User:Michael VR Muse/Notebook/Biology 210 at AU

Final zebrafish observations with complete zebrafish observation table and movement table March 4

Purpose:

The purpose of the experiment was to update the zebrafish observations with the last observation coming on March 4. The purpose of the experiment was to also tie together all of the observations made that are from March 4 as well as all of the other observations that can be seen in the previous 2 zebrafish journal entries. These observations were condensed into an observation chart that can be seen in the data and observations section. The purpose of the experiment was also to test the effects of movement from the zebrafish from the push test, explained in the previous journal entry, as well as a new assay using a disposable pipette called the pipette test. The hypothesis would be that alcohol slows movement in zebrafish.

Materials and Methods:

This materials and methods section is for the entire experiment and some of this was explained in previous journal entries. The experiment started by obtaining three agar plates. One of the plates was filled with 20 mLs of deer park water. Another plate was filled with 20 mLs of 1.5% alcohol. The last plate was filled with 20 mLs of 0.75% alcohol. The 0.75% alcohol plate was obtained by diluting the 1.5% alcohol by pipetting 10 mLs of the deer park water and 10 mLs of the 1.5% alcohol. Then 20 zebrafish for each plate were obtained from a container and were pipetted onto their desired plates. The pipetting was done using a half cut disposable pipette. The zebrafish from each plate were then observed under the dissecting microscope. Then the plates were sealed with tape and put away. The zebrafish were then observed Friday February 20th, 1 after the experiment began, to make sure the fish were living. Then the following Monday February 23rd, 4 days after the experiment began, the fish were observed again. First they were observed solely by eye to check for dead larvae. The dead larvae were then removed from the plates. Then 10 mLs of solution from each plate was pipetted out. Then 10 mLs of new solution, whether it was only water, 1.5% alcohol, or 0.75% alcohol, was pipetted back into its respective plates. For the 0.75% alcohol plate this meant pipetting 5mLs of water and 5 mLs of 1.5% alcohol into its plate. Then each plate received one 1 drop of Paramecium. Then on Thursday, February 26th, 7 days after the experiment began, the zebrafish were observed again. First the dead larvae and the dead semi developed zebrafish were pipetted out of the plates. For each of the groups one dead semi-developed zebrafish was saved by placing it in a small test tube with 5 mLs of formaldehyde. Then three depression slides with 1 zebrafish from each group was made and observed under the compound light microscope. Then, like the previous observation day, 10 mLs of solution from each plate was pipetted out and 10 mLs of new solution was pipetted back in. Then on Monday March 2, 11 days after the experiment began, the zebrafish were observed again. Three depression slides were made with 1 zebrafish from each group and were observed under the compound light microscope. Then the zebrafish in each plate were observed and video taped for thirty seconds. Then the plates were pushed gently by hand, and the zebrafish were again observed and videotaped for thirty seconds after the push. This was named the push test. After all of the observations were recorded 10 mLs of each solution was pipetted out of the plates and 10 mLs of new solution was pipetted back in. Then on Wednesday March 4, 13 days after the experiment began and the last day of observations, the zebrafish were observed again. Three depression slides were again made and each was observed under the compound light microscope. Then a depression slide with the zebrafish saved in formaldehyde was made and observed under the compound light microscope. Then each plate containing zebrafish was observed and videotaped for thirty seconds. Then the push test was performed and the zebrafish were observed for another thirty seconds. Then two zebrafish from each group were gently poked with a disposable pipette and observations were made about their reactions. This was called the pipette test. Then all of the living zebrafish were placed in different containers related to their variable.

Data and observations:

Figure 1. Table of the complete zebrafish observations for all 6 of the observation days.

Figure 2. Table of the results for the different assays testing for movement in the zebrafish.

Figure 3. Image of a zebrafish from the control group on march 4, the last observation day. Notice the fully developed pectoral fins that look like hair on the sides of its body just below the head.

Figure 4. Image of a zebrafish from the .75% alcohol group on march 4. Notice that the pectoral fins are not visible

Figure 5. Image of a zebrafish from the 1.5% alcohol group on march 4. Notice that the pectoral fins are not visible

Other Observations with regard to zebrafish on 3/4:

- All of the control group had fully developed pectoral fins while both alcohol groups seemed to have lost their pectoral fins or had masses grow over them to make them function improperly and not visible under the microscope.

