User:Livvy Shafer/Notebook/Biology 210 at AU

3/19/14 Lab Six: Embryology and Zebrafish Development

Question: What impact does caffeine have on the development of zebrafish embryo. This lab addresses this question by having us observe a group of twenty control embryo, and twenty embryo exposed to caffeine over two weeks and compare the results. The lab looked at fin and eye development, as well as movement and tail length and coloring.

Procedure Retrieve to petri dishes- one control and one test

Fill the control dish with 20 mL of distilled water and put 20 healthy embryos in the dish

Fill the test dish with 20 mL of caffeine (10-50 mg/L) and put 20 healthy embryos in the dish

On each day of observation look at the number of dead eggs, living embryos still in egg casing, living hatchlings and dead hatchlings

On day four remove 10 mLs of water and any of the empty egg casings or dead embryos from both petri dishes, add 20 mLs of water to control, and 20 mLs of caffeine solution to test group

On days four and seven and eleven observe representative embryos from each group under the compound microscope. Take note of the degree of body and tail pigmentation. the eyes and eye movements, the pectoral fin development, the development of the swim bladder, the development of the mouth, and the general movement of the embryos

On day seven fix three larvae from the control and three larvae from the test group by putting them in a tube and adding one drop of tricaine solution per mL of water in the tube, and then have the lab instructor add the paraformaldehyde and store the samples.

On day seven and eleven add 10 mLs of water to the control group, and 10 mLs of caffeine solution to the test group.

Results Test Group Data: File:TESTGROUP.PM

2/22/14 'Lab Five: Objective: The point of this lab was to help me understand and have a new appreciation of the invertebrate organisms that fill niches all over the place, especially on campus. The lab also had the purpose of helping me to see how complex these invertebrates can be, even though they are thought of as simple organisms. This experiment fulfills its purposes by having me examine invertebrates such as worms, and those collected from my transect under the microscope.

Procedure: Procedure One: Look at Planeria, an acoelomate, under the microscope. Observe their digestion of the egg yolk. Look at the nematodes under the microscope, as well as a cross section that demonstrates their pseudocoelomate structure. Observe the coelomate Annelida under the microscope, noting the layers of its muscle and its internal organs. Describe the movements of the organisms. Procedure Two: Select five organisms from the petri dish to identify. Ours had no organisms from our own transect, so we observed those from the West Virginia leaf litter. Fill out the table presented below. Procedure Three: Look at the groupings of vertebrates in the book. Choose five organisms, two of which must be birds, that could inhabit the transect. Determine the classification of all five organisms. Describe the biotic and abiotic features each organisms would need in the transect. Make a food web of the organisms.

Data: The data are presented in pictures and tables below.

Conclusions: Through doing this lab I saw firsthand just how complex invertebrate organisms can be. I saw this by examining several types under a microscope. In the future I would collect a new leaf litter sample so I could examine invertebrates from my own transect, rather than the ones from West Virginia. I would also try to look at more than five types, and I would look at more worms as well.

Lab Questions: Procedure One: Acoelmates: Planeria- gliding motion. Pseudocoelomates: Nematoda- no movement Coelomates: earthworms- wriggling motion

Procedure Two: West Virginia Leaf Litter

The size of the organisms in the West Virginia leaf litter ranged from 75 micrometers to 2.5 cm. The largest was the beetle and the smallest was the arachnid. Below are some pictures of the organisms in the leaf litter.

The first picture is of the pseudoscorpion.

The second picture is of the beetle.

The third picture is of the flea.

The fourth picture is of the arachnid.

The fifth picture is of the lice.

Procedure Three:

L.S.

