We did an experiment to test our hypothesis. If you grow bacteria in a medium with an antibiotic, then after several generations, all of the bacteria will be resistant to the antibiotic.
We washed our hands before and after our work. I always tied my hair back before doing any lab work and we all wore lab coats. We wore goggles, too, and disinfected the bench top before and after any lab work.
On the first day, Amber got our parental culture, Pseudomonas Fluorescens. I used it to inoculate two new cultures. I labeled a test tube of nutrient broth "A" and a test tube of nutrient broth with the antibiotic Kanamycin "B." I lit the Bunsen burner so that I could flame the open mouths of the culture tubes to prevent them from getting contaminated. I opened a sterile pipette package enough to insert it into a pipette pump. Then I opened the parental culture tube. I flamed its open mouth and removed a tenth of a milliliter of the parental culture. I flamed the mouth of the test tube again and then replaced the cap.
Next I opened culture tube A and inoculated the medium in the test tube with the sample I had removed from the parental culture. I flamed the mouth of the tube and replaced the cap. I put the pipette in a beaker of disinfectant. I repeated the procedure for culture tube B. I inoculated it with a tenth of a milliliter from the parental culture following the same sterile technique procedures such as flaming the mouth of the test tubes and putting the used pipette in disinfectant.
In order to see whether any bacteria in the parental culture were resistant to Kanamycin, we plated samples of it on nutrient agar with and without Kanamycin. I labeled the plate with just nutrient agar "one" and the plate with nutrient agar containing Kanamycin "two." I used a pipetter with a sterile pipette tip to remove a tenth of a milliliter of the parental culture. I flamed the mouth of the test tube before and after removing the sample from the culture. Then I deposited the sample of the culture in the middle of plate one. I sterilized a glass rod spreader by dipping it in alcohol and then touching it to the flame of the Bunsen burner. After the alcohol had been burned off, I cooled it briefly and then used it to spread the drop of culture evenly over plate one. I repeated the procedure to make plate two. After I deposited the sample on the middle of the plate, I sterilized the spreader by dipping it in alcohol and burning off the alcohol. Then I used it to spread the culture evenly over plate two. We put the culture tubes and the plates into a 25-degree celsius incubator.
I collected culture A and labeled the nutrient agar plate "three" and the nutrient agar plate with Kanamycin "four."
I followed the same procedure for preparing the spread plates that Amber did three days earlier, but I plated samples from the culture that had grown up in tube A. I used a pipetter and a sterile tip, flamed the mouth of test tube A, removed a tenth of a milliliter sample, flamed and recapped the test tube, and placed the sample in the middle of plate three. I sterilized the spreader and used it to spread the culture evenly. I did the same thing for plate four.
I got culture tube B and labeled the nutrient agar plate "five" and the nutrient agar plate with Kanamycin "six." I followed the same procedure Jim did except that I plated samples from culture tube B.
Then we put plates three, four, five, and six in an incubator at 25 degrees Celsius.
I removed our plates from the incubator and spread them out to examine and record our results.
There was bacterial growth covering plate one. Plate two with Kanamycin showed 49 individual colonies instead of a lawn of growth. Plate three showed a lawn of bacterial growth, too. But plate four with Kanamycin showed 71 individual colonies. Plate five had a lawn of bacterial growth and so did plate six, which contained Kanamycin. How do we explain these results?
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