User:Christine I. Piotrow/Notebook/Biology 210 at AU
Determine the effect of fluoride on developing zebrafish embryos. Because fluoride was observed to affect the pigmentation, tail length, and mobility of frog embryos, I predict that the zebrafish embryos would likewise also exhibit abnormalities in those same aspects when exposed to fluoride. By exposing an experimental group of zebrafish embryos to fluoride and comparing them to a control group without treatment we can observe any differences between the two that the fluoride could have caused.
MATERIALS AND METHODS
1) Read a published paper about how embryos are affected by fluoride (we were provided with one on frog embryos and not zebrafish embryos).
2) Set up two petri dishes labelled control (only water) and fluoride (only treatment).
3) Transfer 20 ml of water to the control and 20 ml 10mg/L fluoride solution to the fluoride dishes.
4) Find at least 40 zebrafish eggs for the experiment and transfer half to the control and half to the fluoride dishes.
5) Take notes on their development every other day and change their water by replacing 10 mls of old water with fresh water. The fluoride dish likewise has 10 mls of treatment changed instead of water. This is to prevent the embryos from drying out due to evaporation.
6) After one week, prepare three larvae from the samples by fixing the in 0.02% tricaine solution which would then be placed in 4% formaldehyde.
Day 1 Observations:
Day 3 Observations:
Day 6 Observations:
One fish from control and two fish from fluoride were sacrificed and preserved.
Day 8 Observations:
Day 10 Observations:
Day 15 Observations:
On average, the fluoride treatment group were smaller in length and paler in complexion and had movements that seemed awkward or impaired compared to the control group. Despite the fluoride group initially having more eggs that were further along in development, they tended to hatch slower and develop structures slower than the control group. One fluoride group egg never hatched. Zebrafish within the fluoride group also died much more consistently than in the control group, although there were more total dead in the control than in the experimental group at the end. From these data we can conclude that the fluoride slowed down normal zebrafish development, altered pigmentation, and affected the fishes’ mobility.
Surprisingly, after the long weekend following Day 10 and leading up to Day 15, both zebrafish groups were almost completely eradicated and only the smallest individuals in the group survived by Day 15. It’s possible that the smallest fish, which would need the fewest food resources, were able to survive while the larger, more developed fish of each group perished.
For future studies, I would be interested in seeing if the amount of fluoride added to developing embryos would exacerbate the effects observed in this experiment. In other words, is abnormal development correlated with higher amounts of fluoride treatment? Additionally, I would even like to repeat this same experiment in an attempt to mitigate the damage the mass extermination had on the sample data.
Analyze and identify the identity of the bacterial genes sequenced from the transects studied in lab. From the two samples derived from the second transect in section 6 (A6-T2-1 and A6-T2-2), the genetic information that was sequenced, the sequences can be run through a database in order to identify the organism that is most likely to have that sequence.
MATERIALS AND METHODS
1) Bacterial samples from transect were transferred to a sterile tube filled with 100 microliters of water.
2) Samples were incubated at 100 C for 10 minutes and allowed to run in the centrifuge.
3) 5 microliters of supernatant were added.
4) The next week, the PCR products were run in an agarose gel and sent off to Genewiz inc
Both bacterial search information match up for the descriptions of the colonies in class, so it is likely that the bacterial samples obtained are in fact these bacteria. While only the genus could be found for A6-T2-1, Chryseobacterium, the species of bacteria for A6-T2-2 was identified. The transect the samples were found in seems to be a fine habitat for these bacteria, as it contains both organic plant matter and sources of water.
Lab 5: Invertebrates
The purpose of this lab was to observe and classify the different types of invertebrates. Using the berlese funnel (set up in Lab 4, one week prior to Lab 5), invertebrates from the transect were collected and then observed. Identifying the different types of invertebrates that inhabit the transect is very useful for a clear understanding of the ecological niche.
MATERIALS AND METHODS
1) 25 ml of 50:50 ethanol/water solution was poured into a 50 ml flask.
2) Screen material was taped into the bottom of a funnel.
3) Funnel placed into the neck of flask with preservative at the bottom.
4) Leaf litter sample collected in step 1 was placed into the top of the funnel.
5) A 40 watt incandescent lamp was placed about two inches above the leaf litter.
6) Leaf litter sample was covered in foil.
7) Leaf litter sample was left to stand for one week.
8) Preservative at the bottom of the flask was transferred to a Petri dish.
9) Five organisms for the transect were identified and recorded in a data table.
10) Further organisms (vertebrates, birds, etc.) were considered and researched.
Arthropod identification sheet handed out in class.
Basic traits of different arthropod classes were used to identify the station samples in jars.
Table of invertebrates identified in Step 9.
In the transect samples, leaf litter organisms appear to be the most common. Some specimens are very small, needing magnification to be seen (soil mite), but others may be seen with the naked eye (millipede). Two samples from West Virginia had to be borrowed from the lab because two other invertebrates could not be found from the funnel sample. The soil mite from the transect was the smallest organism and the largest organism was the beetle from West Virginia.
