User:Sasha A. Weitzman/Notebook/Biology 210 at AU
Invertebrates and Vertebrates
Invertebrates are an incredibly diverse group of organisms, with over 14 different phyla. Sponges, of the Porifera phyla, are the simplest, as they do not form true tissues (the only of animal phyla) and are stationary beings. Also primitive are Cnidaria and Ctenophora, as they are among the few that have radial symmetry, or a sphere lacking a back or sides, instead of bilateral symmetry. Bilateral symmetry, on the other hand can be characterized by the presence of sides as well as head and tail. However, one advantage to the Cnidaria is the presence of cnidoblasts and nematocytes, which allow the organisms to sting. Finally, Cnidaria are missing the mesoderm germ layer, and only have the ectoderm and endoderm. The ectoderm is the outermost layer, forming the skin and nervous system, the endoderm consists of the digestive tract, and the mesoderm is the muscle and circulatory systems, as well as the bones and organs. As organisms gain more sensory cells, cephalization, or the development of a nervous system, begins. While some invertebrates lack a body cavity known as the coelom, others have an incomplete cavity known as a pseudocoelom. Typically, these organisms also have simple circulatory systems.Finally, some of these phyla are considered protosomes (anus forms after the mouth), while vertebrates are deuterostomes (anus forms before the mouth) (Bentley, 2015).
This lab will focus on the vertebrates from last weeks' Berlese Funnel. While most of the invertebrates are arthropods, this phylum also includes mites and pseudoscorpions (microarthropods). The soil also contains many other organisms such as millipedes, centipedes, ants, etc. The Berlese Funnel will be examined to inspect the organisms present.
In this lab, the Berlese Funnel was used to look at invertebrates. The liquid was poured onto a petri dish and observed under a microscope. Using a key, 5 different invertebrates were identified and their length was recorded as well as their description and number in shape. In the next class, the transect was revisited to examine 5 vertebrates, two of which were birds. The vertebrates were classified and a food web was constructed.
This lab looked at several kinds of invertebrates. Of the ones observed, the Arthropoda Diplopoda was the largest (see Table 1) and the Arthropoda Chilopoda was the smallest. Furthermore, the millipedes were most present in the leaf litter. As the vertebrates present in this community were observed, it became possible to see the trophic levels, with the larger animals representing the uppermost levels. Furthermore, as these organisms interact with each other and share resources within the transect, a carrying capacity is reached. If there are too many species within the community competing for the same resources, the carrying capacity can be reached and natural selection will occur. As part of this, some organisms will die while others will reproduce and evolve.
Bentley, M., Knight, S., Zeller, N., Walters-Conte, K. 2015. Exercise V- Invertebrates and Vertebrates "Biology 210 Laboratory Manual"
Freeman, S., Quillin, K., Allison, L. 2014. Biological Science (Fifth edition).
Plantae and Fungi
Over millions of years, single celled organisms slowly developed to form plantae, fungi, as well as animalia. Within plantae, this was first seen through the development of aquatic green algae. As plants reminiscent of the modern day Bryophytes evolved, these organisms adapted to their environment's conditions such as temperature, water availability, nutrient availability, and gamete interactions. Eventually, more species evolved, such as Tracheophytes appeared, which contained a true vascular system and later seeds. This allowed these plants more opportunity to spread deeper onto land from water. Eventually, flowering plants such as Angiosperms evolved, which not only reproduced through gametes, but could spread them via wind, allowing these organisms to exist in many niches of the environment. This lab focused on analyzing plant samples from the transect, noting each plants methods of vascularization(monocot or dicot) and reproduction as well as specialized cell structures. This refers to the presence of any structures, from stomata (pores that allow for gas exchange) to pollen, spores, etc.
