User:Peter To/Notebook/Biology 210 at AU

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Lab 6 The Zebrafish Experiment Purpose The purpose of this experiment was to observe effects of stimulus during embryonic development. Exposure to difference kinds of substances or light can cause damaging changes in zebrafish development. For our experiment we decided to examine the effects of alcohol on zebrafish development. This allows us to see the effects of the environmental conditions (alcohol) on embryonic development. This experiment is also an important model for study in other organisms. For example, we cannot conduct experiments on human females but the results of the zebrafish experiment will give us clues on how alcohol could effect human children and cause fetal alcohol syndrome.

Materials and Methods For this experiment zebrafish eggs were delivered to our lab. We had looked at a study performed by professionals on alcohol and zebrafish development. They concluded that alcohol causes abnormal bodies and changes. We hope to repricate their experiment on a general level. We hypothesize that alcohol will cause an abnormal unproportional body shape. the eyes will be smaller in relationship to the zebrafish in the control. Two petri dishes were set up. One petri dish has 25 ml of water with a small dosage of antibiotics; this is the control group. In the variable group that we are focused on studying, we added 25ml of a 1.5% ethanol solution. Both petri dishes were given 20 zebrafish eggs. Their stages of devlopment were identifiied and rerecorded. Initial observations were performed using the dissecting microscope to make sure all the eggs of both groups are starting out healthy. Dead eggs were discarded. The following day after the experiment was set up, observations were made to make sure all the eggs were still healthy and alive. Observations were taken on mondays and fridays of the study. Progress of growth was observed. Data includes zebrafish size, mortality rates, abnormal body shapes. One drop of zebrafish food was added to the petri dish at each observation day and the solution of the petri dish was replenished.

Data Initial experiment set up On day one, 20 zebrafish in each petri dish. Both groups were in the 20-somites stage of development. On day four, 18 were alive in the control group and 12 were alive in the alcohol group. The control group was in the protruding mouth stage and the alcohol group was in the pec-fin stage. The control group was slightly larger in size at 0.4cm and the alcohol group was 0.3cm in size. The zebra fish in the alcohol group had a slower rate of development. The alcohol group had behavioral differences. They were all swimming faster and look very jittery. The control group looked more relaxed. Both groups had a yellowish tinted transparent body and black dots for eyes. On day seven of observations only 2 were alive in the control group and 8 were alive in the alcohol group. On day 12 all zebrafish in both experiments were dead. It was clear that the control group made in further in development. They had visible tail fins and more developed bodies. However, very few made it this far in the experiment. In terms of the zebrafish in the alcohol, they did not develop as much and most dead ones at the end of the experiment were in the protruding mouth stage.

Conclusions The hypothesis was, if zebrafish are exposed to 1.5% ethanol then successful development would decline. The end results of the experiment did indicate that the zebrafish in the control group were more developed. However, the control group had very few making it past day 7. The alcohol group showed behavior changes and slower growth but they survived longer in the experiment. Overall, all zebrafish were dead by the last day of observations. This may be caused by cross-contamination by other lab studies. When observations were made, our sample was usually found in the wrong section box and in bad shape. There is a clear crack on the petri dish of the control group. Something during the experiment has to contaminate the study because between day 4 and 7 the number alive in the control went from 18 to 2. This is also a bad experimental setup. The lab was performed while other studies with different variables were stored in the same box. Each study should have gotten their own box. There was so much potential for misplacement and cross contamination. In conclusion, our results do not replicate the results of the ethanol experiment in the scholarly article we modeled this experiment after. There should have been a higher success rate in the control group. Our samples were broken and probably had water from other studies seep in because of an un-organized experimental set-up.

Update PT 3/8/15

4.8.15 A data table would improve this lab book entry and some images. SK

PT 2/26/15

Lab 5 Invertebrates

Purpose To observe evidence of invertebrates in our transect.

Materials and Methods The alcohol preservative was removed from our berlese funnel. They were split into two petri dishes for observation under the dissecting microscope.

Data and Observations We identified 1. Springtail, arthropoda, entognatha 2. Proturan, arthropoda, entognatha 3. Springtai 4. Pillbug, arthropoda, malacostraca 5. Soil mite

Conclusions These organisms are part of the ecological community. They live together in balance. Carrying capacity is the population a niche can support successfully. Tropic levels are the levels or organisms in the food chain. All these factors play a role in the existence of the organisms and the transect community.

2.19.15 Good entry. Would be a little more clear and complete if included the data table from the manual. SK

Lab 4 Plantae and Fungi

Purpose This lab was meant to show us the diversity of plants and fungi.

Materials and Methods We walked to our transect and collected samples in a ziplock bag. Five plant samples were taken from our transect.

