User:Nicholas M. Scurato/Notebook/Biology 210 at AU

16S Sequence Analysis (Feb 25)

In Lab 3, we took the genetic information of the bacteria from our transect and multiplied the amount by the process of PCR. This sample of DNA was sent to a lab which was able to get a readout of the DNA sequence. When it was returned, we were able to take the DNA sequence and paste its forward series of nucleotides into a website called BLAST (which stands for Basic Local Alignment Search Tool). This website is able to read the sequence that you paste in and come up the organism or organisms that are closest to this sequence. The two sequences from our transect revealed that we had Flavobacteria and Epilithonimoras. When we look back at the observations we made from the third lab, we can infer which plate had the either identified bacteria. A quick search on the internet reveals that Flavobacteria are gram negative (Wildpro). Based on that information, we can infer that Plate 3 without Tet, which was the only plate with a gram negative bacteria contained Flavobacteria.

References:

Boardman, S. (n.d.). Flavobacterium.

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Lab 5: Invertebrates (Feb 11)

Purpose: The purpose of this lab is to be able to identify invertebrates from our transect and get a better understanding of the group of invertebrates. We collected the invertebrates from our transect by setting up a Berlese Funnel the week before which has the primary function of collecting any microscopic organisms in a sample of ground transect.

Materials/Methods: The major player in this lab was the Berlese Funnel. This was assembled by attaching a funnel to a conical tube. The conical tube would hold 25 mL of a mixture of 1/2 water, 1/2 ethanol. this conical tube with the mixture would later hold the invertebrates as well. The conical tube would be attached to the funnel by parafilm and tape. The funnel would have a screen taped to the bottom to hold the large samples in the funnel but to allow the smaller invertebrates to fall down into the conical tube. With the sample of leaves, soil, etc. in the funnel, the entire apparatus would sit under a light fixture. The purpose of the light fixture is to help the invertebrates travel down and fall into the conical tube. We also used a light microscope to observe the organisms.

Data and Observations: Organsim: 1. Springtail primitive insect. We found one in the sample and it was collected from the top sample. It had very noticeable antennae. 2. Thrips. We found two in the sample and it was collected from the top sample. We noticed wings. 3. Soil Mites. We found four and it was collected from the bottom sample. We noticed it had 8 legs. 4. Beetle Larvae. We found one or two and it was collected from the bottom sample.

Conclusion and Future Directions: With the assistance of a chart that matched the possible appearance of the microscopic invertebrates with a name, we were better able to identify the organisms.

Vertebrates and Niches We noticed, by continual traversing by our transect, that larger, vertebrate animals live and use the transect as a niche. Some vertebrates include, but not limited to: Birds, squirrels, the occasional dog, humans, and possibly little magical night elves that grant wishes and are known for gossiping.

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Lab 4: Plantae and Fungi (Feb 4)

Purpose: The purpose of this lab is to gather information of funtion and characteristics of various plants and fungi. To do this, we have to return to our transect and collect a variety of plants as well as examine prepared slides of plants and fungi.

Materials/Methods: This lab requires about 500 grams of plant material from the transect, or a Ziploc bag full of the leaves, grass, etc. from the transect. In order thoroughly examine the plant cells, we need to use a light microscope. This is so we can observe the xylem and phloem and better determine if the plant is a eudicot or monocot. This lab also requires a dissecting microscope to properly observe the overall structure of a mushroom at a greater magnification.

Data and Observations: Transect sample plants: 1. Long and relatively narrow, brown, large and tough. three inches in width and seven and a half inches in length. Vascular bundles are scattered, and it is a gymnosperm with seeds (monocot). 2. Broad leaf, brown and large. five and a half by five and a half inches for both parameters. A network of veins and is a gymnosperm with seeds (dicot). 3. About a half an inch in size, small, green, patchy, and star shaped. Rhizoids, and alternation of generations (Bryophytes). 4. Similar to number 1 5. Similar to number 2 We observed the leaves were all brown in color, but this is because this is the winter season. We suspect these leaves were green in the spring/summer, and then changed colors to purple, red, or yellow before turning brown as the temperature decreased. The fungi observed under the light microscope (Rhizopus Nigricans, Sporangia) was very interconnected and blue/green in color. This color is most likely from a dye, rather than being the natural coloration of the fungus.

