User:Ginevra Frank/Notebook/Biology 210 at AU

Lab 6 Zebrafish

Intro: Before beginning the Embryology and Zebrafish Development lab, each pair of partners described the experiment assigned. My partner and I are studying the use of Quantum Dots monitoring Zebrafish embryonic development.

Materials and Methods We prepared two samples of 20 Zebrafish embryos each, dropped in with an eyedropper into petri dishes with 20mLs of water. In one petri dish, we injected 0.05 concentration Quantum Dots. One of us checked on Monday and the other on Friday to ensure that our embryos were still alive since our version of Quantum Dots was homemade and not previously tested. So far all of our embryos are still alive. Depending on the effectiveness of Quantum Dots in monitoring embryonic development in the Zebrafish, this method of Quantum Dots could be used in determining phenotype development, disease development, and further details in developmental biology.

Conclusion We will continue to monitor the development of our Zebrafish embryos, and the use of Quantum Dots as visual aids in identifying stages of development. ... After three days, the eggs had virtually all hatched in both dishes. After 5 days many fish in both dishes had died. About ten remained in each, all adult size. On day 7 when we ended our experiment we had one live zebra fish in each dish. We concluded that due to the warm environment of our lab, the zebra fish developed more quickly than anticipated and thus ran out of food from their yolk and starved. However, under UV light and with a magnifying glass, the quantum dots had not successfully entered any of the fish, the live one nor the dead corpses, in the dish. The water had a bit of a red fluorescent cloud that was most likely the quantum dots having had dispersed in the water and not been absorbed by any of the embryos.

Lab 5 Invertebrates + Embryology

Intro: During this lab we were covering Invertebrates and Embryology & Zebrafish development due to the snow days causing us to miss lab the previous week. The invertebrate lab had us look at our Berlese funnel that we had prepared previously and which had been taken down for us during the snow days. We examined the specimens in the Berlese funnel from our transect. In the

Materials and Methods:' When observed in a dish under a microscope, we were only able to find, what we identified as, a flea. Once we identified the flea based on the characteristics we saw and Google images we compared it with, we observed other samples of invertebrates. We filled out a worksheet to help us compare different features among Arachnids, Crustaceans, Millipedes, Centipedes, and Insects. We also looked at round worms and flat worms, making note of their specific features.

Conclusion Our transect yielded very little life, perhaps because it is from a controlled environment. Most of the lab involved observations of embryos and invertebrates.

Lab 4 plants and fungi

Intro The purpose of this lab was to further explore our transects and gather different life samples to study and name. We also prepared a Berlese funnel for leaf litter and soil collected from our transects.

Materials and Measure First, we were instructed to take three plastic bags to our transect to collect samples. One had dried leaf samples, one had dirt mixed into it, and the third had different species of plant life. Once collected, we brought our bags back into the lab and prepared the bags of dried leaves and soil to be placed in a Berlese funnel. In the Berlese funnel we poured 25 mL of a 50/50 ethanol/water solution into a flask. We then cut a piece of mesh screen to place into the bottom of the funnel, taping the sides of the screen so that nothing would fall out of the bottom of the funnel. Finally we placed the funnel into a square-sided bottle. From the Ziploc bag with plant samples, we had found 5 different samples that were hard to confidently define, but through the keys provided we determined as best we could the type of vegetation found.

Data and Observation We tried to take our samples from different parts of our transect in garden beds and in between them. Sample 1: small, green, simple heart tooth shape Sample 2: clover leaf Sample 3: sage Sample 4: a different type of clover leaf Sample 5: most similar to a dried western cedar sample. All of our plants appeared to be vascular, and did not show evidence of flowering or reproductive parts.

Conclusion Our transect is unique in that it is made up of garden plots planted by students. In that way, the maintenance and variety of plants growing there is dependant on humans. Due to the time of year, much of the vegitation in our transect is dead or still underground. It will be interesting to see how things continue to grow there. It was interesting to use the key to try and name the various plants found in our transect. Sage had clearly been planted there in a plot, but the other plants seemed as though they may be indigenous to the transect.

LAB 3:

1. Intro:

Microbiology and Identifying Bacteria with DNA

In lab 3, we observed the bacteria cultivated in our hay infusions from our collected transect samples. We first observed on a macro level the physical characteristics, and noted the smell of our jars that have now been sitting for another week. Later, we calculated the number colonies grown from our transects. We determined antibiotic resistance by observing bacteria grown on our agar plates both with and without tetracyline. Finally, we prepared our samples to be put into a PCR machine so that we could observe the gene sequence of the bacteria by amplifying the rRNA.

