User:Casey M Culhane/Notebook/Biology 210 at AU

Header: Observing Microorganisms 1/27/16

Purpose: The purpose of this lab is to compare microorganism growth in an antibiotic resistant environment. The lab will show that in the microorganisms' growth will flourish on a normal agar plate; however, the microorganisms' growth will be significantly stunted by the antibiotic resistant agar plate. This is important because reactions by microorganisms to antibiotic resistant environments is the basis of medication and treatment of certain diseases.

Materials and Methods: This lab has three procedures. The first procedure included analyzing the agar plates. The agar plates were obtained and examined at the macroscopic level. The number of colonies on each plate were counted and recorded in Table 1. The second procedure involved a patch of growth from the top of the two agar plates (one that just has the nutrient and one that has both the nutrient and the tet). Each was placed on a slide and gram stained. To prepare a stained slide, one had to sterilize a metal loop using a fire. The loop then was used to obtain a small patch of growth. A drop of water was placed on the slide. The slide was passed through the fire and then crystal violet was added to the patch for 1 minute. At 1 minute, the crystal violet was washed off, rinsed with water, and covered again with Gram's iodine mordant for another minute. Again, the patch was rinsed with water, decolorized with 95% alcohol for 10-20 seconds. The safranin stain was placed on the patch for 20-30 seconds, and then the patch was rinsed with water. Finally, excess water was blotted off the slide, and the slide was placed under a microscope. Descriptions of the microorganisms in the slides were recorded in Tables 2 and 3. The final procedure for the lab was to set up a PCR for 16s amplification. After labeling the 2 PCR tubes with transect number, colony identifier, and group number, 20 μL of the primer mixture was added to the PCR tubes and mixed to dissolve the PCR bead. Each of the two bacterial colonies were added into the tube using a toothpick, and the tube was closed and placed in the PCR machine. The machine will run for several hours, and the PCR data will be analyzed next week in lab.

Data and Observations: The following photos are pictures of the nutrient agar plates and the nutrient + tet agar plates. The plates show the growth of the colonies. The number of colonies on each of the eight agar plates was recorded into Table 1. Table 1 is the following picture. . Clearly, the number of colonies of microorganisms were larger on the plain nutrient plates. For example, the 10^3 dilution nutrient agar had 406000 colonies, whereas the nutrient + get agar plate at the same dilution had 113000. The trend carries through all the dilution levels. This indicates that the microorganisms grow better without the antibiotic present. The lack of antibiotic allows the microorganisms to grow and reproduce without any competitors. The antibiotic resistant agar plates have significantly less colonies because the antibiotic resistant agar kills the microorganisms. The exact number of bacteria and fungi that were effected by the tetracycline were not recorded during the lab. Also, the specific species of bacteria that were affected by the tetracycline were not recorded in the lab. The following tables 2 and 3 show descriptions of the cells and motility types of the colonies. . No pictures of the organisms were able to be obtained through the microscopes.

Conclusions and Future Directions: The agar plates showed abundant life on the nutrient plates compared to the growth on the nutrient + tet plates. The hypothesis is supported through the evidence presented in Table 1. Table 1 shows the large amount of colonies grown in the nutrient plates, whereas there is significantly less colonies on the nutrient + tet plates. The tetracycline kills off the microorganisms. CC

Header: Analyzing the Hay Infusion for Organisms 1/20/16

Purpose: The purpose of studying the hay infusion that was set up last week is to see what organisms grew in the infusion. The infusion will grow life; however, the life will not be particularly abundant. The life will only be visible using a microscope.

Materials and Methods: The lab today has three parts. The first part is to use a dichotomous key. One must start by mounting wet slides of a variety of known organisms (for example, haematococcus, tolypothrire, marine diatoms, conceptacle of fucus (male and female), and euglena). The microscopes should be set at either the 4x or the 10x objective. Once the microscope is focused, the organisms should be measured using an ocular micrometer. The organisms should be compared to a dichotomous key to guess the identity of the organisms and then checked against diagrams of organisms. The second part of the lab is to apply knowledge of the organisms to the hay infusions. After photographing the hay infusion, noting the smell, and recording notes about the appearance of the infusion, one should take a sample from the top of the infusion and the bottom of the infusion. The top and the bottom should be mounted onto wet slides and focused on the microscope between 4x and 10x. Then one should identify 3 organisms from both the top of the infusion slide and the bottom of the infusion slide. The organisms should be measured using the ocular micrometer and descriptions of the organisms should be noted. The final part of the lab is to set up a serial dilution. Four tubes are needed. Each tube contains 10mLs of sterile broth containing one of the following: 10^-2, 10^-4, 10^-6, or 10^-8. Four nutrient agar and four nutrient agar plus tetracycline plates are also needed. After labeling the plates, add 100 microliters of the culture to the 10mLs of broth in the 10^-2 tube. The mixture should be stirred. 100 micrometers of the 10^-2 broth is then added a tube holding 10^-4. The mixture should be stirred well. The procedure should be completed similar for the 10^-6 and 10^-8 tubes. Finally, put 100 microliters from the 10^-2 tube onto the nutrient agar plate labeled 10^-3. Spread the sample on the plate using cooled glass. Then place 100 microliters from the 10^-2 tube onto the 10^-3 + tet plate. Spread the sample on the plate using cooled glass. Both procedures should be repeated for the 10^-4 dilution (which is placed onto the 10^-5 plate), the 10^-6 dilution (which is placed onto the 10^-7 plate), and the 10^-8 dilution (which is placed onto the 10^-9 plate). The plates should held at room temperature room for a week.