Conclusion and Future Directions: We were able to get a better understanding of fungi and the plants in our transect (as well as plants in general) after engaging in this lab. Future directions included setting up a Berlese Funnel to collect invertebrates that live in our transect.

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Lab 3: Microbiology and Identifying Bacteria with DNA Sequences (Jan 28)

Purpose: The purpose of this lab is to gain a greater understanding of various aspects of bacteria such as their morphology, movement, structure to function correlation, etc. This lab is also intended to help us understand the relationship between bacteria and antibiotics. I predict that if there are bacterial colonies growing on the agar plates that have Tetracycline, then those are colonies of bacteria that are mutated genetically so that they are resisted to or insensitive to the degenerative effects of Tetracycline. Furthermore, I predict that if there are colonies of bacteria growing on the agar plates without Tetracycline, then it's possible that some of the colonies also have that mutation, while the others do not have that mutation.

Materials/Methods: Originally, we took a sample of a colony from two agar plates with tetracycline and from two agar plates without tetracycline totaling four slides with samples from colonies that were on different plates. with the samples on the slides, we stained the samples using Gram stains. This iss to better distinguish between bacteria that are gram-positive (which will show up blue/purple with the stain) and gram-negative (which will show up pink with the stain). After staining the samples, we observed the slides using a microscope and tried to identify and record the bacteria from each sample. Finally, we prepared for the next lab by setting up DNA sequences from bacteria by process of Polymerase Chain Reaction (PCR). That involved putting a sample of bacteria into a tube filled with 100 μL of water. There will be one tube for each plate that we observed from (the two with tetracycline, and two without). In our case, it will be only three tubes because one plate did not have any colonies of bacteria for us to observe from, so we would not be able to collect any DNA from that sample- or lack thereof. Once there was a sample from all three acceptable plates, those tubes were put into a heat block for about 10 minutes to denature the DNA of the bacteria. After the 10 minutes, the tubes are put into a centrifuge for five minutes, and while this is going on we will add about 20 μL of the primer to a tube with a PCR bead to dissolve the bead. After five minutes of centrifuging, the tubes are removed and five μL of the now isolated and ready DNA from the tubes are removed and placed in the smaller tubes with the primers and PCR beads dissolved. These tubes are stored for next week's lab.

Data and Observations: With the Hay Infusion Culture, we observed the liquid went from dark brown and translucent to dark brown and opaque. There was also a thicker crust still forming on the top of the sample and the rotten swamp smell was still present. The plates had yellowish orange colonies forming and the occasional colony of mold because of the storage conditions and condensation forming inside the closed petri dish. Plate 4 with Tet, we observed from staining that it had gram-positive bacteria which we identified as Sarcina. Plate 3 with Tet showed no colonies so we inferred there was no life growing in plate 3 with Tet. Plate 4 without Tet had similar results as plate 4 with Tet. They both had the gram-positive bacteria Sarcina. Plate 3 without Tet we noticed had gram-negative bacteria which we identified as hypha. Plate 1 w/ Tet had 0 colonies Plate 2 w/ Tet had 0 colonies Plate 3 w/ Tet had 0 colonies Plate 4 w/ Tet had 50 colonies (about 50,000,000,000 colonies/mL)

Plate 1 w/out Tet had 0 colonies Plate 2 w/out Tet had 25 colonies (about 2,500,000 colonies/mL) Plate 3 w/out Tet had 132 colonies (about 1,320,000,000 colonies/mL) Plate 4 w/out Tet had 50 colonies (about 50,000,000,000 colonies/mL)

Conclusion and Future Directions: Future Directions included the set up for next lab with Polymerase Chain Reaction (PCR) to later identify the DNA sequences of each bacteria identified.

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Lab 2: Identifying Algae and Protists (Jan 21)

Purpose: The general purpose (which is common among all the labs) of this lab was to identify the microbial organisms from our transect that grew in the Hay Infusion Culture the lab before.

