coli I will test tetracycline, chloramphenicol, furadantin, nalidixic acid, triple sulfa, and kanamycin. On Bacillus subtilis I will test streptomycin, erythromycin, novobiocin, tetracycline, chloramphenicol, and penicillin. As a side observation, I would also like to see if Bacillus subtillis shows resistance to penicillin. The use of penicillin is being reduced because of the resistance many types of bacteria are developing against it.

Hypothesis

My hypothesis is that penicillin will inhibit the most growth against Bacillus subtilis, and that tetracycline will stop Escherichia coli more effectively than the others.

Rationale

I feel that this experiment is valid because it shows how different antibiotics react to different types of bacteria. It also points out the fact that not all antibiotics work the same or that they work at all on all types of bacteria.

Materials

1 bottle of tryptic soy agar

1 culture of Escherichia coli

1 culture of Bacillus subtilis

4 sterile petri dishes

1 pack of test discs

2 sterile pipettes

2 tubes of physiological saline

1 forceps

1 wax pencil

2 sterile swabs

Method

1. Loosen the cap of the tryptic soy agar to allow it to vent. Place the bottle in a water bath at 100 degrees Centigrade so that the water level reached the level of the medium. The agar will melt in about 20 minutes.

2. Gradually cool the medium to about 45 degrees Centigrade by letting the water bath cool. Then let the medium sit for 10 minutes.

3. Pour two plates for each species of bacteria. The agar should be approximately 5 mm deep. Cover each petri dish immediately after poring to prevent contamination.

4. After pouring the plates, allow the agar a few minutes to solidify. You can check this by tilting the plate. If the agar flows to one side, more cooling time is needed.

5. Add a tube of physiological saline to each of the bacteria culture tubes. Replace the top on the saline tube. Swirl the culture tube to disperse the bacteria in the saline.

6. Using a sterile pipette, transfer 0.25 ml of saline from one bacterial tube onto each of the two plates. With a sterile swab, spread the saline in a thin layer over the entire agar surface. Repeat with the other bacterial tube and plates, using a pipette and swab. Allow 5 to 10 minutes for the liquid to be absorbed into the agar.

7. Use the wax pencil to label each dish so that you will know what species it contains.

8. When the agar has solidified, the antibiotic test discs can be added. Using a forceps flamed between each operation place one of each type of test disc on the surface of each agar plate. The discs should be placed about 3 cm apart. The antibiotic on each disc should be identified by the following abbreviations:

S=Streptomycin, E=Erythromycin, N=Novobiocin, T=Tetracycline, C=Chloramphenicol, P=Penicillin, F=Furadantin, Na=Nalidixic acid, Ts=Triple sulfa, K=Kanamycin

9. After the cultures have been in the incubator for 48 hours. Remove them and check around each disc for an area where the bacterium could not grow. Measure each grow area in millimeters and record them on a table ( see table 1).

Literature Search

Escherichia is a genus of rod-shaped bacteria, in the family Enterobacteriaceae. Named for Theodor Escherich (1857-1911), a German bacteriologist, the only species, Escherichia coli, is found in large numbers as a normal inhabitant of the large intestine of warm-blooded animals. Whenever they leave their usual habitat, these organisms can cause urinary-tract infections, peritonitis, endocarditis, and other diseases. Some strains cause severe gastroenteritis. E. coli has been widely used as a model in molecular biology studies.

Certain rare strains of the bacteria Escherichia coli can cause food poisoning in young children, the elderly, and people with impaired immune systems. E. coli 0157: H7, normally found in the intestines and fecal matter of humans and animals, can survive in meat if the meat is not cooked past 155 degrees F. A 1993 U.S. outbreak of this type of food poisoning, which affected over 450 people, was attributed to contaminated hamburgers that were cooked rare.

