Abstract All bacteria undergo constant change or growth. A bacterial ...

stationary phase, and death phase. In the lag phase the cellpopulation remains temporarily unchanged.1. Lag Phase-Although there is no apparent cell divisionoccurring, the cells may be growing in volume or mass.2. Exponential Phase-is a pattern of balanced growth whereinall the cells are dividing regularly by binary fission. The cellsdivide at a constant rate depending upon the composition ofthe growth medium and the conditions of incubation.3. Stationary Phase-Population growth is limited by variousfactors. Viable cells are being counted, but cannot bedetermined whether some cells are dying and an equalnumber of cells are dividing, or the population of cells hassimply stopped growing and dividing.4. Death Phase-Viable cell population declines. (4)Antibiotics affect the bacterial growth curve. The activity ofantibiotics may be classified as “bacteriostatic” or “bacteriocidal”.Bacteriostatic drugs stop the cells from growing and multiplying, butdo not actually kill them. Bacteriocidal drugs actually kill thebacteria. These drugs are the preferred choice when a person’simmune system is weakened and incapable of destroying theorganism itself. In either case, the antibiotics act by disruptingbacterial cell activity.(4)There are hundreds of antibiotics, most of which belong to eightmajor family groups. The drugs are classified according to their basicchemical structure. Ampicillin is a bacteriocidal, mild broadspectrumantibiotic that belongs to the Penicillin group. Ampicillin isdosed every 4-6 hours. Rocephin is also a bacteriocidal, thirdgeneration cephalosporin that is dosed every 12 hours. Levofloxacinis a bacteriocidal, second-generation quinolone that is dosed every 24hours. (5)The purpose of this research is to determine the death phase of abacterial growth curve after exposure of selected antibiotics. The

three antibiotics used will be Ampicillin (from the penicillin class),Rocephin (a third generation cephalosporin), and Levofloxacin (aquinolone). It is hypothesized that Escherichia coli, a gram negativerod,will have a faster bacterial growth curve death phase whenexposed to Rocephin versus Ampicillin and Levofloxacin.MaterialsEscherichia coliSterile watercalibrated loopsgraduated cylindersterile cotton swabsMueller Hinton agar powderautoclavedistilledTryptic Soy brothsterile salinedisposable pipettespipette dispenserAmpicillinRocephinLevaquinsyringeincubator10ul inoculating looptest tubesparafilmwater baththermometerProcedurePart A1. Crush E. coli Kwik Stik and roll onto nutrient agar plate. Streakfor isolation.2. Incubate for 24 hrs. at 37 o C.3. Make a .5 McFarland’s Standard by touching a sterilebacteriological loop to 4-5 large morphologically similar, well-

isolated colonies from a 24 hr. agar plate containing E. coli (K-12)4. Emulsify colonies in 10ml of autoclaved distilled water. Captightly.5. Vortex suspension 2-3 seconds.6. Make a 1:100 dilution using the serial method.7. Transfer 10ml of saline solution into two tubes labeled A(control) and B (Ampicillin).8. Transfer 10ul from each tube to a nutrient agar plate and streakfor colony count. This will be initial colony forming units/ml.Part B1. Mix Ampicillin and sterile saline according to manufacturersdirections using sterile procedures.2. Transfer .2ml of antibiotic to tube labeled B.3. Set tubes in 37 o C water bath.4. Repeat step 8 (Part A) at 1hr, 2hr, 4hr, 6hr, 8hr, 10hr, 12hr, and24hr. (3 trials each)5. Incubate all plates in 37 o C incubator for 24hrs.6. Count colonies on each plate.Part C1. Dilute Ampicillin at 1:2 (50%) and 1:10 (10%) strength.2. Repeat 2-6 from Part B.3. Repeat Parts A-C for Rocephin and Levofloxacin.Part D1. After growth curve is established, repeat testing between timesof last bacterial growth and no growth to determine a moreprecise time of bacteria death.2. Calculate all colony forming units/ ml by multiplying coloniesby dilution factor.

