User:Karly Brinkman/Notebook/Biology 210 at AU

February 16, 2014 Lab 3 Information (Identifying and Studying Bacteria) Packet Questions

Archaea on agar plates? No, I do not think think that any Archaea species will have grown on the agar plates because Archaea tend to grow in extreme environments, like deep in the ocean or in hot springs or volcanoes. None of the transects at AU are located in 'extreme' environments so I expect that we will mainly find bacteria growing on the plates.

Procedure 1: Quantifying and Observing Microorganisms

Hay Infusion Culture Observations: The liquid in the hay infusion culture is a bit darker, the water level has dropped, it smells less rotten than it did before, and there is no longer a film on top of the liquid (see Figure 6). The appearance and smell is likely to change from week to week because many of the living organisms will have eaten up the dried milk so there is no longer a viable food source--therefore the remaining organisms are likely to either die out, or the heterotrophic organisms may eat each other for food. Either way, a lack of the dried milk will have affected the physical characteristics of the hay infusion culture.

Procedure 2: Antibiotic Resistance

Differences between antibiotic/non-antibiotic plates: The plates without antibiotic contained a much higher number of colonies than the plates with antibiotics--in fact, only one of the three plates with antibiotics had any colonies at all (see Table 2). This indicates that the antibiotics were largely (though not entirely) successful in killing off the bacteria.

Effect of tetracycline on bacteria/fungi: The tetracycline only allowed approximately 90 colonies of bacteria to survive, as well as a few patches of fungi.

Species of bacteria unaffected: We have not yet determined what types of species live on the agar plates.

How tetracycline works and what types of bacteria are sensitive to this antibiotic: Tetracycline works by blocking transfer RNA to messenger RNA in bacteria, effectively inhibiting bacterial protein synthesis. It works against gram-positive and gram-negative bacteria, including anaerobic bacteria, Rickettsiae, Chlamydia, Mycoplasma, and spirochetes.

*The rest of the bolded, red sections in the lab manual fit in nicely with the procedures that I have to mark down, so I will be sure to include the necessary observations, data, etc. in the next section* --KB

February 16, 2014 Lab 3 Procedures (Identifying and Studying Bacteria)

Procedures 1: Quantifying and Observing Microorganisms

Objective: The objective of the first experiment was to observe our hay culture infusions and count the number of colonies that had grown on the agar plates we had prepared the previous week. The week before I had predicted that no bacteria would grow on the agar + tetracycline plates. In addition to seeing whether that prediction was correct, I predicted that more colonies would grow on the less dilute plates.

Steps:

1. My group retrieved our hay infusion culture and observed its appearance (see Figure 6 and Hay Infusion Culture Observations in the Packet Questions above).

2. My group retrieved our agar plates and counted (to the best of our abilities) the number of colonies in each plate. We marked down the numbers counted in Table 2.

3. Using the conversion factor present in Table 2, we calculated how many colonies per milliliter were present in each agar plate.

Raw Data:

Figure 6:

Table 2:

Conclusions: I concluded that the most colonies of bacteria grew in the most diluted plates (according to the conversion factor) and that bacteria were able to grow in one of the tetracycline plates. My prediction about none of the bacteria being antibiotic resistant was incorrect as well as my prediction about less colonies growing on the more dilute plates. Understanding that antibiotic resistance is possible was immediately helpful to me in Procedure 2: Antibiotic Resistance, as that section focuses on the dangers of antibiotic resistance. I now have first-hand knowledge that such resistance is possible.

Procedure 2: Antibiotic Resistance

Objectives: The objective of this procedure was to observe the agar plates without tetracycline and compare them to the agar plates with tetracycline, also using Table 2 to note the differences. We were then supposed to try and determine the effect of tetracycline on the total number of bacteria and fungi, determine how many species of bacteria are unaffected by tetracycline, find out how tetracycline works, and determine the types of bacteria that are sensitive to it. By using all of our agar plates as well as consulting the internet for information on tetracycline, we determined that we should be able to answer all of the questions presented in the lab manual and make inferences about bacteria's ability to resist different types of antibiotics.

Steps:

1. I answered the first bolded question in the packet (see Lab 3 Information Packet Questions above) by comparing the agar plates with the agar + tetracycline plates as well as Table 2.

