User:Yvette T. Mbah/Notebook/Biology 210 at AU

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3/19/2015 Zebrafish lab: Effects of Alcohol on Embryonic Zebrafish Development.

Purpose

The purpose of this lab experiment was to use the Zebrafish which has been determined to be a good model to use in the lab due to its transparency and similarity to other vertebrates to study how environmental conditions affects embryonic development. The conditioned my lab partner and I observed in this lab was the effect of alcohol on embryonic development. In order to determine the effects of alcohol on Zebrafish development, a control (deer park water) and an experimental group (1.5 % alcohol) were set up and observed for a specific duration of time. My lab partner and I observed the zebrafish development every other day and recorded our observations. During the course of our observation, we took out the dead zebrafish, counted them and also fed them. We hypostasized that alcohol would impair the development of zebrafish. From this, we predicted that if alcohol is added to the embryonic zebrafish, then they will have severe physical development.

Materials

For this lab experiment, we used two petri dishes. One for control with 20 embryos and the other for the experiment with 20 embryos. 20 mls of deer park water, 1.5% alcohol, Microscope, Paraformaldehyde and a dissecting scope.

Methods

Day 1

We set up the experiment using 2 petri dishes. In the control, 20 mls of deer park water was placed in the dish and 20mls of alcohol was placed in the experimental dish. 20 healthy translucent embryos were placed in each dish and covered to be observed throughout the week.

Day 4 and 5

10 mls of water was removed from both dishes and 25 mls of water was added. This was done because most of the solution had evaporated over the week. Dead embryos were removed from both dishes and placed in paraformaldehyde.

Day 7

5mLs of water was taken out an5 mls of fresh water or alcohol was added. Dead embryos and cases were removed from both dishes. Two zebrafish from each dish were removed and placed in a tube containing paraformaldehyde. The Zebrafish were feed 2 drops of paramecium.

Day 14

Final observations were made

Data and Observation

Day 1

All fish in experimental group alive, 2 dead in control.

Day 4-5

Control: 3 dead in control group, leaving 15 fish. 2 or 3 unhatched, all the rest hatched and immobile but heart beat visible. 1 mobile fish. About .5cm in length, heart rate at 90 beats/second. Experimental: 2 dead in control group, leaving 18 fish. 2 or 3 unhatched, all the rest hatched and very mobile with heart beat visible. More hyperactive in movement than control. Heart beat at 135 beats/sec.

Day 7

Control: 10 fish dead in control group, leaving 5 fish. All hatched. Mobile with visible heartbeat. About 1.2 cm in length, heart rate at 95 beats/second Experimental: 13 dead in experimental group, leaving 5 fish. One unhatched but dead. Mobile with visible heartbeat, moving in circular motion as opposed to straight. Heart beat at 120 beats/second.

Day 14

All of the fish were dead.

Conclusion

We concluded that zebrafish were a viable model organism and alcohol definitely impairs embryonic development in zebrafish.

YTM


03/ 05/2015 PCR for 16s Sequencing

Purpose

The purpose of sequencing two samples of our unknown bacteria was to help us better understand PCR and use it to analyze our gene sequence from our bacteria. PCR has always been fun for me because you amplify a specific portion of your gene and can better understand and view it why also getting hands on experience. Materials and Methods

Earlier in the semester, we had created a Hay Infusion with leaves from transects and this hay infusion culture was used to create agar plates with serial dilutions. Small samples were taken from the agar plate and placed in a sterile tube and incubated at 100 degrees for ten minutes in a heat block making sure it floated in water at the same time. Next, the samples were put in a centrifuge for 5 minutes at 13,400rpm. 20 micro liters of water was added to the tube and mixed during centrifugation. Finally, 5mcro liters of supernatant from centrifuge sample was transferred to the 16s PCR sequence and the tube was placed in the PCR machine using agarose gel. We selected the most visible gels (vile A and D) for sequencing. We used http://blast.ncbi.nlm.nih.gov to copy and paste our raw sequence in order to identify the bacteria.

