User:Mary Simcock/Notebook/Experiment

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Identifying and Studying Bacteria |- |style="background-color: #cdde95;" align="center"|

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                                                                 Identifying and Studying Bacteria


Purpose

  • The purpose of this experiment was to, in four separate procedures, quantify and observe microorganisms, observe the effects of antibiotics on the colony growth, observe bacteria cell morphology and to set up PCR for 16S amplification. The samples from our hay infusions that were incubated at room temperature on plates with and without antibiotics will be used to observe the previously stated aspects of the procedure.

Materials and Methods

  • To begin this experiment, our first step was to observe the colonies on each of our plates, specifically the number of colonies present for all eight plates. Upon completion of this step, our plates were then observed for any differences between the antibiotic and non-antibiotic plates. The third part of the procedure involved a more intense observation of our bacteria through microscopy. First, wet mounts were made using a sterilized loop, obtaining a small amount of growth from the plate, and mixing it with a drop of water on a microscope slide. A cover slip was then placed on the slide and the sample was observed under the microscope, specifically looking at the cell morphology. This procedure was followed for four bacterial samples – two from tet (+) plates and two from tet (-) plates. We chose our 10-3 and 10-5 plates. The other technique used to observe the morphology of our bacteria was gram stain. Gram staining employs the use of crystal violet, iodine, decolorizer, safranin and water rinsing. Bacteria samples were collected from the same two tet (+) and tet (-) plates used for the wet mounts using a sterilized loop. Once the gram stain had been completed the slides were blotted dry and observed via microscopy for cell morphology. The colonies were described by diameter, color, shape, texture, motility, shape and arrangement and were designated as either gram negative or positive. The final procedure of our experiment involved the preparation of PCR for 16S amplification. For this part of the experiment, one tet (+) and one tet (-) plate was sampled. Two PCR tubes were labeled and 25μL of a primer/water mixture was added to each tube and mixed to dissolve the PCR bead. Then, a sample from each of the tet (+) and tet (-) plates was using a toothpick, added to one of the two PCR tubes, mixed, and placed in the PCR machine.

Data and Observations

  • Procedure 1: Quantifying and Observing Microorganisms
  • Dilution Agar Type # Colonies on Plate

10-3 Nutrient Lawn 10-5 Nutrient About 130 10-7 Nutrient 1 10-9 Nutrient 0 10-3 Nutrient + tet About 200 10-5 Nutrient + tet 1 10-7 Nutrient + tet 0 10-9 Nutrient + tet 1

  • Procedure 2: Antibiotic Resistance

On the tet (+) versus the tet (-) plates we were able to see much more growth on the plates without the tet. Furthermore, there were more types of colonies observed on the plates without tet

  • Colony Label Plate Type Colony Description Cell Description Gram +/- Additional Notes

10-3 (-) tet Elevated, cream colored, circular, smooth, 1.5mm Move quickly, coccus, blue + .5 os on 40x, many, small 10-3 (+) tet 2mm, mustard yellow, circular, smooth Bacillus, no movement, tightly packed - 4 os on 100x, few large masses 10-5 (-) tet Yellow, circular, glossy, 3mm Coccus, bacillus, packed together + No movement, 1.3 os on 100x 10-5 (+) tet Creamy yellow, 4mm, circular, glossy Bacillus dominant, no movement, tightly packed - Large mass, 1.5 os on 100x



Conclusions and Further Directions

  • Based on our observations from both procedures one and two, we can conclude that the addition of the tetracycline antibiotic resulted in a decrease in the overall growth and diversity of the colonies. Furthermore, we can conclude from the results in procedure three that the bacterial colonies grown in the presence of tetracycline are gram negative. This indicates that they have a thin layer of peptidoglycan in their cell walls. To further analyze the cell morphology, I would like to be able to see the cell walls of the bacteria to get a closer look at the difference in thickness of the peptidoglycan layers between gram positive and gram negative bacteria.

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