User:Ricardo M. Rodriguez/Notebook/Biology 210 at AU

March 2, 2015 16s DNA Sequence

Purpose: The purpose of this lab is to identify the unknown species that was growing in the tetracycline agar plates. These unknown species came from the transect we took our sample from, the community garden at American University. We identified these bacteria through DNA 16s sequencing.

Methods: Two single colonies from each of the two plates containing tetracycline and one without were chosen. The DNA from the two selected plates were isolated and amplified through PCR technique for 16s sequencing. After one week, it is expected for the PCR products of these species to be run in a gel and then sequenced. In order to identify the bacteria, the sequence is put into NIH’s “BLAST” online identification system. This program is able to accurately identify species once students paste the nucleotide sequence. Immediately after, students will be able to find the 16s DNA sequence and describe which bacteria are there.

Results:

Figure 1 shows the gel samples of the bacteria tested

From this picture, Samples A and D are in columns 2 and 3.

The sequence for Sample A is:

NNNNNNNNNNNNTNNNNNNNGNNNCTCCTNNNACGNGTCACCGNCTTCAGGTACCCCATNNNNNCCATGGCTTGACGGGC GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGCCATGGCTGATGCGCGATTACTAGCGATTCCAGCTTCATAGAGTCG AGTTGCAGACTCCAATCCGAACTGAGACCAGCTTTCGAGATTCGCATCCAGTCACCTGGTAGCTGCCCTCTGTACTGGCC ATTGTATTACGTGTGTGGCCCAAGGCGTAAGGGCCGTGATGATTTGACGTCATCCCCACCTTCCTCTCTACTTGCGTAGG CAGTCTCACTAGAGTCCCCAACTGAATGATGGCAACTAGTGACAGGGGTTGCGCTCGTTGCAGGACTTAACCTAACACCT CACGGCACGAGCTGACGACAACCATGCAGCACCTTGAAAAATGTCCGAAGAAAAGTCTATTTCTAAACCTGTCATTTCCC ATTTAAGCCTTGGTAAGGTTCCTCGCGTATCATCGAATTAAACCACATAATCCACCGCTTGTGCGGGCCCCCGTCAATTC CTTTGAGTTTCAGACTTGCGTCCGTACTCCCCAGGTGGCTAACTTATCACTTTCGCTTAGTCTCTGAATCCGAAAACCCA AAAACGAGTTAGCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGTCCATCAGCG TCAGTTGTTGCTTAGTAACCTGCCTTCGCAATTGGTGTTCTAAGTAATATCTATGCATTTCACCGCTACACTACTTATTC CAGCTACTTCAACAACACTCAAGACCTGCAGTATCAATGGCAGTTTCACAGTTAAGCTGTGAGATTTCACCACTGACTTA CAGATCCGCCTACGGACCCTTTAAACCCAATAAATCCGGATAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACG GAGTTAGCCGGTGCTTATTCGTATAGTACCTTCAGCTTNCCACACGTGGAAAGGTTNNTCCCTATACNAAAGAAGTTTAN NNCCATNNGNNNTCGNCTTCACGCGGGNTGNTGGNTCAGNTCTCANCCNTGNCNANNTCNTCANNGCTGCNTCCCGNAGN ANTCNGGNCCNNNNNNTCAGNACNNNNNGNGGGGNANNCN

For Sample D

NNNNNNNNNNNNGNNNTTANNNNTGCAGTCGNNCGANNGAGTAGCNCNNNNTNNCGGACGCTGACGAGTGGCGAACGGGT GAGTAATACTATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATACGATCTGAGGAT GAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCA AGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAAC TGCTTTTGTACGNAACGAAAAAGCTTCTCCTAATACNAGAGGCCCATGACGGTACCGTANNAATAAGCACCGGCTAACTA CGTGCCANCAGCCGCGGTANTACGNANGGTGCGAGCGTTNATCGCGANTTTACTGNGCGTAAAGCGTGCGCANGCNGNTA TGTNANGACANNATGTGAAATCCCCNNNNTCTNTCCTGNTNAACTGNCNTTTGTGNCTGCNNGGNTAAANTACGGNAGAG GNGTGNTGGAAT

