User:Naseem Parsa/Notebook/Biology 210 at AU
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Appearance or smell of Hay Infusion Culture might vary week to week depending on the growth of bacteria in the jar.
Appearance or smell of Hay Infusion Culture might vary week to week depending on the growth of bacteria in the jar
Revision as of 17:41, 16 February 2014
Objectives of this lab: 1. To understand natural selection 2. To understand the biotic and abiotic characteristics of a niche
This lab will address the first objective through the observation of the Volvicine line, which is the evolution of the Chlamydomonas cell into a Gonium cell and finally into a Volvox cell. The second objective will be addressed through the observation of a transect at AU, or a niche.
Procedure Volvocine line: 1. prepare slides of Chlamydomonas, Gonium and Volvox 2. add protoslo to the slides 3. observe any characteristics (flagella, size, colonies)
Defining a Niche: 1. Take a sample comprised of plant and soil matter from the transect 2. Create a Hay Infusion Culture with 12 g matter in a jar (500 ml water, 0.1 g dried milk) 3. leave jar unopened in lab for future observation
Raw Data Transect- AU community garden near tennis courts Description- Dry grass, not many growing plants.
Five Biotic organisms in transect: 1. cabbage leaves 2. small green leafy plant 3. wood 4. sage plant 5. larger leafy green
Five Abiotic organisms in transect: 1. metal nails 2. soil 3. stones 4. tennis ball 5. plastic flag marker
Sample was comprised of a leaf with roots still attached from cabbage garden, and soil.
Conclusions Hay Infusion Culture was created with the soil/plant sample from the transect. It will remain in lab until the next week, where observations of its appearance, smell, contents will be made. Transect showed how niches function within an environment. Our transect showed signs of life but not many as it was an environment that doesn't really thrive in the winter months. The objectives of this lab were addressed as the evolution of the Chlamydomonas cell into the multicellular Volvox cell was observed to be a result of natural selection. For future studies, observing the Hay Infusion Culture for a longer period of time might add to our study of what types of organisms grow and live in certain niches.
Very good first entry. Could include more detail and images/drawings if you have them. Ensure to address all red text from protocol. For more instructions see TA notebook. SK
Objectives 1. To understand how to use a dichotomous key 2. To understand the characteristics of Algae and Protists
This lab will address the first objective through identifying different organisms on a wet mount and characterizing them from eight known organisms. The second objective will be addressed through observing our Hay Culture Infusions from the previous week by taking samples from the culture and identifying the various organisms in the sample.
Dichotomous Key 1. Make a wet mount of a sample and observe at 4x objective and then at 10x 2. Characterize the organism found in the sample, measure the organism 3. Match description with organisms from a Key
Hay Infusion Culture Observation 1. Observe smell, appearance of the culture 2. Take a few samples from two different niches, including plant matter 3. Make a wet mount with samples to observe 4. Characterize at least three different organisms in each sample 5. Measure organisms
Preparing and Plating Serial Dilutions 1. Pour 10 ml of distilled water into four tubes, labeled 2,4,6,8 2. Label four agar plates and three tetracycline plates, labeled 10^-3,-5,-7,-9 3. Mix Hay Infusion Culture and take 100 µl sample and add to first test tube 2 4. Take 100 µl from test tube 2 and pipette into test tube 4, etc. 5. Take 100 µl from 10^-2 test tube and pour onto agar plate 10^-3, spread sample. 6. Repeat for tetracycline plate
Sample from Procedure 1 was identified as paramecium. Had a long oval-shaped cell structure and smaller organisms inside.
Hay Infusion Culture Observations- Smells like moss, sour; cloudy appearance; some matter settled to the bottom; floating layer at the top (greenish, brown color); apparent reduction in mixture from the starting point.
Two niches for wet mount- Top layer, bottom layer
Organisms in top layer appear to be paramecium, might be chilomonas or some other outside organism? Looks like a large colony of cells 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 long oval-shaped cells. Looked similar to paramecium.
Conclusions The first part of this lab helped to characterize whether the bacterial growth on the tetracycline plates were antibiotic-resistant or not. Observing the cells more closely on a wet mount allowed for further analysis of the type of cell. Gram-positive and negative analysis were done on the samples from both a nutrient agar plate and from a tetracycline plate. The bacteria found on the nutrient agar plate were found to be gram negative, showing a pink color to the stain. The bacteria found on another sample of nutrient agar was found to be gram-positive, however. The bacteria found on the tetracycline plate were found to be gram-negative, and looked similar to sperillium cells. The final portion of this lab saw the beginning of DNA sequencing of the bacteria found on the plates through PCR.