2. I answered the second bolded question in the packet (Lab 3 Information Packet Questions) by comparing the agar plates with the agar + tetracycline plates as well as Table 2.

3. I answered the third bolded question in the packet (Lab 3 Information Packet Questions) by looking up information on tetracycline and antibiotic resistance on the internet (http://mmbr.asm.org/content/65/2/232.full).

4. I answered the fourth bolded question in the packet (Lab 3 Information Packet Questions) by looking up information on how tetracycline works and the types of bacteria sensitive to this antibiotic (http://www.skintherapyletter.com/treat/rosacea/tetracycline.html and http://www.chm.bris.ac.uk/motm/tetracycline/antimicr.htm).

Raw Data:

See Figure 6 and Table 2 from Procedure 1

Conclusion:

Both from looking up information on tetracycline and observing the effects of tetracycline on the bacteria in my transect, I learned that at least one species in my transect is resistant to tetracycline. I also learned that tetracycline is an antibiotic that works by inhibiting protein synthesis in many species of bacteria, including both gram-positive and gram-negative bacteria, but resistance to this antibiotic has been increasing since the 1950s. I found it surprising and a bit disturbing that bacteria resistant to tetracycline is common enough that it exists in a plant/soil sample at American University, and I will keep this information in mind when dealing with bacteria in the future--and whenever I use hand sanitizer.

Procedure 3: Bacteria Cell Morphology Observations

Objective: The objective of Procedure 3 was to examine three of the agar plates, observing the colonies and specific cells using wet mounts and gram-staining.

Steps:

1. My group chose to analyze plates '2', '4', and 'T3.' Plates '2' and '4' were agar + nutrient plates while plate 'T3' was agar + tetracycline, and was our only tetracycline plate with bacteria growing on it. See Figure 7.

2. We began to prepare our wet mounts, making one wet mount per each plate. a) We sterilized a loop over a flame and used it to scrape up a bit of bacteria on the surface of the plate, then mixing it with a drop of water on the slide. b) We placed a cover slip over the top.

3. We also prepared a separate slide, using the same step as 2a (but without a cover slip since this is for the gram stain). We drew a red circle around the drop so we would be able to find it under the microscope.

4. We observed the wet mount under the microscope, first observing the plates at 40x objective, and then placing a drop of oil over the lens as we moved to 100x oil objective lens.

5. I recorded my observations of the three plates in Table 3, including the colony description, number of colonies per mL of culture, and cell description. I also sketched what I saw under each lens, and at what magnification I saw it (Figure 8).

6. After labeling the three slides prepared for gram-staining, we passed each slide through a flame three times with the bacterial smear side up.

7. After covering the slides with crystal violet for one minute, we rinsed them off with water-filled wash bottle.

8. We then covered the slides with Gram's iodine mordant for one minute.

9. After rinsing off the Gram's iodine mordant, we decolorized the smear with 95% alcohol for 20 seconds, stopping when the solvent flowed colorlessly from the slide.

10. We covered the smears on the three slides with safranin stain for 20-30 seconds, then rinsed it and blotted it with a paper towel.

11. We focused the gram stain at 40x magnification and then used a drop of oil on the microscope lens to observe it at 100x.

12. I marked my observations of the cell's quality (gram-positive or negative) in Table 3.

Raw Data:

Figure 7:

Note: the plate at the top of the picture is plate '2,' the plate towards the right is plate '4,' and the plate towards the bottom is plate 'T3.'

Table 3:

Figure 8:

Conclusion: We were able to examine three agar plates and successfully wet-mount and gram-stain each of them in order to learn more about the bacteria living in each plate. The bacteria on plates '2' and '4' were very similar, while the bacteria on plate 'T3' had some differences. For example, 'T3' was the only plate with evidence of rod-shaped bacteria, and was the only plate with fast-moving bacteria. It makes sense that 'T3' would contain different types of bacteria/organisms than the other plates because only certain types of bacteria are antibiotic-resistant (as we learned in procedure 2), so these bacteria would have to contain special qualities that helped them become antibiotic resistant.

Procedure 4: Start PCR Preparation for DNA Sequence Identification

Objective: The objective of the last experiment was to isolate DNA from the bacteria of two nutrient + agar plates and one agar + tetracycline plate, and then use two primer sequences to selectively amplify the 16S rRNA gene. If successful, we will be able to examine this DNA next week to try and determine why some bacteria are antibiotic resistant and some are not.