2. Procedure: -Using the table provided in the lab, we counted the number of colonies found on the agar plates and converted the number into colonies/mL Based on growth in agar plates with and without tetracycline, we determined which bacterias were antibiotic resistant. -To look at samples under the microscope, we prepared wet mounts on slides with samples from two agar plates without tetracycline, and two agar plates with tetracycline We set the microscopes to 10x and then 40x to observe cell size and shape. - After observing, we prepared samples for Gram stains, following the instructions outlined in the lab printout. -Finally, we prepared a sample for the PCR machine taking bacteria from the plate and putting it into a test tube with 100 microlitres of water to incubate the bacteria for 10 minutes at 100 degrees C We then put the incubated sample in the centrifuge with 5 microlitres of supernatent for the PCR

3. Observations: - At first glance our jar looked less cloudy and more clear, as if more of the previously floating sediment had settled to the bottom of the jar. The smell also seemed to have subsided in intensity.

LAB 2:

1. Intro:

Identifying Algae and Protists

In this lab, we observed our hay infusion jars from the previous week. We observed appearance and smell of the jar solution, and made comparisons to last week. We then used a dichotomous key to compare our observations to, and identify the algae and protists in our sample. At the end of the lab, we prepared serial dilutions to be studied later. Purpose: The reason for conducting this experiment was to practice using a dichotomous key, and prepare serial dilutions for later labs.

2. Materials and Methods: Dichotomous Key Practice: • obtain key for eight organisms • make a wet mount of a sample with known organisms • look at wet mount under microscope at 4x and 10x • focus on one organism, if necessary add ProtoSlo • characterize the organism, and notice the size based on the micrometer • identify the organism using the key, and confirm it with a diagram • repeat procedure and practice with larger key Hay Infusion Culture Observations: • bring the jar to the table carefully • observe the physical appearance and smell of the sample • take a few samples from two different niches within the jar (top and bottom) • note where the samples are from in the jar • with a dropper, create a wet mount and add Protoslo and observe organisms • draw pictures of observations • characterize, measure, and identify six different organisms (three from the top and three from the bottom niches) • choose one of the 6 organisms, and describe how it meets all of the needs of life from page 2 in Freeman textbook Preparing and Plating Serial Dilutions: • obtain 4 tubes of 10mls sterile broth and label them 2, 4, 6, 8 • set a micropipeter to 100 microliters and get tips • obtain four nutrient agar and four agar plus tetracycline plates • label all plus tetracycline plates “tet” • label one plate from each of the two group 10^-3, 10^-5, 10^-7, 10^-9 • swirl the hay infusion culture • take 100 microliters from the mix and add it to the 10mls of broth in the tube labeled 2 (for 10^-2) for a 1:100 dilution and swirl the tube well • take 100 microliters from tube labeled 2, and add it to the tube labeled 4 (10^-4) to create a dilution of 1:10,000 and swirl to mix the tube well • repeat for tubes 6 (10^-6) and 8 (10^-8) • take 100 microliters from the tube labeled 2 and drop it on the nutrient agar plate 10^-3 • spread the mixture onto the agar plate • repeat the same for the tetracycline plate labeled tet 10^-3 • repeat this with tube 4 on the 10^-5 plate, tube 6 on the 10^-7 plate, and tube 8 on the 10^-9 plate • make a drawing/diagram of the procedure and label the diagram the figure

3. Data and Observations:

      Our Hay Infusion was cloudy, with some silt or sand settled at the bottom and a layer at the top of some sort of slime or mold film. The sample smelled like sour wet mold. 

The two niches we identified were 1) The silt on the bottom 2) The slime layer along the top We observed the following organisms from our niches: Bottom: Hypotrich (~150 micrometers), and other very small motile organisms, there were no other identifiable organisms. Top: Chilomonas, Paramecium, and other small motile organisms.

4. Conclusion: Paramecium fits requirements for life because it has evolved over time, it is single celled, it uses energy, and it eats food. If we observed our samples in another two months, I would expect more growth in mold and slime and probably see more organisms possibly larger ones. The selective pressure that played a role in our experiment was the environment. The transect from which our sample came has four garden plots. This soil is tended with specific seeds planted in it and the area is fenced in so as to keep predators out. Our transect is less “wild” than another plot on campus might be, as it is an area where a lot of human/nature interaction takes place.