2/19/14 Lab Four: Objective: The purpose of this lab was to help me understand the different features of plants, and to see that plants are actually very diverse organisms. Another goal for this lab was to help me understand the important roles fungi plays, and why it is important. The experiment we did helped me reach these goals by having me examine different plants from my transect, so I saw the diversity of the plants right here on campus. I also learned about fungi by looking at it under the microscope. Procedure: Procedure One: I went to the transect and collected five different types of plants from different areas of the transect. I put the collected plants into a bag for observation later on. I also took a leaf litter sample, it was about 20% soil and 80% loose leaf matter from the ground. The sample went into a bag and was used later on in the lab. Procedure Two: Observe the lily stem and compare it to the Mnium. Record the height of the lily stem- 24 inches Record the height of the Mnium- 8.5 cm. Procedure Three: I looked at the leaves of the moss, and noted the appearance of the leaves. I also looked at a cross section of an angiosperm leaf, and noted the presence of stomata on the underside of the leaves. Procedure Four: Look at the diagram of the bryophyte reproductive cycle. I identified the male and female gametophytes, and observed the haploid and diploid parts of the life cycle. Procedure Five: I examined the black bread mold under the microscope, and noticed the presence of sporangia. I turned the dish over and found the rhizoids that grow into the agar. Procedure Six: I poured 25 mL of a 50:50 ethanol/water solution into the flask. I then fit a piece of screening material into the bottom of a funnel with tape. Then I put the funnel into the neck of the flask. Raw Data: Pictures of the plants and transect will be below, and the table is written out in procedure one below. Conclusion: Through doing this lab, and looking at the five different plants from my transect I saw the diversity of the plants on campus. I also saw the importance of fungi by examining it under the microscope. In the future I would look at more plant types from my transect, and more types of fungi to learn more about them. Questions from Lab: Procedure One: The first plant we found was a bush, and it had cylindrical parts that resembled a corn dog on the top of the stems. There were no leaves on the plants, and they were seedless. There were around three bushes in the transect and they were located in the center of it. The second plant we found was a plant, and had long thin leaves. The plant was seedless and they were located on the outer edge of the transect. The third plant we found was a tree, and there were two of them at the back edge of the transect. They had small green leaves and red stems, and were seedless. The fourth plant we found was at the back edge of the transect, and it was long, thin and tall. It had little fuzzy flowers on it. The fifth plant we found was a plant, and it was low to the ground and was all over the transect. It did not have vascularization and it had small green leaves, and was seedless. Procedure Two: The stem height of the lily was 24 inches, and the height of the Mnium was 8.5 cm. Procedure Three: The first plant did not have leaves. The second plant had long thin leaves, and they all grew straight up from one area it looked like. The third plant had leaves staggered from each other on the stems, and they occasionally branched off the main stem from a smaller stem. The Fourth plant had small fuzzy flowers covering the stem, so there were no leaves. The fifth plant had a cluster of small, green leaves coming from one spot. Procedure Four: Our plants did not have seeds on them. However, there was one plant that had small fuzzy flowers on its stem. There may have been spores in the horsetail plant that we brought back in the part that resembled a corn dog. Procedure Five: The sporangia contain the spores that are released when the fungi needs to reproduce. So in a sense the sporangia are vital for the reproduction of fungi. The sporangia are small round structures that form from the hyphae that grow upwards. The sample I examined appeared to be a part of the ascomycota division of fungi. I concluded this because it looked like a mold to me, and mold fall under the category of ascomycota.

L.S 2/10/14 Lab Three: Objective: The objective of this experiment was to learn about antibiotic resistance by observing it, learn about bacteria properties, and their DNA sequences and how they can be used to find the species of bacteria. This experiment does this by having us examine bacteria from our agar plates, some were treated with tetracycline, and still had bacteria on them. This allows us to see antibiotic resistance, since the bacteria grew even though there was an antibiotic on the plate. We also saw the characteristics of our bacteria by looking at it under the microscope, and recording them in a table. The last part was fulfilled by collecting the DNA from the bacteria and running a PCR reaction. Procedure: Obtain a prepared slide that has different types of bacteria on it. Look at the stained portion of the slide on lower objectives and then at higher objectives. Move on to the 100x objective and apply oil to the slide. Observe three total species of bacteria We chose two from our tetracycline plates, and one without tetracycline. For each sample prepare a wet mount slide and look at them under all objectives, record any characteristics in a table.

