User:Naseem Parsa/Notebook/Biology 210 at AU

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Lab 1

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


Lab 2

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.

Procedure

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

Raw Data

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.



Lab 3

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.

Procedure

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

Raw Data

-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.



Lab 4

Objectives 1. To understand the characteristics and diversity of plants 2. To appreciate the function and importance of fungi

The first objective will be addressed by collecting plant samples from our transects and observing/characterizing them into a major plant group. Vascularization, types of leaves, and seeds were described in order to properly characterize the type of plant. The second objective will be addressed through observing the fungi from agar plates that were preserved from the microbiology lab.

Procedure

Collecting Plant Samples 1. Fill one bag with leaf litter from transect 2. Collect five different plant samples (from already dead plants)

Plant Vascularization 1. Observe the cross-section of a moss and lily slide 2. Note the vascularization of those plants 3. Note the vascularization of the plants collected from transect

Plant Specialization 1. Examine leaves of moss 2. Observe and describe the shape, size, and cluster arrangement of the leaves collected from transect

Plant Reproduction 1. Study the bryophyte reproductive cycle and identify male and female gametophytes and sporophyte 2. Dissect a lily flower sporophyte and identify the parts 3. Dissect any seeds collected at transect and identify the parts, identify as either monocot or dicot

Observing Fungi 1. Observe fungi on agar plates from microbiology lab

Berlese Funnel Set-Up 1. Pour 25 ml 50:50 ethanol/water solution into a flask 2. Screening material placed at the bottom of the funnel 3. Funnel is placed in neck of flask 4. Leaf litter goes inside the funnel 5. Funnel/flask is placed underneath a light fixture

Raw Data

The five leaves that we collected were identified as two types of clover leaves, a smooth toothed heart-shaped leaf, a sage leaf, and a possible western cedar leaf. Our leaves showed that the plants were vascular. Other details were unable to be determined by the leaves from the transect as there was not much else growth to collect and observe.

Conclusions

This lab helped us learn how to characterize plants based on certain characteristics such as shape, size and vascularization. Although we were unable to find many leaves in our transect, the leaves we did identify showed us that much of our transect was not natural as most of the plants had been planted. This lab also taught us a new method in identifying invertebrates in our transects. The Berlese Funnel will hopefully show the various invertebrates that live in the transect.



Lab 5

Objectives 1. To understand the importance of invertebrates 2. To learn how simple systems (including specialized cells and overall body plan) evolved into more complex systems

The first objective will be addressed through observing acoelomates, pseudoacoelomates and coelomates and how they move. The second objective will be addressed through analyzing the invertebrates collected from the Berlese Funnel that was set up in the previous week.

Procedure

Observing Acoelomates, Pseudocoelomates, and Coelomates 1. Observe Acoelomates and its cross-section 2. Observe nematodes and pseudocoelomates 3. Observe Annelida, the coelomate

Analyzing Invertebrates 1. Contents of Berlese Funnel are transferred to a petri dish 2. Observe contents under the dissecting microscope and identify any invertebrates

Raw Data

The contents of the Berlese Funnel were very few. We found one flea in our leaf litter.

Conclusions

The Berlese Funnel experiment did not produce many results for our group. Only one organism was found, which was a flea. We were unable to create a food web as a result of only finding one invertebrate in our transect. Although our transect was unable to provide a variety of invertebrates to observe, it was helpful to look at other groups' results to get a better idea of what types of organisms live in various settings. This lab was also useful in demonstrating the similarities and differences in various invertebrates.



Lab 6

Objectives 1. Learn the stages of embryonic development 2. Compare embryonic development in different organisms 3. Set up an experiment to study how environmental conditions affect embryonic development

The first objective is covered in the introduction of the lab. The second objective will be addressed through observing and comparing the embryonic development in a starfish, frog and chick. The third objective will be addressed through creating an experiment based on the papers we were assigned that described the factor being used to manipulate embryonic development.

Procedure

Starfish development 1. Observe stages of starfish embryonic development from slides

Frog development 1. Observe stages of frog development from slides 2. Compare with starfish development

Embryogenesis experiment 1. Set up an embryogenesis experiment with zebrafish 2. Create two petri dishes with zebrafish embryos, one control with water and one affected by quantum dots 3. Observe them over a period of two weeks

Raw Data

We set up our zebrafish experiment with quantum dots. One of the petri dishes was the control, so the zebrafish were put in water. The other petri dish was the treated dish, where zebrafish were put in with 0.05 M quantum dots. 20 zebrafish were placed in each dish. Over the course of the first week, the zebrafish grew without food in each dish. The zebrafish in the treated dish seemed to move and grow faster than the zebrafish in the control dish. There were a few dead fish after the first week that were removed from the petri dishes. The water from the control dish and the quantum dot solution were emptied out from the petri dishes and new water and solution were added. 10 fish from each dish were preserved in a solution so that they could be observed in the case that the other fish died by the second week. By the second week, most of the fish had died. Only one fish from the treated dish survived. The preserved fish were observed under a UV light. Neither of the tubes containing controlled and treated fish showed any signs of quantum dot effects. There were some specks of red underneath the UV light, but it was most likely contamination of some sort and not a quantum dot. None of the quantum dots penetrated the fish.

Conclusions

The embryogenesis development stages of various organisms were observed through looking at slides of frog, chick and starfish development. The zebrafish experiment allowed for us to watch the progress of growing fish embryos. Although most fish died and none of the quantum dots appeared in the preserved fish, it was still successful in that one fish managed to survive in the treated dish by the end of the second week.

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