Results

Sequence vile tube 13A MB65 (Variovorax sp. ML3-12 16S ribosomal RNA gene, partial sequence)

NNNNNNNNNNNNNNNNNNGNNNTNNANNNNNNNNNTGCNNCTNNNNNTNNNCTACTTCTGGCAGAACCCGCTCCCATGGT GTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGTGACATTCTGATCCACGATTACTAGCGATTCCGACTTCA CGCAGTCGAGTTGCAGACTGCGATCCGGACTACGACTGGTTTTATGGGATTAGCTCCCCCTCGCGGGTTGGCAACCCTTT GTACCAGCCATTGTATGACGTGTGTAGCCCCACCTATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTT TGTCACCGGCAGTCTCATTAGAGTGCCCAACTGAATGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACC CAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTACGGTTCTCTTTCGAGCACTAAGCCATCTCTGG CGAATTCCGTACATGTCAAAGGTGGGTAAGGTTTTTCGCGTTGCATCGAATTAAACCACATCATCCACCGCTTGTGCGGG TCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTTCGTTACTGA GTCANTGNNNACCCNNNNNNCAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGC TTTCGTGCATGAGCGTCAGTACAGGTCCAGGGGATTGCCTTCGCCATCGGTGTTCCTCCGCATATCTACGCATTTCACTG CTACACGCGGAATTCCATCCCCCTCTACCGTACTCTAGCTATGCAGTCACAGATGCAGTTCCCAGGTTGAGCCCGGGGAT TTCACAACTGTCTTACATAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCNANNNCGCNNCACCCTACGTATTACCGC GGCTGCTGGCACGTANTTAGCCNNTGCTTATTCTTANNNNCCGTCATTAGCCCTCTTATAGAAAAGCCGTTTCNTCNTAN AAGCAGTTANACCCGANNTCATCNGCANGCGGCATGNTGGATCAGCTTTCNCCNTNTN


Sequence vile tube 13D MB66 (Chryseobacterium sp. MH gene for 16S rRNA, partial sequence)

NNNNNNNNNNNNTNNNTGTAGCGNACNNNNNNNGTCTNNTGGATTCGGGCCGCCNNTACTATATAGNGTNGTTGTCTGCC TGTACCAGGAACGGGANNAAACGTCGTATTTNGGNAGATGGGCGCAACCGGAGGTTGGACGAATTTGAATTATAAGGTGN CANTCCNATNGCAAATGAGNCCGGNACTGCAGATNNGATTAGCGCTTCACCGGGAAGTGCNCTGATGTAACTTTGTAGGA GNGTGAGGTCCNATGATCGTTATTATGGATGGNTTGATTGGAATAATGATATGGATTTTAATGATGCANNAGAAACTATT CTTGCTACAACTTAAAGGTTTAAACTAGTGACAGGGGTTGCGCTCGTGTACCGANNTAACCTAACNATNGAAATTACCGG GTGAGGTTTGCATGCCAGGNTNGGGTGCTGCCGCTCCGTGGGGACATTTTCCATACGATAATTTCCTATATTATCCTTGG TAAAGTGCCGCGCGTACCACCTAAATACACCACATAATCCACCGCTNGTGCGGGCCCTCTTCANTTCCNTTGAGTTTCAT TCNNGCNAANGNACTCCCCNNNNNGNNNANTTATCANTTTCNCTAANTCACTGANNCCNAAGANCCNGANNGANNNNNAN NNTNANAANNNNAGTGGACTACCANGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGTCCATCAGCGTCAGTTAAGACA TAGTAACCTGCCTTCGCAATTGGTGTTCTAAGTAATATCTATGCATTTCACCGCTACACTACTTATTCCAGCTACNTCTA CCTTACTCAAGACCTGCAGTATCAATGGCAGTTTCACAGTTTAAGCTGTGAGATTTCACCACTGACTTACAGATCCGCCT ACNGACCCTTTAAACCCAATAAATNCNNANAACGCTNGCACCCTCCGTATTACCGCGGCTGCTGGCACGGANTTAGCCNN TGCTTATTCGTATAGTACCTTCAGCTTTCCACACGNGGNAAGGTTGATCCCNATANNAANANNTTTANNCCCATANGGCN TCATCNTTCANGCNNANGGCTGGATCNGNTCTNACCCATTGNCCANTANTCCTCACTGCTGCCNCCCGTNNNANNNNG

Conclusion

Using the blast website, I found that the bacteria in tube 13A was Variovorax Sp. ML3-12 16S ribosomal RNA gene, partial sequence while the bacteria in tune 13D was Chryseobacterium Sp. MH gene for 16S rRNA, partial sequence. After doing more research I found very interesting information on the Chryseobacterium. According to http://blast.ncbi.nlm.nih.gov Chryseobacterium is a yellow-pigmented bacterium designated strain that was isolated from a lactic acid beverage. The strain had Gram-negative, non-motile, rod-shaped cells. It was strictly aerobic and chemo-organ tropic and grew at 5-30 degrees C and at pH 5-8. The Variovorax was described on the http://blast.ncbi.nlm.nih.gov as part of a study in which soil sample was isolated from a greenhouse. It was described as a gram-positive, non-spore forming, and a rod shaped bacterium with irregular light colonies. The characteristics I encountered for these bacteria are consistent with the characteristics that we observed in lab.