Label the slides Heat fix the air dried slide by passing it through a flame three times with the bacterial smear side up Cover the slide with crystal violet for one minute Rinse the slide with distilled water Cover the slide with Gram's iodine mordant for one minute Rinse the slide with distilled water Decolorize the slide by covering it with 95% alcohol for 10-20 seconds Rinse the slide again Cover the slide with safranin stain for 20-30 seconds and rinse Blot the excess water off and let air dry Focus the sample under the microscope at a low magnification Observe the slide at 40x and then 100x Record Observations in table

Transfer a single colony of bacteria to 100 microliters of water in a sterile tube. Incubate at 100 degrees C for ten minutes and centrifuge Use 5 microliters of the supernatant in the PCR reaction

Conclusions Through this experiment we concluded we had at least three different types of bacteria on our agar plates. Two of the types were resistant to the tetracycline. One of the types was Gram positive, while the other two were gram negative. This tells us about their cell wall and plasma membranes, the Gram positive bacteria has a cell wall with peptidoglycan, and a thin plasma membrane, while the Gram negative cells have an extra membrane that prevents the stain from sticking. All of the objectives were addressed by the experiment, and in the future I would observe more than three types of bacteria. I would try to look at six or seven types of bacteria to see a wider variety of bacteria.

Questions: I don't think any archaea will have grown on the agar plates because they live in extreme environments, such as very salty, cold or hot environments. The appearance and smell of the Hay Infusion Culture can change from week to week because new bacteria can be growing in it, and more plant matter can be decomposing in the culture, changing the way it looks and smells. Table: Dilution Agar Colonies Counted Conversion Factor Colonies/ mL 10^-3 Nutrient 120 x10^3 1200000 10^-5 Nutrient 30 x10^5 30000000 10^-7 Nutrient 4 x10^7 400000000 10^-9 Nutrient 17 x10^9 1.7x10^11 10^-3 Nutrient + tet 23 x10^3 230000 10^-5 Nutrient + tet 1 x10^5 1000000 10^-7 Nutrient + tet 1 x10^7 100000000

There are more colonies on the agar plates that haven't been treated with tetracycline, than those with tetracycline. The tetracycline seems to prevent a lot of bacteria types from growing on the plates, meaning the bacteria is not yet resistant to the tetracycline. Those that do grow on the plates are resistant to it, and can grow anyway. Tetracyclines work by preventing the bacteria from performing the necessary enzymatic reactions they require to live. "Tetracycline - Antimicrobial properties." Tetracycline - Antimicrobial properties. N.p., n.d. Web. 8 Feb. 2014. <http://www.chm.bris.ac.uk/motm/tetracycline/antimicr.htm>

Table Two Colony Label Tet + or NA Colony Description # of Colonies= bacteria per mL of Culture Cell Description Gram + or - x10^-5 NA Circular, dark purple, entire, flat 1 30,000,000 Diplobacilli, rod shaped, fast cilia - x10^-5 T Circular, cream, convex, entire 1 1000000 Brownian Movement - x10^-3 T Small, yellow, circular, convex, entire 1 230000 Streptobacilli, no movement +

LS 2/7/14 Lab Two Objective: The objective of this lab is to find the algae and protists that occupy niches in my transect. This experiment had us take samples from different places in the hay infusion culture from the last lab, and then had us make slides to look at under the microscope. By doing this we were able to find three different organisms from the top of the jar and the bottom of the jar. Procedure: Take a few samples from two different places from the hay infusion culture. Note where the samples came from. Use a dropper to place a small amount of liquid onto a slide and put a cover slip on top. Draw pictures of the organisms, six total, present noting their size. Explain how one of the organisms in the sample meets the requirements for a living organism.