- Deformities were seen in the alcohol groups but none were seen in the control group

Conclusions and future directions: Clearly the hypothesis that alcohol slows movement in zebrafish is supported. In each assay the control groups movement was never effected however in both alcohol groups the movement slowed down during each observation and it became less frequent. By march 4 the alcohol groups were barely moving before or after the two assays. In the future it would be interesting to see what the effect of dysfunctional pectoral fins has on zebrafish but also other fish because by the last observation period the lack of pectoral fins was a major difference between the control group and the alcohol groups. Also an interesting additional experiment would be to test the effects of sanitation on early developing zebrafish in the plates. During the first three observation periods the alcohol groups solution was much cleaner than the control group and they had more zebrafish survive during the early days of development.

• Michael VR Muse 07:33, 19 March 2015 (EDT):

Bacteria Identification February 26

Purpose: The purpose of the experiment was to take four bacteria from the serial dilutions and determine what type of bacteria they were. The hypothesis would be that with a proper PCR and running of a gel the bacteria could be sent of and sequenced properly identifying which bacteria it is.

Materials and Methods: The experiment was a continuation of the January 29th lab when four different bacteria from the agar plates were chosen for a PCR. This occured by transfering the bacteria to a sterile tube in 100 microliters of water. Then the tubes were incubated in 100 degrees celsius for 10 minutes in a heating block. Then the samples were centrifuged for 5 minutes. Then while the centrifugation process was occuring 20 microliters of a primer water mixture was placed in the PCR tube. This tube was mixed until the PCR bead dissolved. then finally 5 microliters from the centrifuged samples were placed in the PCR tubes. The products from the PCR reaction during this weeks lab were then run on agarose gel and compared to a DNA ladder. Of the four bcteria that were run on the gel the bacteria described as yellow without tetracycline and the one described as white without tetracyline formed a think band on the gel next to the DNA Ladder where the 16S gene correlated. These two bacteria were then sent away for sequencing. The two bacteria were then sent back during the week of February the 26th with two codes. The codes were then put into a blast website which gave the DNA sequence. Then the sequence showed what type of specific bacteria it was.

Data and observations:

Figure 1. Image of the gel that was used to detect the 16s gene. Notice the two thick bands.

Data with regard to the codes:

-Yellow without tetracycline: MB32

- White without tetracycline: MB37

Data with regard to sequences from the codes:

-White with tetra colony #3

NNNNNNNNNNNNNNCNANANTGCAGTCGAGCGAATGGATTAAGAGCTTGCT CTTATGAAGTTAGCGGCGGACGGGTGAGT AACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCAT GGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG AGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCG TAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTG GTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCANCCGTGGAGGGTCATTGGAAACTGGGAGACTTGANTGCAGAAGAGG AAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAANGCGACTTTCTGGTCTGT AACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACNATGAGTGCT AANTGTTAGAGGGTTTCCGCCCTTTANTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTG AAACTCAAGGAATTGACNGGGGCCCGCACAAGCNGTGNANCATGTGGTTTAATTCNAANCAACGCGANNACCTTACCAGG TCNTGACNTCCTCTGANACCCTANAGATNNGGCTTNNNCNTCGGGANCAGAGTGACAGTGNNCATGNTGTCNTCANCTCN NGTCNTGAGNNGTTGGTNANNNCNNCAACNAGCNGCAACCCNNNATCTTNNTTGCCNNNNTNANNTNN

This sequence proved a match to the Bacillius cereus strain

Figure 2. Image from the gram stain of white without. Clearly this is gram positive. Bacillius is known to be gram positive.

-Yellow without tetra colony #1

NNNNNNNNNNNNNNNNNNNNNTANNNTNCNNNCGGGGNNCGATAGNANCNTGCTNAAGATTCNNGANNGACGGGTGAGTA ATGCCAAGGAATCTACCTAGGGGTGGGGGACACCTTTTCGAACGGAAACCTCATACCGCATACATCGTACNGGANAGGGC AGGGGACCCTCCGGCCCTGCCCTAATAAATGAGCCCAGGTCGGATTAGCTGGTTGGTGAGGTAATGGCTCACCAAGGCNA CGATCCGTAGCTGGTCTGAAANGATGATCNGTCACCCTGGAACTGAGACACGGTCCNNACTCCTACGGGAGGCNGANTGG GGAATATGGGAGAATGGGTGATCGCNTNNNCCANCCCGCCCGCGTGTGTGANNAAGGTCTTCNGATCGTTTTGACNTTNA NANANGATNNAACGCGTGAAATTCNGANCGTACTGTTTTAAAATACACCNNNNAACACCGAGCCCTNNNCCNNGTNCCAC CANNNGGTNGTAACNTTNTNCGGATTTCTTTTGTTCANAGTTTCTGGACGTGGAGCNGNATANNTGNNGAAATATCTTAN TNTNANATCACCCTGCCANNNATANNGNCTGTCTTGANAAATTACAAGCGNCAGGATGAAGTAGTGTANCGGAGATCCGC NTAGATANTACTGANAACANNNATTGCGAAGGGAAGTCGCTATGTCNTAACGGGACCCCCGATAGCACAAN

This sequence proved a match to the pseudomonas strain.