A berlese Funnel was set up to collect invertebrates. a screen was placed in the funnel. The funnel was sealed into a flask with alcohol preservative. Our dead plant matter (leaves and wood) were placed into the funnel. They were covered in tin foil and placed under lights.

Data and Observations Berries: The berries were on a stick and came in a whole bunch. They were red in color and spherical. We determined the to be gymnosperm seed plants. It is also dicot because it had two cotyledon inside. Moss: Moss was found on a rock.. It was green and healthy looking. Moss is mono cot. It has no taproot, instead a fiberous root system. Tree Bark: Vascularization is xylem and phloem. It was found on the ground next to a tree. Leaf: This leaf was found in a pile of dead leaves. Vascularization is xylem and phloem. Long Leaf:This leaf was picked up from the ground. It had a long shape with parallel veins. We determined the leaf to be dicot. It looked dead and dried out. Vascularization is xylem and phloem.

We also dissected a flower lily to look at mechanisms of plant reproduction. We isolated the petal, sepals, stigma, anther, stamen, stem, and ovary. The anther is where the pollen grains are. The ovary contained the ovules.

Examination of Fungi Samples. Fungi Sprorangia are important because it allows alternation of generations. When the sporangia open they release spores. They are (n+n) diploid cells that can land and start growing into a new plant. When the fungi go into alternation of generations haploid cells are released so they can undergo meiosis. We examined a mushroom under the microscope. There were also other fungi samples available. They are important because they produce some of our antibiotics used to fight infection. In other cases they are healthy edibles we can consume.

Conclusion There is great diversity in plants and fungi. We saw multiple different species from our transect.

-PT 2/12/15

2.11.15 Good notebook entry. Some of the information is in the wrong section eg. some information in purpose section should be in results. All of the results should appear in the data and observation section and then conclusions at the end. It would be nice to have some images. To make a table try making it in excel and then using to convert it into OWW format and then copy and pasting it into your notebook. Nice to see you're using references. I suspect that archaea aren't present in your transect. You should also include the PCR setup to amplify the 16s gene. SK

Lab 3 Microbiology and Identifying Bacteria with DNA Sequences

Purpose The objective for this lab was to apply the knowledge we learned about bacteria cell walls from lecture. We learned the the thick cell wall causes gram stain to stick therefore bacteria that turns purple are gram positive. Pink bacteria are gram negative. We observed different colonies of bacteria found on our plates. We also wanted to observe antibiotic resistance. In some groups plates that were treated with antibiotics still had bacteria colonies.

'Materials and Methods Cell culture plates were prepared in the last lab. These plates had nutrient rich agar. They were prepared at different concentrations and with or without antibiotics. To prepare the slides we took samples from the visible bacteria colonies with a sterilized loop and smeared the onto slides. After observing live and moving bacteria we prepared fixed slides. Sample were placed on the slide and passed through a flame. After flame passage we used the gram staining method to color our bacteria for further observation.

Data and Observations Do you think any Archaea species will grow on the agar plates?... I think it is possible. Archaea is extremely diverse and some archaea species are found in extreme environments. Last Observation of Hay Infusion Culture- The water turned very dark. It didn't smell as bad. I conclude that the microorganisms in the culture has decomposed and used the resources in the culture. That's why the water turned darker. Everything was broken down and digested.

Table 1 Dilution: Agar Type: Colonies Counted: Conversion Factor: Colonies/mL 10^-5: nutrient: 150: x10^5: 150,000,000/mL 10^-7: nutrient: 4: x10^7: 40,000,000/mL 10^-9: nutrient: 0 10^-5: nutrient+ tet: 0 10^-7: nutrient + tet: 0 10^-9: nutrient + tet: 0

There were no colonies of bacteria in our plates with antibiotics. This indicates there were no variant strains of bacteria that could survive the antibiotic. We only observed fungus growth on the tet plates. The effect of tetracyline on the bacteria was they killed it.

Information from US National Library of Medicine. Tetracycline works by inhibiting protein synthesis in bacteria. Without proteins to carry out functions they die (Chopra). Tetracycline attacks both gram positive and gram negative bacteria. However, there are some bacteria that are now resistant to this antibiotic (Chopra).

References Chopra, I., & Roberts, M. (2001). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol Mol Biol Rev., 65(2), 232-260. Retrieved from US National Library of Medicine National Institute of Health database.

Bacteria Colony Data: Bacteria slides from Colony 1 was peach colored and pale yellow, they were round and moved by brownian motion. They are gram - and were determined to be Spherical Coccus or Coccobacillus. Bacteria slides from Colony 2 was also pale yellow, they were smalled and had a slight rod shape. They were gram + and were determined to be Fusobacterium. Bacteria slides Colony 3 was orange and round, they appeared to be swimming. We are not sure if they are gram + or - and were unable to come to an agreement of what kind of bacteria they were.