The first part of this lab involved collecting 5 plant samples and a leaf litter from the transect. To do this, the plant samples were extracted (noting the extraction site) and placed in a ziploc bag. For the leaf litter, a second ziploc bag was filled to the brim with leaves and plants scattered across the ground, excluding soil. Next, each group returned to the lab, and using Table 1(see below), recorded specific characteristics of the plant samples. Finally, the leaf litter sample was used to create a Burlese Funnel, a mechanism for collecting invertebrates. To accomplish this, 25 mL of ethanol was placed into a plastic tube and taped to the bottom of the funnel. A screen was then taped to the inside bottom of the funnel, to prevent the leaf litter from falling into the ethanol tube. Finally, the leaves were poured into the funnel, placed under a light source, and covered with aluminum foil until next lab.
Table 1: Characteristics of Plants Collected form the Transect
Crategus crus Galli
As the results indicate above, there is quite a bit of variability of plant life within our transect. The Hibiscus, for example, was the largest of the samples, and as a result more of the plant characteristics could be observed. The Hibiscus utilizes many large stems to supply the plant with water and nutrients. In addition, the spores and megapores, methods of reproduction, were observed within the middle of the flowers. Contrastingly, the Crategus crus Galli, had smaller, white flowers, many of which were still budding. These plants appear to be dicotic, a characteristic observed among all of the samples. Interestingly, spores and pollen were observed on the Paper Birch, a surprise given the plants' very small vertical nature. For many of the plants, it was difficult to locate specific parts of the cell without observing a cross section. However, within the Malus Hupehensis, the calyx, an upper part of the stalk, was noted. For each plant, many of leaves were oval green shaped. After the plants were observed, the Burlese Funnel was set up for next lab.
Bentley, M., Knight, S., Zeller, N., Walters-Conte, K. 2015. Exercise IV-Plantae and Fungi "Biology 210 Laboratory Manual"
Identifying and Studying Bacteria
Unicellular organisms can be seen in each of the 3 domains of life, Bacteria, Eukarya, and Archaea. Within Archaea, there are Euryarchaeota and Crenarchaeota, although these organisms are typically seen within very extreme conditions. For that reason, lab this week will mostly focus on the prokaryotes of Bacteria, which fall under six categories. As bacteria grows, the cells multiply to form millions of organisms grouped together in a colony, which when combined with other colonies forms a lawn. For this reason identifying bacteria can get quite tricky; however, through using a gram stain, identification of bacteria becomes possible. A gram stains causes gram positive organisms to retain the crystal violet dye within the peptidoglycan cell walls, while gram negative organisms do not have this ability. In addition, each prokaryote has different characteristics, such as bacillus (rod-shaped), coccus (sperical), and spirillum (twisted spiral) shapes. Furthermore, by using a Polymerase Chain Reaction (PCR), one can better understand the organisms DNA and find its species, as well as related ones.
The agar plates from the Hay Infusion were collected and observed, noting signs of bacteria growth through colonies as well as lawns. If colonies were scarce enough, they were counted and recorded in Table 1 below (see Results). Next, the gram stains were prepared, using 2 samples from the nutrient only agar plate, and 2 samples from the Tetracycline containing plates. Individual plastic loops were then used to scrape a piece of growth from the agar plate and transfer it onto a slide containing a drop of water. On the underside of the slide, the plate type (T or N, 10^__) was recorded, and a small circle where the bacteria was placed was drawn. The bunson burner was ignited and each slide was carefully passed through the flame until it was heat fixed, indicated by the water drop vaporizing and the bacteria achieving a fluorescent tint. Each slide was then stained with crystal violet for a minute, followed by a water rinse. This procedure was replicated again, this time with Gram's iodine mordant. The slides were then decolorized using 95% alcohol for 10-20 seconds, rinsed, and stained with safranin for another 20-30 seconds. The samples were then air-dried and placed in the microscope. As they were analyzed, bacteria sightings were recorded, making note of cell characteristics as well as gram positivity or negativity, indicated by the present of crystal violet within the cell walls. Finally, PCR reactions were set up, using 2 colonies (one tetracycline, one nutrient agar only) for the samples. Loops were used again to add a colony from the agar to a sterile tube containing the master mix and specially designed primers. The tubes were then labeled and placed in the PCR machine until next lab.