In 1928, Alexander Fleming noticed that growth of the pus-producing bacteria, Staphylococcus aureus, had stopped around an area in which an airborne mold contaminant, Penicillium notatum, had begun to grow. Fleming determined that a chemical substance had diffused from the mold, and named it penicillin. The small, impure amounts he initially extracted lacked potency, yielding disappointing results in early attempts to treat human infections with penicillin. In 1939, Ernst Boris Chain, Howard Walter Florey, and Edward Penley Abraham at Oxford University began to study the possibility that purer, more stable penicillin preparations might be effective. In 1941 the partially purified material was administered to a policeman suffering from osteomyelitis. Dramatic improvement ensued, but the supply was exhausted before a cure could be effected, and the patient died. Nonetheless, the matter obviously deserved further exploration, and the outbreak of World War II added an element of urgency. The war, however, interfered with attempts to make penicillin in England on a large scale. Chain therefore hand-carried a vial of the mold to the United States, where the necessary industrial capacity was available for mass production. Application of beer-brewing technology yielded large amounts of mold liquor, from which partially purified penicillin could be laboriously recovered for clinical use. The first batches became available for military use in 1943. The material was so scarce that patients’ urine was collected and the excreted penicillin recrystallized to be used again.

Meanwhile, Rene Dubos at the Rockefeller Institute had been pursuing Pasteur’s original train of thought. Observing that microbial populations in soil held one another in check, he isolated and purified an antibiotic from a soil bacterium in 1939. It and similar substances subsequently isolated were effective when applied to superficial wounds but proved too toxic for systemic administration. By 1944, Selman Abraham Waksman and his colleagues had isolated streptomycin from a soil microbe and proved its effectiveness against the tubercle bacillus. Between 1945 and 1960, a systematic search was carried on for antibiotics derived from bacteria and molds found all over the world. Many hundreds of antibiotics were discovered, and dozens were screened for antibiotic activity and toxicity. Many were eventually marketed, and prescription use accounted for hundreds of tons annually.

In 1957, penicillin was synthesized in the laboratory. Complete synthesis of penicillins proved prohibitively expensive, but harvesting the basic molecules of penicillin from Penicillium molds and then tacking on diverse molecules proved feasible and led to a large number of tailor-made penicillin variants. The 1960s witnessed a veritable explosion of so-called semisynthetic (part mold-made, part synthetic) penicillins, each designed to deal with the increasing problem of penicillin-resistant bacteria, to achieve better absorption and higher concentrations in the body, or to broaden the penicillins’ effective antimicrobial spectrum.

Conclusion

My conclusion is that my hypothesis was only partially correct. In my hypothesis I stated that penicillin would inhibit bacterial growth best in Bacillus subtilis. This section of the hypothesis was correct. However, I also stated that tetracycline would inhibit bacterial growth best in Escherichia coli. This section was incorrect. The antibiotic that worked the best on Escherichia coli was nalidixic acid.

Results

In my experiment I received the following results:

Bacillus subtilis (gram positive)

Growth Area:

Tetracycline:12 mm

Novobiocin:9 mm

Chloramphenicol:18 mm

Strepomycin:11 mm

Penicillin:21 mm

Erythromycin:15 mm

Escherichia coli (gram negative)

Growth Area:

Chloramphenicol:17 mm

Kanamycin:11 mm

Triple sulfa:9 mm

Nalidixic acid:17 mm

Furadantin:14 mm

Table 1

Escherichia coli Bacillus subtilis

Antibiotic Width in mm Antibiotic Width in mm

Chloramphenicol 12 Tetracycline 12

Kanamycin 11 Novobiocin 9

Triple sulfa 9 Chloramphenicol 18

Nalidixic acid 17 Strepomycin 11

Furadantin 14 Penicillin 21

Tetracycline 10 Erythromycin 15

Bibliography

United States Pharmacopeia. Complete Drug Reference.Consumer Reports Books, Yonkers, New York. Copyright 1994. pp 45, 970, 1489, 1686

Facts and Comparisons. Drug Facts and Comparisons. Facts and Comparisons Inc, St. Louis. Copyright 1991. pp 120, 1374, 1598, 1895

USP DI. Health Care Provider. United States Pharmacopeial Convention. Rockville, Maryland. Copyright 1985. volume 1, pp 145, 1967