According to NIH BLAST, sample A is "Chryseobacterium sp. C” 16s ribosomal RNA gene and Sample D is identified as “Uncultured bacterium clone”16s RNA gene

Conclusion

Chryserobacterium indologenes is a yellow pigmented, Gram-negative filamentous, non-motile rod and can be found in soil, plants, foodstuffs and water sources including those found in hospitals (Hsueh, P.R., 1996). This makes a lot of sense because our sample was taken from a number of things such as soil, plants and dead leaves. This bacteria is also known to resist antibiotics such as tetracycline. This is very intriguing because this bacterium was taken from the plate that had tetracycline and it grew despite the presence of it. However, Sample D is unidentified. From what was researched, it was found that it is considered and uncultured bacteria.

References

Hsueh, P. R., Hsiue, T. R., Wu, J. J., Teng, L. J., Ho, S. W., Hsieh, W. C., et al. (1996). Flavobacterium indologenes bacteremia, Clinical and Microbiological Characteristics. Clinical Infectious Diseases , 23, 550-555. doi: 10.1093/clinids/23.3.550

R.M.R

February 18, 2015 Lab 5: Invertebrates 2.0

Purpose

Now that we have studied and identified different types of life in transect #4, our final element for our study is to consider the vertebrates that inhabit and pass through our transect.

Methods

The observations of the transect were made at 4:30 in the afternoon on February 12, 2015. The day was cloudy and windy with the temperature being at about 25°F.

Results

The organisms we saw that day were: Deer footprints and droppings. The footprints were similar to deer hooves. Odocoileus virginianus Squirrel, Sciurus carolinensis Robin, Turdus migratorius Dog, Canis familiaris Cardinal. Cardinalis cardinalis Earthworm. Lumbricina

Discussion & Conclusion

The fact that we had a farm land for our transect means that we would attract many organisms since it is a great source for food. The biotic characteristics such as the vegetables growing are very beneficial to the organisms such as the deer and the squirrel. The abiotics characteristics such as the fertile soil are beneficial to the earthworm because it supplies shelter to them. In addition, the cardinals can also benefit from the soil since it’s a good platform for seeds that they can feed of. The dog was there with its owner but it could use many of the transects resources such as soil to dig around and wood chips to play with.

This diagram is a food web based on all of our organisms we observed.

R.R

February 18, 2015 Lab 5: Invertebrates 1

Purpose

The purpose of this lab is to appreciate and understand the surprising complexity and beauty of the invertebrates and to learn how the vertebrate organ systems evolved from these simple organisms. I hypothesize the invertebrates we will find will most likely be worms or beetles since our sample includes a lot of rich soil and vegetables.

Materials and Methods

The materials we used for this lab included, Planaria sample, Dissecting scope, Nematodes, Annelida, Preserved example organisms, Berlese funnel prepared during Lab 4, and a Dichotomous Key.

Procedure 1: Observe the Planaria with the dissecting scope and note the type of movement these worms use and how it reflects the simplicity of this organism. Additionally, examine the cross section of the whole mount of the Planaria with the microscope. We do the same for the nematodes. We observe them and a cross sectional slide of their pseudo-coelomate structure and describe their movement.

Procedure 2: We observe the example organisms from each of the five major classes: arachnida, diplopoda, chilopoda, insect and crustacean. Each of these five classes are These classes are defined by body parts, body segments, and number of appendages.

Procedure 3: We break down the Berlese Funnel and gently pour the top 10-15 mLs of liquid and organisms into one petri dish. We then try to identify the class of any Arthropoda invertebrates we observe.

Data & Observations

The movements of Planaria were pretty smooth. The tended to slide across the tray, which is very predictable since they are worms. This might be due to their bodies being cylindrical and elongated, with no signs of muscles. The nematodes moved like the Planaria but slower. We compared their movements to somewhat of a worm because they would squirm in place. Their bodies are wide in the front and become more tapered at the end, which is why their movement is more, concentrated on their upper body. The annelids are very long and thin with pink rings of muscle in the surroundings. When they move they tend to wiggle and twist with their body sort of imitating a slinky.