YTM


02/18/2015 Invertebrates Lab

Purpose

This lab was done to understand the importance and evolutionary history of vertebrates. The purpose of the lab was to understand the complexity of the invertebrates and to learn how the vertebrate organ systems evolved from simple organisms. To do this, we looked at the simplest animal invertebrates which are sponges (Phylum Porifera) that do not have true tissues. We also learned about Cnidaria and Ctenophora which are among the most primitive of invertebrate organisms with a radial symmetry and have tissue organization. We also studied the tree germ layers of tissues: ectoderm (forms skin and nervous system), endoderm (forms lining of digestive tract) and mesoderm (forms circulatory system).

Material and Methods

For this lab, we observed acoelomates, Planaria using a dissecting scope. Next we observed the nematodes and coelomates Annelida to study the body movement and structure of the worms.

Data and Observations

After observing the three species of worm (Acoelomates, Pseudocoelomates and Coelomates), we took down our Berlese Funnel and observed the invertebrate organisms that were collected in the tube under a microscope. We identified at least five invertebrates and studied their structure. We found that some of the organisms were motile while others were stationary. File:Invertebrate table.docx


File:Vertebrate table.docx

Conclusions and Future Directions

After observing the organisms from transects 2 under the microscope. We found out that the most common invertebrate was a fly. It was kind of difficult to differentiate between the different types of flies. The only think I can advise to do in the future is to build the Berlese funnel from leaves from a wider area of transect so as to have a diversity in the invertebrates that we can observed.

YTM


02/12/2015 Plantae and Fungi

Purpose

The purpose of this lab was to observe the characteristics and diversity of plants such as moss, Mnium, angiosperm and Lilium order to determine their Vascularization. Materials and Methods For this lab, we obtained two Ziploc bags and proceeded to transect where we collected five different species of plants and put in the first Ziploc. Next we collected leaf filter consisting of dead leaves and soft soil and filled the second Ziploc about ¾ full. Photos were taken for the collected trees, pine cones, flowers etc. found in transect to use for cataloging. We then observed fungi (in this case mushroom) under a microscope. Next, we used leaf litter to set up a Berlese funnel so that we could collect vertebrates to observe in the next class. To set up Berlese funnel, 25ml of 50:50 ethanol/water was mixed into a 50ml tube. The tube was tapped at the bottom to prevent ethanol from evaporating. A screening material was placed at the bottom of the funnel to prevent the leaves from falling into the tube. The funnel set up was then placed under a 40watts lamp and covered with foil paper to observe next week.

Data and Observations

File:Plant Sample Chart.docx File:Bacteria Characterization.docx

The five plant we collected from transect were identified as follows. Samples 1, 3 and 5 were Angiosperms (flowering plant), sample 2 was a lycophyta (seedless plant) and sample 4 was a non-vascular plant (Bryophyta) also known as moss. Since we could not identify most of the plants we collected from the transect we went on the web and found a website (Britannica kids leaf chart) to help us identify the plants.

Conclusions One thing I noticed with our plants was that though they were found in the same place, they relatively different in characteristics. They differed in shaped and sizes. We brought by red berries from transect but could not determine if the seed was a monocot or dicot because you can’t know until you look into the seed.

YTM


02/05/2015 identifying bacteria and observing antibiotic resistance

Purpose

The purpose of this lab experiment was to observe prokaryotic bacteria and the growth of bacteria on the agar plates and also to understand the effects of antibiotic (tetracycline) resistance.

