At the peak of antibiotic drugs, there were considered “magic bullets.” But as more and more antibiotics are used, whether as a medicine for people or crops, many antibiotic resistant cases have been discovered and antibiotic resistant organic beings now pose a serious problem to man kind. The principles behind the appearance of antibiotics are rather simple. Microorganisms such as bacteria have gone through a miniature evolution. Bacteria have shorter life span than human and thus undergo many generations within a single human generation. As Charles Darwin pointed out in his books, time is the essential gradient as any kind of selection that leads to evolution require a long time (in respective to the bacteria). There are two fundamental ways in which an organic being can develop resistance to antibiotic drugs; first is through prolonged exposure to the antibiotic drugs; secondly the population has resistant genes to begin with. A native organic population, whether be E. coli bacteria or insects is most susceptible to the new a type of foreign invasion because the organic population has previous experience with the new force and thus has no defense against it. However, each individual within species has exchangeable genetic information but with subtle differences in the genetic information within each individual to allow slightly different interpretations of the genetic information (how wonderful). A result of this difference is the variations between individuals within single specie (as Darwin pointed out also). If a native bacteria population has been exposed to penicillin, some individuals within this population will have slight variations in genes that allow them to counter the penicillin and survive. Those that survived will have different genes that offered protection against penicillin than those that did not survive. These genes are called polymorphic genes and are slowly being identified by scientists/biologists. Those that survive are mostly able to reproduce and these genes that are resistant to penicillin are preserved and accumulated in a directional selection towards new pure strained specie (maybe not quite as new specie) that is immune towards penicillin. And this my friend, is the principle of The Fittest Survives, this is Natural Selection at work. In the second scenario, the population injected with antibiotic drugs already has immunity within the population due to previous experiences with these particular antibiotic drugs. The third possible explanation of antibiotic bacteria is mutations. However, this process is least likely to happen. In most cases, mutation tends to destroy organic beings but on rare conditions, mutation can destroy a particular gene that controls the production of penincillinase, which maybe produced in large quantities to destroy penicillin.
Problem:
What effect does penicillin, an antibiotic drug has on different groups of E. coli bacteria?
Hypothesis:
Native E. coli, such as those that are found on feral animal excretions are extremely sensitive to any antibiotic drugs and therefore most susceptible to any antibiotic drugs. However, those populations that already contain resistance to antibiotic drugs, or those that have survived prolonged exposure to antibiotic drugs will have a stronger resistance against penicillin.
Prediction:
According to past experiments, those E. coli bacteria populations that have weak or no resistance against penicillin will have the lowest diameter of the zone of inhibition because the drug will prohibit the bacteria to grow and thus limits the size in the petri dish that the bacteria will occupy. Any E. coli population with the strongest antibiotic resistance against penicillin will have the largest diameter of the zone of inhibition because the antibiotic resistance will allow this population to overcome the antibiotics and therefore will occupy a larger area in the petri dish. Any population that has intermediate resistance to antibiotics will have a diameter of zone of inhibition between that of the strongest resistance population and the weakest resistance population.
Experimental Design:
There is a huge problem with this experiment. Last time I checked E. coli is gram-negative bacteria and penicillin does not work well against it! This means all E. coli population has already in possession a form of resistance against penicillin and as result, the zone of inhibition produced will not be as clear cut if the bacteria tested were gram-positive bacteria. Nevertheless, predictions, hypothesis and all the variables still hold true within the circumstance within this experiment. The major manipulated components of this experiment are the populations of E. coli bacteria that have different susceptibilities to antibiotic drugs. As described before, native E. coli bacteria is most susceptible to any antibiotic drugs and therefore be used as the least strongest of E. coli population. As described in this background information, E. coli bacteria populations that have strongest resistance to penicillin are capable of producing sufficient amount of penincillinase to counter with penicillin. Finally, three different intermediate types of E. coli will be used to compare with the strongest and weakest E. coli population. The controlled variables in this experiment would be factors/conditions that may effect E. coli growth. First, all E. coli bacteria used must be pure strains, which means a E. coli population only has E. coli and nothing else. To achieve this effect, the sterile method is used in preparing the population. Bacteria need food to grow on. Each testing E. coli population will be separated into separate petri dishes containing the identical amount and type of mediums. To quicken the bacteria growth, the bacteria petri dishes are incubated to speed up bacteria growth. Finally, the responding variable will be how much space does each population of E. coli occupies.