In this table we have the measurements of each organism taken from our Transect (farmland) with the ocular micrometer.

Conclusion

The organisms lengths ranged from 2-4mm long and the longest was the springtail measuring at 4 mm with the arthropod being the smallest measuring at 2 mm. The most common one we found were worms (nematodes) since there were 10+ of them in our sample.

RMR

February 09, 2015 Lab 4: Plants and Fungi

Purpose/ Hypothesis

The purpose of this lab is to understand the characteristics and diversity of plants and to also appreciate the function and importance of Fungi. Furthermore, we will learn the function and responsibility of fungi in its environment. My hypothesis is that unlike fungi, our transect wouldn’t be able to grown in extreme conditions.

Materials and Method

For this lab, we used moss, Ziploc bags, agar plate of black bread mold (Rhizopus stolonifer), 50:50 ethanol/water solution, a funnel, screening material, and a 50 mL conical tube.

Method 1: For this first procedure, we had to get three Ziploc bags and proceed to our transect. Once we get there we pick up dead leaves and soil and only put about 500g of leaf litter into one bag.

Method 2: Now we observe the moss. While comparing the moss provided with the angiosperm (lily), we compare and contrast their xylem and phloem and examine the cross section of a lily stem and find the xylem and phloem.

Method 3: Once we are done with the previous procedure, we then examined the moss with a dissection scope and noted the cuticles that are shown by its smooth surfaces.

Method 4: For this method, we exam the Polytrichum. While doing this we identify the male and female gametophytes and sporophyte. We then dissect a lily flower and identify the anther, pollen, stigma, and style. With this, we observe and record information on the plant samples taken from our transect and record their location in the transect, size and shape, vascularization, specialized structures, and mechanisms of reproduction.

Method 5: Lastly, we pour 25 mL of the 50:50 ethanol/water solution in the 50mL conical tube. We fit a piece of the screening material into the bottom of the funnel and tape the sides. Next, we dump the leaf litter sample in the funnel and set up the funnel on a ring stand so that it's held in the tube with ethanol. Wrap parafilm around the spot where the tube and funnel meet to prevent the ethanol from evaporating and put it under a 40 watt lamp. Cover everything with foil then leave the Berlese funnel on the lab bench for one week.

Data and Observations

This image is a drawing of our transect where we took our samples from.

This table shows the observations made about each plant sample taken from the transect. We believed sample #1 to be of the genus Brassica. Sample #2 is also the genus Brassica. Sample #3 is the genus Trefolium. Sample #4 is Lycophyta. Sample #5 is Lactuca. As you can see from the table, sample 1 has leaves that were ruffled and spread out and itt was very close to the ground when we picked it. Sample 2's leaves were rolled up pretty tight in small balls, and attached to a 2.5 foot-high stalk and had spores on the underneath it. On sample 3, the leaves were grouped in a cluster of three, and spread out evenly from one another and stood about 2 inches off the ground. Sample 4's leaves stood by themselves, apart from each other, and were dispersed sparsely on the plant's woody stem. The plant was about 2 feet tall. Last but not least, the leaves on sample 5 were all clustered in a large, loose ball, about 5 inches off the ground.

This image is what i drew of the fungi we observed under the microscope.

Conclusion

It was a very simple lab overall, but my lab partners and i had trouble finding the seeds of some of some of our samples. But this could be due to the fact that we only took samples from vegetables and the dead leafs on the ground. With our sample though, each plant sample was identified as either dicot or monocot using the appearance and that was pretty easy to do. My hypothesis was supported through this lab as well. Our samples were taken from a very mild condition. I think that if it was warmer outside however, we would have more options since more plants would be growing at the time.