Figure 3. Image of the gram stain of yellow without. This is gram negative. Pseudomonas is recognized as a gram negative bacteria.

Previous observations about morphology of the bacteria

- White without tetracycline- Rod Shaped

- Yellow without tetracycline - streptococci

Research about Bacilius cereus: Bacilius cereus is gram positive and rod shaped (Hussey et al., 2005). They also move by swarming (Salvetti 2011).

Research about Pseudomonas Stutzeri: Pseudomonas Stutzeri is gram negative rod shaped and moves with flagella (Lalucat 2006).

Conclusion and future directions: Based on what the blast website provided, all of the data recorded from the previous experiment during the 4th week of laboratory, and the research done by other scientists it is clear that the hypothesis can be supported. The pictures the different stains are great representations of what is expected from the two types of bacteria. While the motility observed in the bacteria does not completely match up with the research on the bacteria, motility is very hard to observe in bacteria and should not be a factor in limiting the successful identification of the bacteria. I would not change anything about the experiment except the urgency to get the sequenced samples back quicker.

References:

Hussey, M., Smith, A. (2001). Gram Stain Gram: Gram Positive Rods. American Society for Microbiology. 1: 1.

Lalucat, J., Bennasar, A., Bosch, R., García-Valdés, E., & Palleroni, N. J. (2006). Biology of Pseudomonas stutzeri. Microbiology and Molecular Biology Reviews, 70(2), 510–547. doi:10.1128/MMBR.00047-05

Salvetti, S., Faegri, K., Ghelardi, E., Kolstø, A.-B., & Senesi, S. (2011). Global Gene Expression Profile for Swarming Bacillus cereus Bacteria. Applied and Environmental Microbiology, 77(15), 5149–5156. doi:10.1128/AEM.00245-11

• Michael VR Muse 14:53, 5 March 2015 (EST):

Zebrafish experiment continued February 26 and March 2 2015

Purpose: The purpose of the experiment was to continue further collection of data and observations about the zebrafish. The purpose was to see how they survived and developed over the last week. The purpose was also to create different assays that could qualitatively and quantitavily provide data that could support a hypothesis. Based on last weeks results, where more fish in the alcohol concentration survived, the hypothesis has become that zebrafish survive longer in alcohol concentrated environments compared to just water. Also reactions to different stimuli were observed therefore the purpose of the experiment is to observe the differences in reactions of the zebrafish to a stimulus.

Materials and Methods: The methods were like the previous entry. The water and ethanol were replaced accordingly. Different pictures of the zebrafish were taken. The fish were observed for motility. The difference in the March 2nd observations is that the zebrafish were observed when the plate that housed them was tapped. The fish were then recorded on a cell phone for 30 seconds.

Data and Observations:

Figure 1. Image of the chart of observations for the Zebrafish completed.

Figure 2. Image of the zebrafish in the tubes used for the formaldehyde process

Figure 3. Image of a zebrafish from the control group

Figure 4. Image of a zebrafish from the .75% alcohol concentration

Figure 5. Image of the zebrafish from the 1.5 % alcohol concentration.

Data about how each plate reacted to the tap:

Control Group: All of the zebrafish immediately reacted. Continuously swam for the full 30 seconds after the tap.

.75% ethanol: All of the zebrafish reacted but was slower than the control groups reaction. On average they stopped swimming about 20 seconds after the tap.

1.5% ethanol: All of the zebrafish reacted but even slower than the .75%. They stopped swimming about 10 seconds after the tap.

Additional observations:

- Control groups water seems to be dirtier than the ethanol groups. Perhaps the ethanol is killing the bacteria and preventing growth.

Conclusion and future directions:

Nothing can be concluded from these two observations time periods. However the reactions to the tap was very interesting. Perhaps the ethanol is starting to slow down muscle function in the zebrafish. Because this addition on the tap test had such great results during the next observation time period the fish will be physically touched with a disposable pipette and then observed for reactions. This is type of test during the next observation period is possible because all of the fish are now swimming.