Materials:
x5 standard petri dish (size of all three plates must be the same, the size depends)
20 ml Penicillin (more is preferred)
x1 Bunsen burner with gas supply
x1 box Matches
x1 Incubator (large enough to fit five standard petri dish)
sufficient E. coli culture that has strongest resistance against penicillin
Sufficient E. coli culture that is not as strong resistant against penicillin as the previously mentioned but not as weak as the next mentioned E. coli, in other words, intermediates
sufficient E. coli culture that has no or little resistance against penicillin
x5 Paper like material that sucks liquid (suck penicillin solution very easy)
x5 Sterile Cotton Swab
1 Bottle of 70% isopropanol
x1 metal tweezer
Caution:
- this lab is a microbiology lab and can be potentially dangerous. It is very important to follow the safety guidelines given by the instructor strictly
- do not open the petri dish after incubation
- discard biohazardous waste in directed wastes
- tie hair back and no loose cloth or papers on the lab station or near the fire source
- no food or drink near the lab station
- wash your hand thoroughly after the experiment
Procedure:
- clean workstation with 70% iso-propanol
- sterilize tweezer with Bunsen burner
- use tweezer to pick up sterile cotton swab to transfer strong resistant E. coli bacteria to one petri dish
- label this dish medium 1, write date, your signature on the petri dish
- sterilize the tweezer
- use a new sterile rod to repeat step 3-5, except substitute strong resistant E. coli with weakest resistant E. coli
- repeat step 4 but substitute medium 1 with medium 2
- use a new sterile rod to repeat step 3-5, except substitute weakest resistant E. coli with intermediate resistant E. coli
- Repeat step 3-5, but medium 2 is replaced with medium 3
- repeat step 3-5 with different two remaining types of intermediate E. coli
- Repeat step 4 for all petri dishes in step 10, but medium 3 is replaced in a consecutive manner of medium 4 and 5
- soak one liquid sacking paper with penicillin
- gently press this soaked paper down medium 1
- wait for 5-10 seconds to drain the penicillin
- seal medium 1 petri dish
- repeat step 12-15 each time with new liquid sucking paper
- place all five petri dishes in the incubator at 36 Celsius for 24 hours
- After 24 hours in the incubator, take all dishes out and measure the diameter of zone of inhibition in millimeter (do not ever open the petri dish!!!!!)
- record the diameter of zone of inhibition in millimeter
- it is strongly recommended that the experiment be repeated as in gram-positive bacteria and penicillin
- it is also recommended that the experiment be repeated with different bacteria and different antibiotics
Observation Table #1: (recommended)
Works Cited
“The Bacterial Cell Wall.” Introduction To Clinical Microbiology. 1995. University of Texas - Houston Medical School, DPALM MEDIC. 7 Feb. 2006 <http://medic.med.uth.tmc.edu/path/00001438.htm>.
“Escherichia coli.” Wikipedia. 4 Feb. 2006. 7 Feb. 2006 <http://en.wikipedia.org/wiki/Escherichia_coli>.
“The Gram Stain.” The Oral Environment. University of Newcastle, UK. 7 Feb. 2006 <http://http://www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/gramstain.htm>.
Karl Bettelheim, and Gavin Thomas. “E. coli Antibiotic Resistotypes.” The E. coli Index, Home of Echobase and colibase. Bacterial Pathogenesis and Genomics Unit at the University of Birmingham, UK. 7 Feb. 2006 <http://http://ecoli.bham.ac.uk/path/antir.html>.
“Student Activities, Measuring Antibiotic Resistance (Kirby-Bauer).” Howard Hugh Medical Institute. 7 Feb. 2006 <http://www.hhmi.org/biointeractive/disease/pdf/antibiotic_resistance/antibiotics_activities.pdf>.