February 4, 2015 Microbiology and Identifying Bacteria with DNA Sequences

Purpose

The purpose of this lab is to understand the characteristics of bacteria and to observe antibiotic resistance. Ultimately, we will use that to understand how DNA sequences are used to identify species. Since we already observed the unicellular organisms from the Eukaria domain in our Hay Infusion cultures last week, it is now time we study the prokaryotes that are grouped in the Domain Bacteria. My hypothesis for this lab is that i do not believe Archaea species have grown on the agar plates because our Hay Infusion Culture sample was not taken from an extreme area. A

Materials

For this lab, we used a variety of things such as bacterial colonies plated last lab (8 plates), sterile tube, light microscope, heat block, prepared slides of bacteria, centrifuge, loop,PCR tube, Bunsen burner, primer/water mixture, crystal violet stain, PCR machine, Gram's iodine mordant, 95% alcohol, and safranin stain.

Methods

PROCEDURE 1: For this first step, we had to observe the growth on the agar plates that were taken from our Hay Infusion Culture. With this information, we will count the total number of colonies on each plate. If there is a large number of bacteria, all the colonies will grow together and therefore form a lawn.

PROCEDURE 2: For this second step, we observed some prepared slides of bacteria and then observed both a native wet mount preparation and a gram stain of two well defined colonies from the nutrient agar plate and two from the tetracycline plate. In order to do this we first had to make a wet mount and observed it using the 10X and then with a 40X objective. To make a wet mount, we first sterilized a loop over a flame and used it to scrape up a little amount of growth from the surface of the agar. We mixed it into a drop of water on a slide and covered it with a cover slip.

PROCEDURE 3: This procedure got a bit more complicated. This was when we had to start the Gram Stain procedure. For this we first sterilized a loop over a flame and used it to scrape up a little amount of growth from the surface of the agar again. Add a drop of water, then circle the area underneath the sample with a red wax pencil. We then heat fix the air dried slide by passing it through a flame three times with the bacterial smear side up. Next, we put it on a staining tray and covered it with the crystal violet solution for 1 minute then rinsing it with water. We repeat the previous steps but instead of pouring crystal violet on the slide for 1 minute then rinsing it off. The next step includes decolorizing the sample. We did this by flooding the bacterial smear with 95% alcohol for 10-20 seconds. We then cover the smear with safranin stain for about 30 seconds then rinse it off. With our newly stained sample, we use it to observe it under 40X and the 100X oil immersion objectives.

PROCEDURE 4: Now that we have characterized four bacteria from our Hay Infusion Cultures, we then had to select one from each of the two plates that had the most complete characterization, isolate the DNA and use the PCR to selectively amplify the 16S rRNA.

Data and Observations This time, our Hay Infusion Culture smelled worse like a sewage. The water wasn't as high as it used to be and more things were collected at the bottom. As stated in my hypothesis, i do not think any archaea species will have grown on our agar plates. Archaea grown in extreme environments and our Hay Infusion Culture sample was not taken from a place under extreme environment. Also, I think the reason for why the appearance or smell of our sample might change from week to week is because of their frequent use of metabolism and the fact that these microorganisms keep multiplying.

This picture is a good example of how fast these microorganisms multiply. With this table we were able to realize how the samples with Tet had less colonies than those without Tet. Furthermore, the colonies without the antibiotic are lighter and less opaque. The colonies with tetracycline are opaque (yellow colored), larger, and more sparse than those without it.This might be the cause of the antibiotic and the microbial being insensitive towards it. Tetracycline sparked the development of many chemically altered antibiotics, so has proved to be one of the most important discoveries made in the field of antibiotics. It is used to treat many Gram-positive and Gram-negative bacteria. Like some other antibiotics, it is also used in the treatment of acne (WHO, 2013). Back to the topic, The colonies picked for characterization were 10^-3 with tetracycline part of colony A, 10^-5 with tetracycline in colony B, 10^-7 without tetracycline in colony C, and 10^-3 without tetracycline in colony D.

From my observations, the cells without yet are more mobile. Here is a better description on our findings....

Conclusion

I found this lab in particular very difficult. Not only were the instructions very vague, but we didn't know how careful we had to be when handling some of the samples. Although we did burn one sample during the gram staining, we were able to do it again and succeed. On the other hand, we did walk of this lab with a better understanding of the concept behind what we were doing.