Steps:

1. My group transferred a single colony of bacteria to 100 micro liters of water in a sterile tube, and incubated this tube at 100 degrees Celsius for 10 minutes.

2. We repeated step 1 for two more tubes. In total, we used one tube from plate '2,' one from plate '4,' and one from plate 'T3' (the last of which was an antibiotic resistant plate).

2. We then placed the three tubes in a centrifuge and turned on the machine.

3. We used 5 micro liters of supernatant in the PCR reaction.

Raw Data: NONE

Conclusion: After finishing the procedure, we had to wait a week in order to determine whether or not the amplification was successful. If it was, we would be able to determine why some bacteria were antibiotic resistant and some were not. A week after that, we would be able to analyze the DNA sequences. --KB

February 8, 2014 Lab 2 Procedures (Identifying Algae and Protists)

Procedure 1: How to Use a Dichotomous Key

Objective: The objective of the first procedure was to be able to observe several species of protists under a microscope and identify them using a dichotomous key. I hypothesized that by observing the size, shape, movement, and colors of the organisms under the microscope, I would be able to use the dichotomous key to correctly identify the different types of protists.

Steps:

1. I prepared a wet mount of the sample containing the protists, making sure to insert the pipette towards the middle of the sample where more protists were more likely to be.

2. I examined the wet mount at 40x magnification and observed several different motile organisms.

3. I located one organism in particular and focused the microscope to 10x to observe it more closely.

4. I followed the instructions of the dichotomous key to match the organism I saw under the microscope to an organism in the key.

5. I located a picture of the protist I thought I had identified to see if it resembled what I was looking at under the microscope.

6. I wrote down the name of the organism, recorded the size, and drew a picture of it (see Figure 2)

7. I repeated steps 3-6 for a different organism (see Figure 2).

8. I repeated steps 3-6 for a different organism (see Figure 2).

Raw Data:

Figure 2:

Conclusions: I concluded that though it was difficult to keep track of the different organisms under the microscope because they moved so quickly, I was fairly accurately able to identify three distinct protists using the dichotomous key. I identified a Peranema, a Euplotes, and a Paramecium aurelia (see Figure 2). I will be able to immediately use these skills to observe and identify different protists in the Hay Infusion Culture (Procedure 2). I will also be able to use dichotomous keys in future labs or other experiments that require me to identify different organisms using dichotomous keys. I addressed the objective by accurately using size, shape, color, and other indicators to identify protists.

Procedure 2: Hay Infusion Culture Observations

Question: What kinds of organisms (specifically protists) have been growing in transect three? I hypothesized that by extracting samples from different parts of the hay culture infusion, my group would be able to use the dichotomous key to identify different protists from different niches in the hay culture infusion. My group's observations of organisms in the hay culture should give us valuable insights as to what different species of protozoa live in transect three.

Steps:

1. I made observations of hay culture infusion, including its coloration, smell, and whether or not life like mold or green shoots existed in the liquid (see Lab 2 Information: Observations AND Figure 3).

2. I used a pipette to extract samples from the film on top of the culture and the plant material in the middle. I made a wet mount of each of these samples.

3. My group and I observed three organisms from the sample of the film on top of the hay infusion culture and three organisms from the plant material in the middle of the hay infusion culture.

4. We wrote down the size and name of the different organisms, and drew pictures of them (see Figure 3 and 4). We used this information to try

Raw Data:

Figure 3:

Colpidium from Plant Material:

Conclusions: We identified a Colpidium and Pandorina in both samples from the hay infusion culture, implying that these niches were very similar to one another. The film sample had a Paranema sp. and the plant material sample had a Paramecium aurelia, so those two are different. This implies that these protists are common to different kinds of niches, or that the film and plant material niches are very similar. We could infer that the protists we found in the hay infusion culture are similar to the organisms in our transect since the hay infusion came from the transect. Of course, other organisms from the classroom could have entered the culture since we did not have a lid on it. Through our identification of different protists, we answered the question about what kinds of organisms exist in transect three. Again, obtaining even more practice with dichotomous keys will be helpful in future experiments or observations when I am expected to try and identify microorganisms.

