s with flagella. Paramecium are protists that have cilia for movement; have two nuclei; reproduce asexually and sexually. Organisms in bottom layer appear to be chlamydomonas cells. Small, round cells with one or two long flagella.
We were unable to identify more than one clear organism in our sample. There is a potential that one of the organisms might be bacteria of some sort.
If the Hay Infusion had been observed for another two months, we might have seen a significant growth in organisms in the sample. The composition of our samples was affected by selective pressures such as: winter conditions, animals such as squirrels.
Conclusions The structures and characteristics of various algae and protists were observed in the first part of the lab. We were able to identify multiple paramecium cells. Our Hay Infusion Culture was observed to be clearer in color than the previous week, with more settled matter. Samples were taken from two different niches (top and bottom layer) and paramecium were identified in the sample. For next week, we created serial dilutions from the Hay Infusion Culture and treated four nutrient agar plates with dilutions and four agar + tetracycline plates with the dilutions as well. For future studies, it might be helpful to have other Hay Infusion Cultures available in the case that organisms are unable to be identified.
Objectives 1. To understand the characteristics of bacteria 2. To observe antibiotic resistance 3. To understand how DNA sequences are used to identify species
The first objective will be addressed through observing the nutrient agar and agar + tetracycline plates that were made in the previous lab, and counting the colonies formed on the plates. The second objective will be addressed through observing and comparing the agar + tetracycline plates to the nutrient agar plates; tetracycline plates with growth indicate antibiotic resistance. The third objective will be addressed through PCR of antibiotic resistant bacteria and nutrient agar bacteria to amplify the rRNA gene to observe the gene sequence.
Quantifying and Observing Microorganisms 1. count/estimate the number of colonies on each plate 2. convert number of colonies using conversion factor in Table 1 to determine number of colonies per ml of solution
Antibiotic Resistance 1. Note which tetracycline plates had growth, which would indicate antibiotic resistance
Bacteria Cell Morphology Observations 1. Take a sample of bacteria from two plates of nutrient agar and two plates of agar + tetracycline 2. Make a wet mount sample of each by mixing sample with a drop of water 3. Observe wet mount sample under 10x objective and then 40x objective 4. Note cell shapes, characteristics 5. Prepare a slide for gram stain by making a wet mount but letting it air dry before use 6. Heat fix slide over a flame three times 7. Cover slide in crystal violet for 1 minute. Rinse. 8. Cover slide in Gram's iodine mordant for 1 minute. Rinse. 9. Decolorize slide in 95% alcohol for 10-20 seconds. Rinse. 10. Cover slide with safranin stain for 20-30 seconds. Rinse. 11. Dab excess water with paper towel 12. Observe slide under low objective and then 40x.
Start PCR Prep for DNA Sequence ID 1. Add colony of bacteria from plate to 100 µl water in a test tube 2. Incubate at 100 degrees C for 10 minutes and then place in centrifuge 3. Use 5 µl supernatant for PCR
-Appearance or smell of Hay Infusion Culture might vary week to week depending on the growth of bacteria in the jar.
-Colonies in plates without antibiotic have more growth, colonies in plates with antibiotic have less growth but still significant. Have large fungi growth on one of the tetracycline plates.
-Tetracycline inhibits bacterial growth in most cases; however, it is becoming more common that bacteria is resistant to tetracycline. Some of these tetracycline-sensitive bacteria include both gram-positive and gram-negative cells, such as chlamydia.
-Bacteria from sample from tetracycline plate were small circular cells, all located near each other. Bacteria from sample from nutrient agar plate were