Colony Forming Units Per MilliliterThe Ampicillin reduced bacterial counts by 45% within 1 hour andhad negative plate cultures at 6hrs and 15 minutes; however itreappeared at 10 hours.The ampicillin at the 50% recommended strength reduced thebacteria by 48% at the 1 hour testing and had negative cultures by 6hrs. and 15 minutes; however it began to reproduce again at 8 hours.

tntc = too numerous too countdark bars represent tntcThe ampicillin mixed at the 10% strength of the recommended dosereduced the bacterial by 36% at the 1 hour testing but began toreproduce at the 4 hour testing.The bacterial colonies had reduced by 34% at the 1 hour testing andcontinued to reduce upon negative plate cultures at the 1 hour testing.

The bacteria reduced by 29% at the 15 minute testing and had negative platecultures by the 1 hour testing.The bacterial colonies continued to reproduce with a slight reduction of 15%at the 4 hour testing .

The bacteria reduced by 88% within 15 minutes and had negative platecultures by 30 minutes.The bacterial colonies reduced by 51% and had negative plate culturesby 30 minutes.

The bacteria reduced by 39% at the 15 minute testing and had negativeplate cultures by the 30 minute testing.ResultsThe 5mg/1ml (100%) Ampicillin reduced the bacterial populationfrom 2.92 x 10 6 cfu/ml to 1.61 x 10 6 cfu/ml after 1 hour (45%); howeverthe Rocephin and Levofloxacin killed all bacterial colonies within 1hour. The ampicillin eradicated the bacteria within 8 hours; howeverit started to reproduce between 8 and 10 hours and continued toreproduce. The 5mg/1ml (100%) Rocephin and Levofloxacin culturesstayed negative. The .5mg/1ml (10% dilution) Ampicillin nevereradicated the E. coli. The 2.5mg/1ml (50% dilution) Rocephin stayednegative while the Levofloxacin begin to reproduce at 24 hours. Allantibiotics diluted at the 10% strengths (.5mg/1ml) grew bacterialcolonies within 24 hours.Conclusion and ApplicationsThe hypothesis was not supported. The death phase occurredsooner after the bacteria were exposed to the Levofloxacin. Rocephineliminated all bacterial colonies by 1 hour, but the Levofloxacineliminated the bacteria within 30 minutes. The death phase occurred

in all antibiotics at the 5mg/1ml (100%) and the 2.5mg/1ml (50%)dilution; however at the .5mg/1ml (10% recommended) dilution theAmpicillin did not completely kill the E. coli.Antibiotics are prescribed for illnesses caused by bacteria, not byviruses. Prescriptions are written to cover the time needed to helpyour body fight all the harmful bacteria. If you stop your antibioticearly, the bacteria that have not yet been killed can restart aninfection. If you take antibiotics unnecessarily you may contribute tothe development of antibiotic resistance. Antibiotic resistancecontinues to be a problem in the U.S. If your plan of treatment is notfollowed it will contribute to this growing problem.As seen in this study bacterial infections may or may not be curedwith one dose of antibiotics. Hospital laboratories use similartechniques to determine bacterial susceptibility to differentantibiotics.

Bibliography1. Deacon, Jim. Institute of Cell and Molecular Biology, TheUniversity of Edinburgh. “The Microbial World: BacterialColonies, Gram Reaction and Cell Shapes”.2. Deason, Carl. Pharmacist Interview. Grove Drive InPharmacist. October 2, 2005.3. Thomas, Dr. Robert DO. Interview. Integris Grove Hospital.October 10, 2005.4. Kenneth Todar University of Wisconsin Department ofBacteriology. “Growth of Bacterial Populations. (Online)http://textbookofbacteriology.net/growth.html5. Levy, Stuart. “The Antibiotic Paradox.” ( New York: PlenumPress, 1992).6. Medline Plus. (Online)www.nlm.nih.gov/medlineplus/druginfo7. Reynolds, Jackie. Richland College, Biology Lab Manuel.“Determination of Bacterial Numbers” pg. 141-143.8. Roche Pharmaceutical Laboratories. Rocephin PackageInsertion., 2006.9. Tortora, Gerard, Funk, Berdeli, Case, Christine. AnIntroduction to Microbiology, 1999.