Serial Dilutions: Obtain four tubes with 10 mL of sterile broth and label the tubes 2, 4, 6, and 8. Obtain four nutrient agar plates and four tetracycline plates and label them X10^-3,-5,-7 and -9. Swirl the Hay Infusion Culture to mix the organisms up, and then take 100 microliters from the mix and add this to the 10 mLs of broth in the tube with the label 2. Then take 100 microliters of the broth from the "2" tube and put it into the tube labeled 4. Repeat this process for the remaining tubes. Take 100 microliters from each tube and place it on the nutrient agar plates in their respective orders. Repeat this procedure for the four tetracycline plates. The plates will incubate at room temperature for the next week. Conclusions: Through performing this experiment we found that there were several types of organisms in our sample that fulfilled different niches based on their locations. There were five different organisms including- brown algae, Didichium, Chlamydomonas, Spirostomum, and Colpidium. The first three were from the top of the jar, and the last two and the brown algae were from the bottom of the jar. In the future I would take more samples and take one from the middle of the jar to see if we could find other organisms that fulfilled different niches in our transect. Smell: The jar smells bad, and has a faint odor similar to peas. Appearance: The water is cloudy, and has moldy dirt on the sides, and algae on the top and bottom of the water. Top Water Sample Organisms 1) Algae- brown, and immobile. 2) Didichium Cyst- spinning, and bean shaped- 30 micrometers 3)Chlamydomonas- 2.5 micrometers, small black dot with two spikes

Bottom Water Sample Organisms 1) Spirostomum- 2 micrometers, small longer shaped and dark colored 2) Brown Algae 3) Colpidium- 62 micrometers, small with dark coloring, and things sticking out of it everywhere The brown algae meets the requirements for life because it gets and uses energy. It is also made up of cells and has genetic information that it processes. The brown algae also reproduces or replicates itself and it evolves as a population. If the hay infusion culture was observed for two more months I would expect there to be more bacteria and algae to be living in the jar. I would also expect the jar to smell worse because of the decomposing plant matter in the water. Food source and competition could be an example of a selective pressure that affected the composition of my sample. Below are some pictures from my lab notebook: L.S. 1/23/14

Lab One Obejective: The purpose of this lab was to look at different niches that organisms satisfy on campus. My transect was by Kogod, and we had to find different organisms that satisfied their own niches in our transect. I predicted that there would be several types of bacteria and microorganisms in the transect that all perform different functions for the transect. This experiment will help us find the organisms that fill niches through the soil and vegetation sample that we take, and the hay infusion.

Experimental Protocol 1) Observe the transect- noting the location of the transect, and the topography, draw a picture of the transect 2) Find four biotic and five abiotic factors 3) Take notes on the surroundings 4) Take a soil sample and vegetation sample in a 50 mL conical tube 5) Weigh 10-12 grams of the soil sample and place in the plastic jar with 500 mLs of purified water 6) Add .1 gms of dried milk and gently mix it up for ten seconds 7) Take off the top off and place the jar 8) Label the jar with group name and number

Raw Data Abiotic Factors: 1) Big Rocks 2) Little Rocks 3) Pebbles/Gravel 4) Brick 5) Woodchips/soil

Biotic Factors: 1) Cat tails 2) Red Bushes 3) Grass 4) Weeds

Notes: The area has pretty heavy traffic, since it is next to the walkway by Kogod. Concrete, and buildings and roads are in the surrounding area. There is also a drain nearby for water runoff. The area is on a slight slant, which increases the water flow through the transect.

Conclusions: In the future I would collect more samples from different areas in the transect. I wasn't surprised by the biotic factors or abiotic factors found in my transect. Through this experiment I found different biotic organisms that filled different niches.

Below are some pictures of the transect.

LS

Good start. No need to rewrite protocol instructions. Address red text and give details on procedures that you followed any results, reasons and conclusions. Include volvocine line part of lab 1. SK

1/23/14 I have successfully entered things into my lab notebook. LS