• Michael VR Muse 14:53, 5 March 2015 (EST):

Zebrafish experiment February 19, 20, and 23 2015

Purpose: The purpose of the experiment was to learn about the different steps in embryology of a developing zebrafish. The purpose of the experiment was also to learn how to conduct an experiment properly with materials made available and different independent variables to choose from. Ultimately, by choosing alcohol as the independent variable, the main purpose of the experiment became understanding the effects that alcohol had at different concentrations on a developing zebrafish embryo. The hypothesis for the experiment was that the zebrafish that were put in the highest concentration of alcohol will develop slower than the zebrafish placed in the lower concentration of alcohol and the zebrafish placed in only water. In order to successfully conduct the experiment observations were made in regard to the zebrafish development during non laboratory hours.

Materials and Methods: The materials used were live zebrafish, graduated pipettes, disposable pipettes, plates, deerpark water, and alcohol.

The start of the experiment was to read a published paper on zebrafish development. An experiment was then designed to test one of the independent variables provided by the lab. Alcohol was chosen. Three plates were obtained and one was filled with 20 m Ls of deer park water. Another plate was filled with 20 m Ls of 1.5% alcohol. The last plate was filled with 20 mLs of 0.75% alcohol. The 0.75% alcohol plate was obtained by diluting the 1.5% alcohol by pi-petting 10 mLs of the deer park water and 10 mLs of the 1.5% alcohol. Then 60 zebrafish, 20 for each plate, obtained from a container were pippeted onto the desired plates. The pipetting was done using a half cut disposable pipette. Then the plates were sealed with tape and put away. The zebrafish were then observed Friday February 20th (1 after the experiment began) to make sure the fish were living so that the experiment could proceed properly. Then the following Monday February 23rd (4 days after the experiment began) the fish were observed again. First they were observed solely by eye to check for dead larvae. Then 10 m Ls of water was removed as well as the dead larvae. The dead embryos were saved in paraformaldehyde. Also 25 mLs of the proper variable was added to its respective dish. Then samples from each dish was observed with the compound light microscope. Depression slides were made with the organisms.

Data and Observations:

Figure 1. Image of the stages of embryo development in Zebrafish.

Figure 2. Image of the Zebrafish Observation Chart in regard to number of dead eggs, living embryos still in egg cases, number of hatchlings, and number of dead hatchlings. Columns for day 7 and 11 are not filled because they have not occurred.

Figure 2. Image of some of the zebrafish from the control group four days after the experiment.

Figure 3. Image of a close up of a zebrafish from the control group four days after the experiment.

Figure 4. Image of some of the zebrafish from the .75% alcohol concentration group four days after the experiment.

Figure 5. Image of a zebrafish from the 1.5% alcohol concentration group four days after the experiment.

Observations after 4 days (based only on the fish that are swimming):

-Fish seem to be almost fully grown

-Eyes are almost fully developed. There is eye movement.

-Hard to see a heart rate

-Fin is developed in most

- Yolk size is about 2.5 nm

- swim bladder is developed in most

- move by moving fin back forth. Some move very quickly

Conclusions and Future Directions:

The data and observations show that most of the zebrafish have survived and are developing. With that being said all three groups have developed in a random manner. There are wide ranges of embryonic development in each of the groups therefore the hypothesis that the zebrafish in the highest concentration of alcohol will develop the slowest can not be supported at this point in the experiment. While there is no significant difference in the three groups at this point in time the 7th day observation could change that. The only thing I would change about the experiment is to store the dishes in a safer area than just in a bucket.

(Forgot to sign it with the date but I did it last week before lab)--Michael VR Muse 10:05, 3 March 2015 (EST)

2.24.15 Excellent notebook entry. Detailed descriptions of Vertebrates and Invertebrates. SK

Vertebrates in Transect 1 February 12 2015

Purpose: The purpose of the experiment was to become more familiar with the vertebrates found on a specific transect at American University. The purpose was also to understand how to classify species. Also the purpose of the experiment was to understand the food web within the specific transect. Finally the purpose of the experiment was to further tighten the understanding of the specific transect to properly write a lab report about it.

Materials and Methods: The only materials used were a pencil, paper, and the Freeman textbook. The experiment started by drawing upon all of the organisms previously found in the transect. Then 5 vertebrates were identified in the transect. Then the five vertebrates were classified. And finally a food web of the transect was created.