Data and Observations: The following photo are drawings from organisms that were identified using a dichotomous key. The hay infusion's smell was not very strong. The smell was similar to wet dirt. The liquid in the hay infusion was darkly tinted but not difficult to see through. There was lots of big straw sticking out of the infusion. There was a film of mold on the top of the infusion. Nothing was moving but there was a likelihood of life inside the infusion. The following photos are pictures of the hay infusion. The two niches examined from the hay infusion was the top and the bottom of the infusions. There is plant matter in both niches; however, the plant matter is more prominent in the top of the infusion. Three organisms found from the top of the hay infusion are peranema (100μM), arcella (90μM), and oedogonium (70μM). The peranema are mobile but do not perform photosynthesis, so therefore they are protozoa.The peranema meets all the requirements of life by taking in energy in the form of capturing yeast or other bacteria. The organisms are unicellular creatures who replicate using binary fission, and they evolve into other more complex forms of bacteria. Finally, the peranema takes in information from its environment using sensors. The arcella are not mobile and do not perform photosynthesis, so therefore they are also protozoa. The oedogonium are not mobile and do not perform photosynthesis, so they are an algae. The following pictures are from the top of the hay infusion and include the protists identified as described above.

From the bottom of the infusion, colpidium (50 μM) and oedogonium (70 μM) were found. The colpidium were motile but did not perform photosynthesis, so therefore the are a protozoa. The oedogonium were non-motile but did perform photosynthesis. If the hay infusion grew for another two months, more complex organisms would be present; however, due to the constraints of the community, the organisms would not advance to be multicellular. The following picture is from the bottom of the hay infusion and includes the protists identified and described above.

The final part of the lab was to set up a serial dilution. The following photo is a diagram of the serial dilution performed in lab.

Conclusions and Future Directions: The hay infusion showed life. The life was not apparent just by looking at the infusion; however, the microscope slides showed a diversity of algae and protozoa from the top and bottom niches of the infusion. The hypothesis is supported because a microscope was needed to see the life from the infusion. CC

Header: Viewing the Volvocine Line and a Transect at AU 1/13/16

Purpose: The purpose of studying the Volvocine Line is to analyze the evolution of one line of organisms. The purpose of studying the transect at AU is to see the biotic and abiotic factors of an ecosystem. The chlamydomonas will be the most evolved in the Volvocine Line with gonium as the second most evolved and volvox as the least evolved. The transect at AU will be contain more biotic factors than abiotic factors.

Materials and Methods: To view the chlamydomonas, one must mount a wet slide with the chlamydomonas and place the slide under the microscope. After adjusting the lens to 40x, one can count the number of cells, measure colony size, determine the presence of specialized cells, determine the mechanism of motility, determine if it is isogamous or oogamous, and draw a picture of the organism. After analyzing the chlamydomonas, take the wet slide off the microscope. Then, one should prepare slides for the gonium and volvox and repeat the procedures. To view the transect, one must walk to his or her transect location. At the location, one should draw the transect, explain the components of the transect, and collect a sample of materials from the transect. The collection of materials will be used to make a hay infusion. Back in the lab, 12 grams of the collected materials from the transect will be added to a plastic jar. 500 mLs of water from deer park will be added as well. Then 0.1 gram of died milk will be put into the jar. The jar will be mixed by shaking for 10 seconds. The jar is left without a lid on the jar for a week.

Data and Observations: The following are pictures from the transect. The following photo is an aerial diagram of the transect. The following are biotic factors located in the transect: low brush, medium sized trees, big trees, bushes, and bugs. The following are abiotic factors located in the transect: concrete (sidewalk), light pole, cigarette bud, rock, and plastic layers under the dirt. The transect is on a slight hill and is located in between Bender gym and Hughes Hall. There are two benches located in the transect. There are two light poles located in the transect. The concrete circles the low and medium sized brush/ trees. Cigarette buds are scattered along the concrete sidewalks of the transect. Although the bugs are not typically seen, there are organisms living in the soil and brush. Underneath the dirt of the transect is a plastic sheet. The following is a picture of Table 1. Table 1 shows the evolutionary specialization of the volvocine line.

Conclusions and Future Directions: The data proves the chlamydomonas to be the least evolved, the gonium to be the second most evolved, and the volvox to be the most evaluated. The hypothesis is rejected except for the part that marks the gonium as the second most evolved. The transect at AU did contain more biotic factors than abiotic factors, so the hypothesis is supported. CC