Materials/Methods: We would look through prepared slides of various organisms, algae and various basic protists, that could possibly be in our transect and practice identifying them by use of a dichotomous key (a list of morphological features that narrow your search of the identity of the organism you are observing). aside from the prepared slides, we also observed the Hay Infusion Culture that stayed in the lab for a week. We looked at the appearance of the culture in the jar and noted its smell as well. After observing the Hay Infusion Culture, we prepared for the next lab. This involved gathering samples from the culture, both on the top and bottom of the jar, and spreading the sample onto agar petri dishes. four of the eight dishes would have tetracycline while the other four would not.

Data and Observations: We observed the appearance and noted the smell of our sample garnered from our transect. After a week in the lab, mold was growing on the top, and there was a crusty layer on the top. The water turned to a dark brown but still translucent. Not only did it look "swampy", but it also smelled distinctly "swampy". When we took a sample of the water in the jar, we observed the possible organisms living in the sample. With the help of the dichotomous key, we noticed Paramecium Candatum, Colpidum, and Volvox. The plates included four sets of two identical concentrations (one had Tet and one did not). The sets had dilutions of 10^-3 (or 1:1,000), 10^-5 (1: 100,000), 10^-7 (1: 10,000,000), and 10^-9 (1: 1,000,000,000).

Future Directions: Mentioned previously, we prepared for the next lab by using our sample from the transect from the first lab. Our next lab would involve us growing colonies of bacteria from our Hay Infusion Culture onto petri dishes. To be able to observe the colonies by next lab, we would need to properly spread the bacteria onto the agar plates during this lab.

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Lab 1: Biological Life at AU (Jan 14)

Purpose: The Purpose of this lab involves two parts. The first part, where we observed three specifics organisms from the Volvocine line, was to support the theory of evolution by examining organisms with different reproductive mechanisms and form. the second part, where we split up into small groups and observed specific locations of nature called transects, was done as a requirement for other labs to come and to understand the possible organisms that would live in these areas or niches. In terms of the first part of this lab, I hypothesize that the more complex the reproductive mechanisms and form are of an organism is, the more evolved it is.

Materials/Methods: For the first part of the lab- We used microscopes and prepared slides for each of the three organisms to observe the form of the cells. The three organisms include Chlamydomonas, Gonium, Volvox. For the second part of the lab- We were put into groups of three or four people and assigned a specific location or transect about 400 sq meters in size containing both abiotic and biotic elements. My transect was number 2, called the Wildlife reserve. When in the transect, we were supposed to record and collect at least five abiotic (or non living components) and at least five biotic (or living components) that make up that particular ecosystem. When we finish collecting both abiotic and biotic components, we head back to the lab and start a Hay Infusion Culture. Hay Infusion Culture involves putting about 10 to 12 grams of the sample you collected into a jar with 500 mLs of bottled water. After that, we add roughly 0.1 grams of dried milk as a nutrient for any microbial organisms gathered in the sample and gently mix for 10 seconds.

Data and Observations: For the first part of the lab- File:VolvocineNick.jpeg

For the second part of the lab- File:Nicktransect2.jpeg This is a picture of my transect. File:Drawn.jpeg This is my interpretation of my transect.

Conclusion: This lab helped support my hypothesis for the first part of the lab, where we looked at three specific organisms from the Volvocine line of Chlorophyta. My hypothesis was that the organisms with the more complex reproductive mechanisms and form are more evolved. The Volvox- considered the most evolved of the three had a multicellular structure with oogamous reproductive mechanisms. In contrast, Chlamydomonas- considered the least evolved of the three is unicellular in structure with isogamous/asexual modes of reproduction. Future Directions: The Hay Infusion Culture will continue to grow, and will be used as an integral part for later labs.

Transect #2: Biotic: 1. A tree 2. Moss 3. Tall bushy grass 4. Grass 5. Bush

Abiotic: 1. Rocks 2. Dirt 3. Snow 4. Bench 5. Sign

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1/21/15

This is Nick Scurato. I have successfully submitted to OWW.

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