Data and Observations:

5 vertebrates that inhabit the transect:

Raccoon: Phylum: Chordata; Class: Mammalia; Order: Carnivora; Family: Procyonida; Genus: Procyon; Species: P. lotor

Red Shouldered Hawk: Phylum: Chordata; Class: Aves; Order: Ciconiiformes; Family: Accipitridae; Genus: Buteo; Species: B. lineatus

Mourning Dove: Phylum: Chordata; Class: Aves; Order: Columbiformes; Family: Columbidae; Genus: Zenaida; Species: Z. macroura

Eastern Chipmunk: Phylum: Chordata; Class: Mammalia; Order: Rodentia; Family: Sciuridae; Genus: Tamias; Species: T. striatus

Eastern Gray Squirrel: Phylum: Chordata; Class: Mammalia; Order: Rodentia; Family: Sciuridae; Genus: Sciurus; Species: S. carolinensis

- The raccoon would benefit from the biotic factors such as all of the living organisms it eats. These include chipmunks, squirrels, and hawks. The abiotic factors that would benefit them would be the rocks. These could provide them shelter.

- The hawk would benefit from an increase in organisms they eat which are biotic factors. These include chipmunks and mourning doves. The abiotic factors that would benefit them are dead sticks and leaves that would help them make nests.

-The squirrel and the chipmunk would benefit from the biotic factors of more live plants. The abiotic factors that would benefit them are also the rocks which could provide shelter.

- The dove would benefit from an increase in the invertebrates in the transect which are biotic factors. It would also benefit from the sticks and dead leaves to make nests which are abiotic factors.

Figure 1. Image of the food web for the transect.

Observations:

- The food web represents the concept of a community. A community is all of the species within an area and how they act with each other. The food web shows how each species interacts specifically showing how each species relies on each other as a source of food. A quick example would be the raccoon that eats the hawk that eats the dove that eats the millipede that eats the bacteria that eats the dead leaves.

- The food web also represents carrying capacity. If there is a shortage of food sources at the bottom of the web that will ultimately lower the amount of organisms at the top of the web. Carrying capacity shows how many of the types of organisms can survive within the habitat. The amount at the bottom directly effects the amount at the top. For example if there is a shortage of dead leaves there will be a decrease in bacteria which will decrease the millipede population which will decrease the mourning dove population, which will decrease the Red Shouldered Hawk population, which will decrease the Raccoon population.

- The food web also represents different trophic levels. There are 5 different trophic levels in this food web. They include primary producers, primary decomposers/ consumers, secondary consumers, tertiary consumers, and quaternary consumers. An example of each trophic level in the food web would be Dead leaves as primary producers, soil mites as primary consumers, centipedes as secondary consumers, Eastern Gray Squirrel as tertiary consumers, and Raccoons as quaternary consumers.

Conclusions and Future Directions: The data shows that there are many different organisms that represent different levels of the food web within the transect. Also the data shows that this transect represents different ecological concepts such as community, carrying capacity, and trophic levels. Also the data definitely enhanced the understanding of the transect for future writing.

• Michael VR Muse 15:34, 19 February 2015 (EST):MVRM

Observing Invertebrates February 12 2015

Purpose: The purpose of the experiment was to observe different invertebrates to further understand their differences. Movement and structure were observed to understand the main differences. The purpose of the experiment also helped characterize one of the types of organisms in transect #1. Part of the lab was used to peer review the introductions for the upcoming lab report which was used to help better our writing.

Materials and Methods: The materials used were the transecting microscope, a funnel, screening paper, a plate, and the samples from the funnel.

The laboratory started by peer reviewing different introductions from different groups and then recieving feedback and giving feedback to the corresponding group. The next part of lab was dedicated to observing invertebrates. First three types of worms were observed. One of the worms was observed using the dissecting microscope while the other two were observed by eye. Then the samples from the Berlese funnel set up during the previous lab were observed. The set up for the Berlese funnel was noted in last weeks notebook but it included 25 mL of the ethanol/water solution being poured into a conical tube. A piece of screening paper was placed into the bottom of the funnel. Then the leaf litter was put on the top of the funnel. The funnel was then placed on a ring stand positioned under a lamp. This week the samples at the bottom of the funnel were observed.

Data and Observations:

Figure 1. Image of earthworms observed.

Figure 2. Image of planaria observed in clear jar.

Figure 3. Image of nematodes observed under the microscope.

Observations:

Earthworms- move like a slinky. Have incompletely linked body cavity.

Planaria- No body cavity. Move in a swimming fashion

Nematodes- move in a wave like fashion. looks like ice

Figure 4. Image of a soil mite found in the Berlese Funnel

Figure 5. Image of the millipede found in the Berlese Funnel

Figure 6. Image of the sprintail found in the Berlese Funnel

Figure 7. Chart showing the three invertebrates found from the Berlese Funnel. (Important to note that 5 invertebrates were not found therefore the whole chart could not be completed.)

Observations:

- Only three invertebrates were identified

- The size range was from .5 mm to 1.0 mm

-The millipede and the springtail were the largest

-The soil mite was the smallest

- The soil mite was the most common organism.