Procedure 3: Preparing and Plating Serial Dilutions

Objective: The objective of this procedure was to prepare for the next week's lab by applying the hay infusion culture to agar petri dishes. The colonies would then be able to grow and replicate faster so that we could observe them for our next lab. Applying them to both agar/nutrient petri dishes and agar + tetracycline plates would allow us to determine whether any of the bacteria was antibiotic resistant. We hypothesized that no colonies would grow on the tetracycline plates because the bacteria would not be antibiotic resistant. We also hypothesized that more bacteria would grow on the less diluted plates.

Steps:

1. We marked four tubes of mld sterile broth as 2, 4, 6, and 8 respectively.

2. We then labeled four nutrient + agar plates as 2, 4, 6, and 8. 2 corresponded to 10^-3, 4 corresponded to 10^-5, 6 corresponded to 10^-7, and 8 corresponded to 10^-9. We labeled three tetracycline + agar plates as T3, T5, and T7. T3 corresponded to 10^-3, T5 corresponded to 10^-5, and T7 corresponded to 10^-7.

3. We swirled the hay infusion culture and and extracted 100 microlitres (using a micropipeter), adding this to the tube labeled 2. The solution inside of this tube became 10^-3 because of the 1:100 dilution.

4. We extracted 100 microlitres from tube 2 and added it to tube 4, creating a 10^-4 dilution.

5. We extracted 100 microlitres from tube 4 and added it to tube 6, creating a 10^-6 dilution.

6. We extracted 100 microlitres from tube 6 and added it to tube 8, creating a 10^-8 dilution.

7. We extracted 100 microlitres from tube 2 and aseptically placed it on the surfaces of agar plates 2 and T3.

8. We extracted 100 microlitres from tube 4 and aseptically placed it on the surfaces of agar plates 4 and T5.

9. We extracted 100 microlitres from tube 6 and aseptically placed it on the surfaces of agar plates 6 and T7.

10. We extracted 100 microlitres from tube 8 and aseptically placed it on the surface of agar plate 8.

11. We left the plates to incubate for one week at room temperature, until our next lab.

Raw Data:

Figure 4:

Figure 5:

Conclusions: Since we left the agar plates to incubate for a week, we could not immediately verify whether or not our hypotheses were correct. After preparing the agar plates, we maintained our predictions that more bacteria would grow on the less diluted plates, and that no colonies would grow on the tetracycline plates. --KB

January 31, 2014 Lab 1 Procedures (Biological Life at AU)

Procedure 1

Objective: The objective of the experiment was to study the Chlamydomonas, Gonium, and Volvox algae of the Volvicine line to see how selective pressures cause the different algae to specialize over time. By analyzing the functional and reproductive specialization of the cells, I predicted that the Chlamydomonas would be the least specialized of the three cells, followed by the Gonium, and that the Volvox would be the most specialized.

Steps: 1. I prepared a slide by using a pipette to drop a single drop of solution containing Chlamydomonas onto a slide. I then covered the slide with a cover slip. 2. I took the prepared slide to a microscope and tried to find a Chlamydomonas cell on the lowest magnification possible (40x), gradually increasing the magnification so I could study it further. 3. I recorded my observations in Table 1, including the number of cells, colony size, and description of any functional or reproductive specialization. 4. I repeated steps 1-3 for the alga Gonium. 5. I repeated steps 1-3 for the alga Volvox.

Raw Data: Table 1

Conclusions: I determined that my data supported my hypothesis since the Chlamydomonas was unicellular, only 10 micrometers long, and reproduced through isogamy, while Gonium contained between 4 and 32 cells, was 20 micrometers long (though also reproduced through isogamy), and Volvox contained tens of thousands of cells, was between 350 and 500 micrometers in length, and reproduced using oogamy. The Volvox was also visibly more developed than the Gonium or Chlamydomonas as it was spherical in shape and contained anterior and posterior poles of cells. This experiment only involved a few of the algae on the Volvicine line, but there are many more. In future experiments, I will obtain more than the three algae I examined here and compare the differences between the three algae to better observe how selective pressures work.

Procedure 2

Objective: My objective was to observe transect three and prepare a hay infusion culture to be studied at a later date. By obtaining samples from the transect, I hoped to collect organisms that I would later be able to study to better understand that specific transect.