Conclusions We were able to identify bacteria as gram positive or negative. Our observations lead us to the conclusion that bacteria is very diverse. We found 3 different kinds and they came from the same transect. Antibiotic resistance was observed in other groups. Our particular plate had no bacteria. This must have been just a lucky or outlier plate.

-PT 2/5/15

2.4.15 Excellent notebook entry. Pictures are rotated and too large. Try saving the images as smaller files and uploading them. SK

Lab 2 Identifying Algae and Protists Purpose The Objectives for this lab was to understand the structure of protists. Protist samples were prepared for practice on identifying protists based on their look by using a dichotomous key. Identifying skills were then used to examine algae and protists from transect samples. The samples are from the Hay Infusions Jars prepared in the previous lab. Materials and Methods

The Hay Infusion Culture: Prepared last week. Samples from the transect was added to a jar with water and milk powder. Mile powder acted as an energy source for organisms to grow.

Sample Protists: Sample protists were pre-made in lab. We added a few drops to a slide to make a wet mount slide.

Data and Observations Protist practice observations: -Oval Shape and elongated -4 ocular spaces which makes it 40 micrometers -Dichotomous key determined it as Euglena

Hay Infusion Culture Jar Data: -Smelled like a sewer -It was a opaque brown color with dirt settled at the bottom. There was a mold like film on the top of the liquid. Samples were taken for observation from two different niches -Organisms close to plant matter may be different because they have more resources. They might be in a symbiotic relationship with the plant matter.

-Samples from two different niches Surface sample had white moldy stuff Bottom sample had dirt, twigs, and very dirty water. -Samples were taken from the very bottom of the Hay Infusion Jar and the top of the hay infusion jar -Wet mount from two different niches results: 1. Bottom Sample, 5 ocular spaces or 12.5 micrometers. Ameoba 2. Bottom Sample, swimming around and colorless, 5 micrometers, Chlamydomonas 3. Surface Sample, 150 micrometers, Paramecium aurella 4. Bottom Sample, Arcella, 40 micrometers 5. Surface Sample, Slow and fast Movements, 70 micrometers, Colpidium 6. Surface Sample, 70 micrometers, Paramecium Bursania Images shore A serial Dilution of the bacteria sample prepared for next week The drawings of the organisms from the Hay Infusion Culture and a picture of the Hay Infusion Culture Serial DilutionsPeterToAUBIO.jpg [[Image:Organism DiagramsPeterToAUBIO.jpg Hay Infusion 2PeterToAUBIO.jpg

Conclusions Paramecium Bursannia meets all the requirements of life. It has an energy source in the hay infusion culture to metabalize energy to substain life. It has a membrane to separate itself from the outside world.

If the Hay Infusion Culture Grew for another two months there would be a decline in the organism because there would not be enough resources. Some organisms that are larger and faster will be able to compete for longer and they will be the only survivors. Selective pressure would affect the weaker less fit organisms. They will die off and will not be able to reproduce offspring.


1.27.15 Good first lab book entry. Need to include more detail, addressing all red text in lab manual and organize into Purpose, Materials & Methods, Data & Observations and Conclusions. Also need to include diagram of transect and list 5 biotic and 5 abiotic features. SK

Lab 1 Biological Life at AU 1/15/15

This lab was performed to observe characteristics of a niche. Different niches were looked at by different groups. A niche is an environment that includes biotic life and abiotic materials. There is interaction between the biotic life and the abiotic materials to create an ecosystem. Dirt and plant samples were collected from each respective transect for further observation and study for future experiments. The area of each transect was 20 by 20 and was predetermined by the Lab instructor.

My Group Transect Characteristics The area was a 20 by 20 with tall grass throughout the area. The AU president house and Cassell Hall and Hughes Hall surround the transect. This area is considered North Campus. Biotic features included insects, moss, grass, trees and fungus. The abiotic features included benches, rocks, metal, dead leaves, and wood. Locations The fungus was growing on the trees. Moss, grass and insects were found throughout the transect. There was a rock/stone path on the side of the transect closer to South campus. There were two benches next to the rock/stone path. There was a metal sign in the corner of the transect. There were also dead leaves throughout the transect.

A 50ml conical tube was filled with a dirt sample and vegetation sample. Back in the lab a Hay Infusion culture was prepared.


First Time on Open Wet Ware Notebook 1/22/15 12:58pm Data about niche and lab 1+2 will be entered shortly. -PT