Table 100 fold Serial Dilution Results
Gram Stain 1
Gram Stain 2
As seen in the results above, there was a huge variation between the agar plates. While most of the bacteria maintained a spherical shape and a yellow tint, some plates such as the 10^-3 nutrient only plate actually grew quite a bit of fungus. We expected to see this on the Tetracycline plates given a contamination error last class, and were surprised to see it on the nutrient plate. Overall, our results were perplexing, as despite one plate from the nutrient only set, the tetracycline plates actually had more bacterial growth, potentially caused by the contamination. According to the NIH, tetracycline is used against a large variety of prokaryotes from the Bacteria and Archaea domains, including gram-positive and negative bacteria (Chopra, Tetracycline antibiotics). A possible explanation for the confusing growth on the T plates could then be the presence of antibiotic resistant bacteria in our transect. If this is the case, that would explain why there is so much growth, despite being treated by antibiotics. Furthermore, while some of the bacterial cells initially exhibited some motility, the majority of the bacteria were non-motile. In addition, all shapes of bacteria were observed, indicating a diverse bacterial population.
Bentley, M., Knight, S., Zeller, N., Walters-Conte, K. 2015. Exercise III- Identifying and Studying Bacteria "Biology 210 Laboratory Manual"
Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 2001;65:232–260.
Freeman, S., Quillin, K., Allison, L. 2014. Biological Science (Fifth edition).
Identifying Algae and Protists (07/02/15)
Last week each group collected a small sample from their respective transect to create a hay infusion. This week, the sample was explored under a microscope to look for biotic life. A dichotomous key was used simultaneously, which follows a series of morphological questions to identify organisms. We used this method to attempt to identify unicellular prokaryotes and eukaryotes from two niches of the hay infusion. Prokaryotes differ from eukaryotes in that eukaryotes contain a membrane bound nucleus and more advanced organelles. In addition, there are two kinds of eukaryotes, algae, which photosynthesize, and protists, which consume nutrients to get energy. By observing the life forms in the hay infusion, each group can gain a better understanding of its hay infusion's ecosystem.
Initially, the hay infusion jar was retrieved and two wet mounts were made from two niches of the jar, in this case the top and the bottom. The wet mounts were made by pipetting a small drop onto the glass mount and covering it with a cover slip. The samples were then examined under a microscope and the dichotomous key was used when an organism was spotted. We were instructed to find 4 organisms, and record their characteristics such as shape, motility, size, etc. After 4 organisms were identified, in our case 3 due to time constraints, serial dilutions were prepared for next weeks lab, in order to allow bacteria to grow and to look at the hay infusions microbiology. To make a serial dilution, 4 tubes containing 5 mL of sterile broth were collected and labeled 10^-2, 10^-4, 10^-6, and 10^-8. 50 mL of the mixed hay infusion was then added to the first dilution (10^-2). The tube was then swirled, and 50 mL was added to the next dilution, and so on until each test tube contained 50 mL of the previous dilution, becoming more diluted each time. For this lab, two kinds of agar plates will be used to grow the bacteria. The first contains a regular nutrient agar, while the second contains tetracycline, an antibiotic. Four plates of each were obtained, labeled based on the dilutions (10^-3, 10^-5, 10^-7, 10^-9), and treated with it's respective dilution (10^-8 dilution was plated on 10^-9, and so on). It's important to start with the most diluted solution on the nutrient agar only plate to prevent contamination of both more concentrated bacteria as well as the tetracycline. The agar plates were then left to incubate until the next class.
Notes on the Hay Infusion: Foul smell or mold and dirt. A thick layer of dirt was present at the bottom and a thin layer of potential mold (small dots) was seen just below the surface. The water was mirky with a light brown/green tint and residue could be seen on the wall of the jar. The green tint and residue on the top surface indicate potential life.