Table of possible vertebrates that inhabit the transect niche from Step 10. Rodents.
Table of possible vertebrates that inhabit the transect niche from Step 10. Birds.
Diagram of a food web with transect species .
When identifying the different types of invertebrates that inhabit our sample, leaf litter dwelling arthropods were very common. This makes sense, because the transect being observed is covered in brushes and has dead plant matter all over the ground. However, because five organisms from the transect could not be found, two organisms from West Virginia had to be substituted into the data. Of the organisms found, their sizes ranged greatly; the soil mite needed to be observed under the dissecting microscope, but the beetle could be clearly seen by the eyes. Different vertebrates such as birds and rodents are very likely to use the transect as their ecological niche.
In the future, a way to reliably obtain worms and/or insect grubs from the transect would be a point of interest. Because of the limitations of the berlese funnel, worms and insect grubs cannot be reliably collected, and therefore a representative sample of all the invertebrates of the transect could not be observed. Examining the transect at a different type of year might allow for observation of migratory and/or hibernative species that use the niche that could not be seen in winter.
Lab 4: Plantae and Fungi
The objective of this lab was to learn how to identify the differences between plant diversity and how to classify different types of plants by their defining characteristics: Vascularization, Specialized structures, and Reproductive methods. Then, using these identification techniques and applying them to the transect, plant samples were collected, observed, and described.
MATERIALS AND METHODS
1) ~500g samples from the transect were collected from the transect and put into one plastic bag.
2) Plant samples from five plants in different areas of the transect were collected and put into a separate plastic bag.
3) Any seeds, flowers, or berries from the transect were collected and put into a third plastic bag.
4) The three bags were taken back to lab.
5) Table 1 was filled out with descriptions of the five plant samples.
6) Fungal samples were observed under a dissecting microsope.
Map of the transect. Areas where plant samples were obtained in Steps 1-3 are shown by the numbers.
Data from Table 1 used in Step 5. Numbers of the transect map match the numbers of the plants in the table.
Each plant sample clearly had a vascular system. Plant 1 was supported by thick, dark wood. Plant 2 was also supported by wood, although lighter in color and less thick than plant 1. Plant 3 was a short bush and had small, thin, woody branches. Plant 4 had a very tall stem, however it is unknown if it was a bush or a tree. Plant 5 was from a bushel of grass, with a thick base and the grass blades were tall, long stems.
Plant 1’s leaves were covered with a noticeable cuticle, and were spiked at the edges. Overall, they were rather small and arranged at the edge of the branches of the tree. Plant 2’s leaves were slightly angled at the edges and were slightly smaller than plant 1’s leaves. Plant 2’s leaves seemed to attach in pairs at the end of a branch. Plant 3’s leaves were large, oval shaped and covered in a cuticle. Leaves were densely clustered in a small area. Plant 4’s leaves were very small and shaped like butter knives. Pairs of leaves occurred downward along the branch of the plant. Plant 5 had long, thin, densely packed blade-like leaves in a cluster.
Plant 1 could be identified as a dicot because of the broader shape of the leaf and the network of veins running throughout the leaf. Berries were found in association with plant 1. Plant 2 could also be identified as dicot because of the vein network. Small flowers were found in association with plant 2. Plant 3 had broad leaves and a large vein network, thus was also a dicot. Plant 4 had narrow leaves, but the vein network identified it as a dicot. Plant 5 was the only monocot, because it had long, narrow leaves and parallel veins spanning the length of the leaf. No evidence of any spores were found.
Drawing of sample fungi from Step 6.
Spores in Fungi are produced in the sporangia. When the sporangia open, the spores are released. Dispersal of the fungal spores by wind, water, and animals is essential to its reproductive survival.
The fungal sample observed under the dissecting microscope appeared to be a zygomycetes fungi. The mycelium was clearly visible and various hyphae joined together to form, dark, globe-like sporangia.
From the transect, using how to identify plants based on vascularization, specialized structures, and reproductive methods, it can be concluded that there are a diverse array of angiosperms in the niche. The plant samples all varied in shape and size and even displayed differing types of reproduction (monocot grass vs. dicot bushes and trees). Most samples were only found with leaf litter, but some had reproductive structures found in association with them (berries and flowers for plants 1 and 2 respectively). A fungal sample from the previous was identified as a zygomycetes because of its dark, obvious sporangia.
In the future, it might be helpful to return to the transect in the spring when the plants have green foliage and when flowers (if any) are more likely to be visible on the plants. This way, the leaves can be observed while they are alive and healthy and any reproductive methods of the plants can be more easily determined. For example, plants 3 and 4 might have flowers in bloom in the spring. If they had flowers, their genus might be able to be identified for the transect.
2/6/14, lab 1 notes
Great job!! Remember what we talked about this week for future labs (to have each lab have its own entry). Also, start working on building a map of your transect to detail your land and where your samples are taken from. We will talk about this more Wednesday. Awesome job!!