Conclusion and Future Directions: Based on the data invertebrates share many characteristics but are also diverse. Also from the data it is clear that morphology is a very important characteristic when determining what type of invertebrate it is. In the future data should be able to be shared between groups with regard to invertebrates found in the samples. While three invertebrates were found in my groups sample it would have been nice to observe a few more.

• Michael VR Muse 15:34, 19 February 2015 (EST):MVRM

2.19.15 Very good notebook entry. Data and observations should include some text as well as pictures. SK

Observing Plantae and Fungi February 6 2015

Purpose: The purpose of the experiment was to observe the differences in plants and fungi. The main structures to observe were the reproductive structures in plants as well as fungi but also specialized structures in plants used for vascularization and photosynthesis. The purpose of the lab therefore enhanced the knowledge of two other phylogenies in Eukaryotes. The purpose of the lab was also to set up a Berlese Funnel for next weeks lab. The last part of the lab was used to run a gel to see if any of the bacteria chosen from the culture last week had the 16S gene.

Materials and Methods: The materials used were 3 Ziploc bags, leaf litter sample, 5 plant samples, the compound light microscope, pipet-men, loading dye, DNA Ladder, and an electrophoresis chamber.

The first part of lab was to collect a leaf litter sample from the transect with a little bit of soil and put it into a Ziploc bag. Then 5 plant samples were collected from the transect and placed in a different Ziploc bag. Each plant sample was observed and data was collected on the observations. Then pre-made slides of a moss and an angiosperm were observed. Then pre-made slides of Fungi were observed under the dissecting microscope. After everything was observed 4 different bacteria samples were prepped to run a gel. 5 micro liters of the PCR sample for each bacteria was placed into a separate tube tube. Then 3 micro liters of the loading dye was placed in each tube. The tubes were then mixed. Then the DNA ladder was added to the electrophoresis chamber. Then each of the four tubes were placed in 4 different wells. The electrophoresis chamber ran for 15 minutes and the results were recorded. Then the Berlese funnel was made. 25 mL of the ethanol/water solution was poured into a conical tube. A piece of screening paper was placed into the bottom of the funnel. Then the leaf litter was put on the top of the funnel. The funnel was then placed on a rind stand positioned under a lamp.

Data and observations:

Figure 1. Image of the map of the transect with the numbers correlated to the plant samples.

Figure 2. Image of plant same #1.

Figure 3. Image of the plant sample # 2.

Figure 4. Image of the plant sample # 3.

Figure 5. Image of the plant sample # 4.

Figure 6. Image of the plant sample # 5.

Figure 7. Image of the chart redrawn from the lab manual for all of the data observed about the different plant samples.

Figure 8. Image of the drawing of the fungi observed under the dissecting microscope.

Conclusions and Future Directions: Based on the data and observations clearly there were different plant organisms identified. Certain structures such as xylem and phloem were able to be identified under the microscope showing the vascular structures in plants. The pre made slide of the fungi showed that fungi have different mechanisms of reproducing. The Gel showed that the white bacteria with Tetracycline and the yellow without Tetracycline had the 16s gene. For future directions I would say that even though running a gel is a fairly simple process it would have been nice to see the proper technique used to perform a successful gel.

--Michael VR Muse 05:51, 12 February 2015 (EST)MVRM

2.10.15 Excellent thorough notebook entry. Includes all data generated and detailed methods of the lab. SK

Bacteria and Its DNA January 29 2015

Purpose: The purpose of the experiment was to better understand the differences in bacteria. More specifically the purpose of the experiment was to understand that differences in bacteria can be seen from their morphology, their staining characteristics, and their DNA. The dilutions from last weeks lab provided the necessary samples to study the morphology and the staining characteristics under the compound microscope. The dilutions also provided the samples to undergo a PCR reaction that will be examined in next week's laboratory.

Materials and Methods: The materials used for were different samples from the serial dilutions, disposable pipettes, disposable slides, the compound light microscope, a bunsen burner, and metal rod, small plastic test tubes, enzymes, crystal violet dye, Gram's Iodine, alcohol, safranin stain, and oil.