R.M.R

"WHO Model List of EssentialMedicines". World Health Organization. October 2013. Retrieved 04 February 2014.

January 26, 2015 Identifying Algae and Protists

Purpose

The purpose of this lab is to practice using a dichotomous key to identify unknowns, understand the characteristics of Algae and Protists, as well as examine them from our transect. It is important to have this set of skills because it’ll help us to further understand our ecosystem. Hypothesis: Because we have such a diverse amount of soil and vegetation in our 500mL beaker, we will find a lot of different bacteria.

Materials and Methods The materials used for this lab were:

Light microscope, dichotomous key, known organism, Hay Infusion Culture, micropippetor, four nutrient agar plates, and four nutrient agar plus tetracycline plates.

Using the Dichotomous Key

Method 1: We make a wet mount of known organisms and observe them under a microscope and measure its size.

Method 2: Afterwards, we get a hold of the dichotomous key and try to identify the organism using the information we collected from the microscope.

Hay Infusion Culture Observation

Method 1: With our culture in front of us, we note the smell of our sample (putrid smell) and described its appearance.

Method 2: We take samples from two different niche as well as including plant matter in our observation. We make a wet mount from the two different niches and determine what protists and algae are present with the help of our dichotomous key and draw pictures.

Preparing and Plating Serial Dilutions

Method 1: Label four tubes of 10 mL sterile broth with 10^-2, 10^-4, 10^-6, and 10^-8. Set a micropippetor to 100 microliters. Obtain four of each kind of agar plate--label all of the tetracycline plates with "tet." Label each of plate with their respective numbers, and add your lab group's info on the plate. Swirl the Hay Infusion Culture--with the lid on-- to mix up the contents.

Method 2: Add 100 microliters from the culture to the tube labeled 10^-2. Mix the tube thoroughly. Add 100 microliters of broth from this tube to the 10^-4 tube and swirl to mix well. Repeat this twice to make the 10^-6 and 10^-8 test tubes. Pipette 100 microliters from each tube onto their respective nutrient agar plate (10^-2 goes on the plate labeled 10^-3, etc.), and spread it around the plate carefully.

Method 3: Repeat this procedure for each nutrient agar plus tetracycline plate. We then place the agar plates in a safe spot in the lab where they will incubate at room temperature. Image:Serialdilution.jpg

Data and Observations

As told in the procedure, the smell coming off of our culture was putrid. It smelled like spoiled veggies and cheese. With the life forming on top of it, brown and gooey, with a thin skin on top and soil resting on the bottom. There was a skin-like structure on the top of the liquid with vain like appearance. As you can see from the image below, there are some apparent life growing on top of the liquid such as mold and green shoots.

Image:Screen Shot 2015-01-29 at 12.34.29 PM.png

Here is a more detailed drawing of our observation: Image:Hayinfusion.jpg

Sample 1: Image:Sample3.jpg From our observations, we noticed that this sample contained a non-motile species that we had trouble identifying with the dichotomous key. It had some green parts, leading us to believe it was a species that performed photosynthesis. Its size was 50 micrometers.

Sample 2: Image:Sample2.jpg This sample contained a diverse amount of organisms including colpidium, at least two different types of paramecia, and an organism we suspected to be some kind of worm (very long with worm like structures and a moving tail). The colpidium were about 50 micrometers in size, and the paramecia ranged from 70-100 micrometers in size.

Sample 3: Image:Sample1.jpg Just like sample 2, this sample contained more colpidium and various paramecia.

Conclusion

My hypothesis was correct to a certain point. All though we did find a variety of organisms within our sample, we only found 3 out of the 20 on the dichotomous key. This maybe a result from the limited resources we had to pick out a sample from. Maybe if we chose different samples from different areas on campus, we would have gotten a wider variety of organisms. Aside from that, the protists found in the hay infusion culture were both heterotrophs and autotrophs. It was very hard to determine the name of some of the organisms we observed. This could be solved by having a dichotomous key that would be more precise. The one we had was very broad and a lot of the descriptions on it could have been applied to many other organisms.