From the top layer, Euglena was observed, and had a green color indicating photosynthesis. The euglena was motile with a size of approximately 35-55 um. Chlamydomas was also observed with a rounder shape and 2 flagellum. These are considerably smaller than the euglena, measuring only about 7 um and lacked the green color of euglena. Finally, we observed Didinium, which were relatively large at 20 um. These were seen across the samples, but were not motile and did not appear to be photosynthesizing, indicating it could be a protist.
This was the first step in looking at the organisms from the Hay Infusion. This gives more insight into what kinds of unicellular species live in our transect. Interestingly, more organisms were found on the top layer of the jar than the bottom. Furthermore, the euglena was found on the top surface, indicating that algae and other photosynthesizing organisms might be more successful attaining energy from the sun closer to the surface where there is more oxygen. After two months, one could predict that the top of the jar would be teeming with mold and algae. In addition, after two months, the variability of the infusion would greatly change. For this lab, we were only able to attain 3 species of organisms, potentially because the infusion only sat for a short time. After two months, a mini ecosystem would exist, potentially including predatory organisms such as amoebas or more species in the deeper parts of the jar. It could even contain entirely new organisms as the open jar interacts with bacteria in the surrounding environment. However, as resources within the jar become more scarce, I predict that photosynthesizing organisms would become more scarce as other organisms need their energy to survive. Eventually, this ecosystem would fail as a result of lack of sustainable resources. That being said, it is already clear that an ecosystem is forming. From looking at the euglena, one can see that it already meets the needs of life. The Euglena obtains energy through photosynthesis, is a unicellular organisms and therefore comprised of cells, is a eukaryote and therefore contains its DNA inside a membrane bound nucleus, reproduces asexually, and will eventually evolve.
Bentley, M., Knight, S., Zeller, N., Walters-Conte, K. 2015. Exercise II - Identifying Algae and Protists "Biology 210 Laboratory Manual"
Freeman, S., Quillin, K., Allison, L. 2014. Biological Science (Fifth edition).
Examining Biological Life at AU (06/30/2015)
Life can be seen all around us, from large organisms such as humans to the smallest organisms such as protists and other unicellular eukaryotes. As these different species interact with each other as well as the surrounding environment, ecosystems form which allow for evolution and natural selection to occur . Over time, natural selection creates biodiversity through the extinction of some species and the formation of others. In this lab, we will be looking at biodiversity through studying a small transect of land and it's containing ecosystem (Bentley, Knight, Walters-Conte, Zeller, 2015). Through this process, we will hopefully gain a better understanding of ecology as well as biodiversity.
This lab contained two projects, Initially, the class was divided into groups of 4 and told to find and describe a 20x20 meter transect of land on the Au campus. Transects were to be chosen based on the likelihood of biodiversity and high biotic and abiotic life. For this reason, we chose to conduct this research within a transect of land located between Hurst Hall and the East Quad Building, and next to Nebraska Ave. Next, we noted the nature of the transect and took a small sample containing soil and some biotic life (grass) for the second part of today's lab, the hay infusion. To make the hay infusion, we combined approximately 10 grams of our sample with 500 mL water and .1 g of dried milk. The jar containing the aforementioned ingredients was then shaken and labeled, then placed in the back of the room with the lid off.
The area is relatively flat and is mostly comprised of different species of grass. In terms of biotic life, the transect also contains several trees, flowers, as well as life such as birds, squirrels, fungi growing adjacent to the trees, worms, and flies. In addition, our transect also contains a lot of abiotic life, through rocks, soil, buildings, the sprinklers for the grass, etc.
This is the first part of an ongoing exploration of our transect. Most of this lab was devoted to finding and describing the transect as well as creating the hay infusion. In the coming weeks, the transect will be more closely studied, looking at the ecosystem from small organisms (protists) to larger invertebrates and vertebrates. In the next lab, we will be studying the organisms present in the hay infusion.