The first part of lab was to observe 4 different types of bacteria produced on the agar plates; one of which had to be from a plate with tetracycline. Using a metal rod each of the 4 different bacteria chosen was placed on 4 separate slides. These slides were then observed under the compound microscope specifically looking at their shape and mobility. These slides were observed using the oil immersion objective at 100x so a small drop of oil was placed on top of the slide. Then 4 different Gram Stains were produced for the four different bacteria. The process was the same for each but it involved sterilizing the metal rod loop over the flame and scraping the bacteria from the agar. The bacteria was then transfered to a slide mixed with a drop of water on it. Then the slide was air dried by passing the slide through the flame three times. The bacteria was then covered with crystal violet for one minute in a staining tray and then washed off. Then the bacteria was covered with Gram's Iodine for 1 minute and then washed off. Then 95% alcohol was put on the bacteria for 20 seconds. Then the bacteria was covered with safranin stain for 30 seconds. And finally after the slide dried the stains were observed under the oil immersion objective. Finally at the end of lab each of the four different bacteria were used to set up a PCR. This occured by transfering the bacteria to a sterile tube in 100 microliters of water. Then the tubes were incubated in 100 degrees celsius for 10 minutes in a heating block. Then the samples were centrifuged for 5 minutes. Then while the centrifugation process was occuring 20 microliters of a primer water mixture was placed in the PCR tube. This tube was mixed until the PCR bead dissolved. then finally 5 microliters from the centrifuged samples were placed in the PCR tubes.

Data and Observations:

Last Observations on Hay Infusion Culture: - No more smell - Nothing floating on top - Water Level lowered again Based on these observations a hypothesis that all all of the protists in the culture are using up all of the cultures resources could be made.

Figure 1. Image of the Table of the results for the serial dilutions.

Figure 2. Image of the Table for the observations made for the wet mounts of the Bacteria.

Figure 3. Image of the Table for the Bacteria Characterization.

Figure 4. Image of the different bacteria formed on the agar plates.

Observations: -The plates without tetracycline have a lot more growth -This indicates that some bacteria from the Hay Infusion are resistant to Tetracycline. -Tetracycline ultimately lowered the amount of Bacteria and Fungi on the agar plates. - There were three different species unaffected by tetracycline

Tetracycline works by stopping protein synthesis by preventing the attachment of the amino-acyl tRNA at the acceptor site. Some Gram positive and some Gram negative bacteria are sensitive to Tetracycline (Chopra et. al., 2001).

Figure 5. Image of the Gram Stain of the Yellow Bacteria without Tetracycline.

Figure 6. Image of the Gram Stain of the Orange Bacteria with Tetracycline.

Figure 7. Image of the Gram Stain of the White Bacteria without Tetracycline.

Figure 8. Image of the Gram Stain of the White Bacteria with Tetracycline.

Conclusion and Future Directions: The experiment definitely strengthened the understanding of the differences in bacteria. Seeing the differences in Gram positive and Gram Negative stains was very important in seeing a real example of the different ways peptidoglycan effects bacteria. The experiment clearly demonstrated the differences in shape, motility, and cell structure of different bacteria. Now all that is left is bacteria and the PCR that was started at the end of lab should fully show this. This was a very interesting lab and nothing could be changed given the time constraints.

References:

Chopra, I., Roberts, M. 2001. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol Mol Biol Rev. 65: 232-260.

--Michael VR Muse 11:40, 5 February 2015 (EST)MVRM

2.4.15 Excellent notebook entry. Good detailed description of Hay Infusion and protists identified. A couple of the photos (1&3) didn't show up when I loaded the page but that may have been a quirk of the internet speed. SK

Identifying Different Species in the Hay Infusion Culture January 22 2015

Purpose: The purpose of the experiment was to observe different protists in a Hay Infusion Culture. The experiment was also used to regain familiarity with the compound light microscope and its different components such as the ocular micrometer. Typically students do not think about small organisms such as protists on a day to day basis but they are far more abundant than humans and play a vital role on the survival of life on Earth. Therefore another purpose of the experiment was to come to a realization of the frequency and importance of these organisms. Finally, the last part of the experiment was to set up a serial dilution for next week's laboratory.

Materials and Methods: The materials used were two different samples from the hay infusion culture, disposable pipettes, disposable slides, the compound light microscope, a dichotomous key, a bunsen burner, a clear rod for spreading, 6 agar plates, and a P100.

The first part of Lab was to observe wet mounts of known organisms under the microscope. Then observe the organism and make notes of their size, shape, color, and special structures used for motility. Then, using the dichotomous key, answers to different questions confirmed the identity of the organism. Then samples from the Hay infusion culture were taken from the top of the culture and the bottom. Each sample was then mounted on a wet mount and observed under the microscope. Then, like earlier with the dichotomous key, organisms were identified and recorded. Finally the last part of lab was preparing a serial dilution. 10 microliters of broth were placed in 3 test tubes. Then 10 microliters of the hay infusion were put into the first test tube. This was swirled around then ten microliters from test tube 1 was put in test tube 2, and then another 10 microliters was taken from test tube 2 and placed in test tube 3. Then 100 microliters was taken from tube 1 and plated on 2 different agar plates. One of the plates was only agar and one was agar and tetracycline. The sample was spread around with the clear rod and then sterilized with the bunsen burner for its next use. These steps were repeated from tube 2 and tube 3 by taking the 100 microliters and plating them on plates with agar and plates with agar and tetracycline.