Steps:

1. I observed abiotic and biotic features in transect three (see the section entitled Lab 1 questions above).

2. I sketched a topographical view of transect three (see Figure 1).

3. My group collected a 10 gram sample from the transect that was 50% soil and 50% plant.

4. We returned to the lab and placed the soil/plant sample in a plastic jar with 500mls of deer park water. The final weight was 11.03 grams.

5. We added 0.1 gms of dried milk to the sample and mixed it for about ten seconds.

6. We took the top off and left the jar to sit for a week.

Raw Data: Figure 1

Future Plans: My group successfully observed transect three and created a hay infusion culture that we could observe the next week to see what life forms existed in the sample. We predicted that adding the dried milk to the sample would allow organisms like protists and bacteria to grow so that we could observe them at a later date. Our work mainly involved setting up a future experiment so it did not involve a hypothesis or prediction of any kind. That will come later.

--KB

January 29, 2014 Lab 2 Information (Identifying Algae and Protists) Packet Questions Procedure 2: Hay Infusion Culture Observations

Initial Observations: The liquid is milky and a film floats on the top. It smells dirty, kind of like a sewer. There are grasses throughout most of the liquid and the leaves have settled at the bottom. The sides of the glass show that the water level has dropped within the last week. I see no signs of life on top of the liquid, only the film.

Samples: My group took one sample from the film on top and another from plant material in the middle. The majority of organisms closer to the plant matter are probably heterotrophic and the plants act as a source of food for them. The organisms in the top film of the culture are probably more autotrophic in nature and therefore do not need to be near the plants.

Pictures:

Characterizations: Sample 1 (Film on top): My group found a Colpidium approximately 80 micrometers in length. Colpidium are mobile protozoa that move by using their flagella. We also found a Pandorina, a photosynthetic colony of cells that each have two flagella they use to move, that was about 40 micrometers in length. We also found a 50 micrometer long Paranema Sp. that moves by using one long flagella and are heterotrophic.

Sample 2 (Plant material in middle): My group found another Colpidium here, this time about 60 micrometers long. We also found another Pandorina about 80 micrometers long and fast-moving. Finally, we found a Paramecium aurelia about 120 micrometers long. Paramecium aurelia are mobile protozoa that move by using cilia, and are heterotrophic.

Meeting all Needs of Life: The five needs of life described in the Freeman text are energy, cells, information, replication, and evolution. The Pandorina meets its energy needs through photosynthesizing, receiving its energy from sunlight. Pandorina are made up of multiple cells, approximately 8 to 16. It stores information in its cells' nuclei and replicates both asexually and sexually. Pandorina are part of the Volvicine line that we learned about in our first lab, somewhere between Gonium and Volvox.

Observing for another Two Months: Had we observed the hay infusion culture for another two months, I think that the heterotrophic organisms would have become more dominant since the autotrophic organisms cannot photosynthesize without sunlight. I think this also means that the plants would have become much more broken down since they are the only food source for the heterotrophic organisms.

Selective Pressures: I think the main selective pressure was the absence of sunlight for photosynthetic organisms. The addition of dried milk also may have been more beneficial for some organisms than others, meaning that organisms unable to use the dried milk as a food source would be unable to grow and reproduce as quickly as the other organisms (or at all). --KB

Excellent start. Great descriptions with nice images and analysis of procedures and results. Add new information to the top of the page to move older entries down. Newest entries at the top of the page. SK

January 29, 2014 Lab 1 Information (Biological Life at AU) Packet Questions Procedure 2: Defining a Niche at AU

General Characteristics of Transect Three: Location: American University Campus in between Hughes Hall and Butler Pavilion Topography: low ground cover throughout most of the area with a sidewalk in the upper left side. There are three high bushes in the middle and several low bushes at the very front. There are some high grasses in the upper right hand corner, and two light poles on either side of one of the three high bushes.

Abiotic and Biotic Components of Transect Three: Abiotic Characteristics: 2 metal light poles, litter/garbage, sidewalk, mulch, soil Biotic Characteristics: 2 squirrels, 1 bird, shrubbery, ground cover plant, tall grasses --KB

January 23rd, 2014 Username entered, able to enter date into lab notebook. --KB