Materials and Methods

For this lab experiment, we used agar plate samples that were diluted the previous week, water, microscope oil, gram iodine, 95% alcohol, safranin stain, crystal violet, Kim wipe, slide-cover slip. For this experiment, two procedures were used namely wet mount and gram stain procedure. For the wet mount experiment, we sterilized a lope over a flame and used it to scrape a portion of bacteria from agar plate. Mixed it with drop of water on the slide. Placed a cover slip and observed it under the microscope using the 10x and then the 40x objective. For the gram stain experiment, a loop was sterilized over flame and used to scrape a portion of the bacteria from agar plate onto slide. A drop of water was added to sample. Slide was slowly passed over flame still water dried. Covered bacteria with crystal violet, let it stay for a minute and rinse stain with distilled water. Covered bacteria smear with gram’s iodine for a minute and rinsed with distilled water. Covered bacteria smear with 95% alcohol for 10-20 sec. covered smear with safranin for 20-30 sec and rinsed using distilled water. Used Kim wipe to clean excess water and allowed to dry. Places cover slip on slide and viewed under microscope using low magnification and then used 40x and the 100x oil immersion objectives.

Data and observations

Do you think any archaea species will have grown on the agar plates? Why or why not? I do not think any Archaea species will have grown on the agar plate because the environment is not suitable for living. Archaea prefer to live in extreme environments such as hot springs or at the bottom of the ocean. Looking at my Hay Infusion Culture, I observed that there is less water in the jar after a week and is smells less than it did the previous week. There were more bacteria/colony on the plates without antibiotics than they were on the plates with antibiotics. This indicates that tetracycline prevented the growth of bacteria. I observed that two species of bacteria are unaffected by tetracycline. According to Tetracycline Antibiotics article, tetracycline kills inhibits protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. If bacteria are unable to undergo protein synthesis, this could account for why we only had gram negative bacteria and not gram positive. File:Yvette Serial Dilutions.docx

File:Tables.docx


Conclusion

One of the challenges that I faced in this particular lab was viewing the organisms under the microscope. It was extremely difficult to actually see the bacteria which made filling the chart difficult. In the future, I propose to manage time wisely and for students to use prepared slides because it was difficult to view anything using the wet mounts slides, and to also try to understand the concepts of the lab and not just do it for the sake of doing a lab because when you know what you are doing, the experiment is easier to conduct.

YTM


I/22/2015 Identifying Unknown Organisms Using a Dichotomous Key

Purpose

The purpose of this lab experiment was to use a dichotomous key in order to identify unicellular eukaryotic organisms in the Hay Infusion Culture. There will be more organisms at the top of the Infusion than at the bottom. If you take a sample from the top of the infusion and view it under the microscope, you will realize that there are more organisms at the top than at the bottom.

Materials and Methods

  • Made a wet mount from the Hay Infusion Culture.
  • Observed wet mount under microscope and focus on one organism at a time.
  • Used dichotomous key to identify Unknown organism
  • Used diagrams from dichotomous key to confirm identification of organisms viewed under microscope

Data and Observations'

My hypothesis was not supported. I thought there will be more organisms at the top of the Hay Infusion Culture than at the top but that was not the case. There was not a diversity in organisms. The only organism observed was the Paramecium. The only difference between the top and bottom Paramecium was their size.

Conclusions and Future Directions

The only organism that I observed from my Hay Infusion Culture was the Paramecium. In the future, I will collect wet/cold soil sample from different locations because some organisms might be found in wet soil than warm soil.

YTM


01/15/2015 Natural Selection and Observing a Niche at AU

Purpose

The purpose of this lab was to study a specific niche at American University (AU) in order to understand the diversity of organisms ans natural selection.Organisms are more likely to be found in conditions favorable to them (natural habitat) than to be found in conditions foreign to them. If an organism prefers the cold, then you are not likely to find it in warm weather and the reverse is true.

Materials and Methods

  • Observed a 20 by 20 meter area of transect # 2.
  • Described characteristic of transect.
  • Observed the different living and non-living life form.
  • Collected soil sample from transect 2 and placed it in a 50 ml tube.
  • Made a Hay Infusion Culture by placing 10-12 g of soil sample into plastic jar with 500 mls of deer park water.
  • 0.1 gm of dried milk was added to jar and mixed for 10 s.
  • labeled jar, removed lid and placed in a safe place in the lab.

Data and Observations

For transect 2, we needed to identify five biotic (living) and five abiotic (non-living) organisms. Biotic organisms observed included: leaves, plants,trees, moss and shrubs. Abiotic organisms included: stone, bench, info sign, and and dirt.

Conclusion and future directions

Transect 2 located by Hughes and McDowell Hall across from shuttle bus stop. i noticed that students were often cutting through the transect which could account for why there were not many organisms found there. In the future, i will collect soil samples from many different places. My hypothesis was supported because we mostly found only paramecium and not many other organisms.

YTM

01/21/2015

I hope OpenWetWare.org will get easier to navigate as the semester continues.

YTM