Data and observations:

Figure 1. Image of the side view of the Hay Infusion Culture.

Figure 2. Image of an aerial view of the Hay Infusion Culture.

Observations in regard to the Hay Infusion Culture:

1.) Smell is indescribably bad

2.) Water has changed to a murky brown color

3.) Parts of leaves and grass float on the surface

4.) Small build up of sediment on the bottom

5.) The water level has dropped from the original amount

Figure 3. Image of the drawing of the protist Colpidium sp found from a sample taken from the bottom of the Hay Infusion Culture.

Figure 4. Image of the drawing of the protists Chlamydomonas (top) and Peranema sp (bottom) found from a sample taken from the surface of the Hay Infusion Culture.

Observations with regard to organisms found from the two samples:

- Only 1 protist was found from the bottom sample while there were two different types (6 total) of protists found from the sample from the surface. This distribution could be because different protists obtain their energy in different ways. Some eat decayed matter which would make sense for the protist at the bottom and some get their energy from the sunlight which would make sense for the protists found on the surface near the plant matter. Being on the surface does not completely mean these protists are photosynthesizing it just means they are likely to be.

-Based off of where the samples were taken I would predict Colpidium sp to be protozoa and Chlamydomonas and Peranema sp to be algae.

- All of the protists were motile

Chlamydomonas: - This organism fits all of the needs of life because...

1.) It acquires and uses energy from photosynthesis.

2.) There were 5 of this specific protist found so it definitely replicates.

3.) It is made up of a single membrane bound cell.

4.) It encodes and processes information to realize that the light comes from above so it naturally moves in that direction. Also the 5 different Chlamydomonas found were very similar in size shape and motility therefore genetic information was passed down.

5.) It is only one species of protist showing that there are other species in the same phyla proving evolution.

Figure 5. Image of the drawing of the serial dilutions.

Conclusions and Future Directions: Based on the data and observations protists are definitely more abundant than a typical student would think. After just observing two wet mounts 7 protists were identified. If I were to make an adjustment for the experiment I would have provided a better dichotomous key. The dichotomous key provided was good but lacked some information on the size of certain protists which was crucial for correctly identifying it. Finally if the Hay Infusion culture grew for another 2 months I would predict that the water level would keep dropping, food sources would diminish, and the niche would reach carrying capacity. --Michael VR Muse 12:43, 29 January 2015 (EST)MVRM

1.27.15 Excellent first lab book entry. Well organized, clear and nice diagram. Images could be a bit smaller. SK

American University Transect January 15 2015

Purpose: The purpose of the experiment was to observe different species in a designated transect on campus. This provided real life examples of biodiversity and helped strengthen each students understanding of the different components of evolution. The experiment also provided a sample from the transect for a Hay Infusion Culture which will be used for future experiments. The last part of lab was spent observing the Volvocine line to once again strengthen the understanding of evolution and to practice using the microscope.

Materials and Methods: The materials used were simply a tube and a flashlight. The methods started with finding the transect. Then determining its boundary. Then observing the different biotic and abiotic components of it. Then drawing the transect. Then finally collecting the sample.

For the second part of lab the compound light microscope was used to observe the Volvocine line. This started with first observing Chlamydomonas, then Gonium, then Volvox.

Data and Observations:

Figure 1. Image of the drawing of the transect with a key of the different components observed.

Biotic - grass (north) -tall grass (east) -red bush (west) -red flower (east) -cottontail plant (east) -organisms in the soil (north,west,east,and west) Figure 2. List of Biotic components

Abiotic -stand (east) -drain (southwest) -Dunkin Donuts cup (west) -straw (southwest) -rocks (north west east and south) Figure 3. List of abiotic components

Figure 4. Image of data table for observations of Volvocine Line

Conclusions and Future Directions: Based on the data and observations clearly there are many different biotic and abiotic components in the transect. The data and observations gave a great real life example of biodiversity and definitely strengthened the understanding of evolution. Also the Volvocine clearly demonstrated how evolution takes place. If I were to do the experiment again I would have tried to get in the areas that were tougher to see to observe more biotic and abiotic components of the transect.

--Michael VR Muse 18:50, 21 January 2015 (EST)MVRM