In this experiment, our objective was to determine a niche on campus here at AU. To do this, we were assigned a 20x20 foot transect to collect a sample from. This sample is the primary means by which we will study the various organisms that inhabit the niche.
1. Take a sample from the 20x20 foot transect with a 50 ml conical tube that provides an appropriate representation of the niche.
2. Make a hay fusion culture with 10 g of ground sample, 500 ml of water, and 0.1 g dried milk, all mixed together for 10 seconds and covered for one week.
3. For the subsequent week, record observations and take a few wet mounts to look at under the microscope. Try to identify and draw findings.
4. Prepare serial dilutions for the samples (10-3,10-5,10-7, and 10-9) and spread them in four separate petri dishes for each serial dilution with nutrient agar. Repeat this for three other petri dishes (10-3,10-5, and 10-7) that have tetracycline. Allow cultures to divide for one week.
5. The following week, make visual observations on the colonies in the petri dishes. Take 3 samples (Agar 1, 10-7; Agar 2, 10-7; Tet 1, 10-5) to make oil mounts to observe under the microscope.
6. From the same labelled colonies above, take small samples and run under a flame to prepare a gram stain. Cover the labelled smear with crystal violet, wait for one minute, rinse off, decolorize with 95% alcohol for 20 seconds, cover with safranin stain for 30 seconds, rinse, and gently dab dry with a paper towel.
7. Take gram stains under microscope, observe and draw findings.
General characteristics of the transact we observed were that it was a (man-made) garden, cement walkways, small trees and bushes with copious amounts of dead plant material.
Biotic and abiotic components include: male cardinal, soil, dead plant material, topsoil, bird’s nest, spiders, worms, bushes, small trees (bare), straw, berries, steel, wax paper soft drink cup, concrete, humans, other birds?
Smell and appearance of Hay Infusion culture was a slightly musty but otherwise neutral smell. Top liquid has plants and a slightly green-ish hue.
When taking samples from different parts of the Hay Infusion culture, it is important to note that different organisms live in different areas of the small ecosystem. Organisms that photosynthesize will be more likely to live at the top of the jar than the bottom.
One of the organisms I observed was a peranema. It is a single-cell organism, capable of movement by flagella, thus it must use energy. It lived with other organisms of the same type, meaning it is capable of replication, thus is capable of processing hereditary information, and by extent a product of evolution.
If I were to predict future changes in the culture, I might say that majority of the plant matter will have sunk to the bottom and begun to decay. It might even look something like a miniature swamp. Organisms that help to decompose plant matter would likely live very close to the bottom and the water might turn a greenish-brown color (perhaps yellow at the top?).
Changes in the composition of the culture might affect how the organisms that live in it survive or die. For example, if there are plants in the culture to begin with it might include organisms that feed off of plant material. But, if the plants all die and cease to become a resource, the environment might select for organisms that can metabolize energy from different sources besides plants.
Because we know that Archaea tend to grow in extreme environments, we may expect them to have grown on the agar plates with tetracycline, because it is a potent antibiotic. The most hardy of Archaea may be able to survive in a harsh environment if they have traits that are favorable for an antibiotic environment.
Hay Infusion has a slightly sour smell. Plant material is dead and decaying, giving the water in the jar a greyish, green/brown color. The bottom is swampy and looks as if it has sludge. A reddish-brown colored film is at the top of the water.
When we looked at our colony plates, there was surprisingly little difference between the plates with and without tetracycline. While there was no growth on the antibiotic plates with very low (10-7) concentration and there were fewer colonies on the 10-5 antibiotic plate, the other two plates were very similar. In the nutrient agar 10-9 plate there was no bacteria, but possibly a fungal growth. This may indicate that antibiotics were already in the environment we took the Hay Infusion sample from and the organisms that live there had already adapted to them. Fungi seemed much more susceptible to the antibiotic than any bacteria. While we were only able to find one species of bacteria for certain (yellow-orange in color) there may have been another present that did not seem to adapt well to the tetracycline (milky white-yellow in color). It is unknown if this other “bacteria” was actually a fungi or not.
Tetracycline is very potent against streptomyces bacteria, and is provided over the counter to treat acne, certain STIs, Lyme disease, and bacterial eye infections.
No motility was observed in the oil mounts.
From the above observations, we see that there are clearly a diverse array of different microscopic organisms that inhabit the transect we sampled from. Some organisms in the mix are resistant to antibiotics. The various plant materials that consolidated the sample have begun to decay and are producing slightly musty, sour odors as well as changing the color of the water in the sample.
For further study, we may try to observe what other kinds of antibiotics the natural sample may be resistant to with other serial dilutions.
The Hay Infusion culture that was observed had multiple areas of habitation, with many different species of organisms that lived in the 50 ml sample we took from the transect. Therefore, we successfully addressed the objective of determining a niche for our spot on AU campus.
Today I successfully entered text into my lab notebook!