User:Jose E Muniz Gomez/Notebook/Biology 210 at AU

Title: Identifying and Studying Bacteria Date: 2/4/2016

A) Microorganisms Observation and Quantification, Antibiotic Resistance, Bacterial Cell Morphology Observations and PCR Amplification for 16s rRNA gene:

1) Purpose: The purpose of the Microorganisms Observation and Quantification, Antibiotic Resistance Evaluation, Bacterial Cell Morphology Observations and PCR Amplification for 16s rRNA gene is to get a deeper approach and a better qualitative and quantitative understanding of the cultivation's that was worked with at the lab. This research methods will help to identify the action mechanism of the bacteria's' that we are working with, the specie and kind of bacteria that was founded and through the PCR technique, ratify the specific bacteria that was obtained from the ecosystem where the hay was developed.

2-Methods/Materials:

a) Quantifying and Observing Microorganisms:

During this process eight cultivation plates were evaluated to identify any bacterial colonial presence, or maybe any fungi development. Four plates were exposed to tetracycline and the other four were not exposed to tetracycline in their agar gel plate. From the four plates that were exposed to tetracycline, (10-3 +T) was the one that more colonies presence had with seventeen, (10-5 +T) showed the development of a huge fungi in the center, (10-7 +T) just had one colony and (10-9 +T) do not presented any bacterial growth. This suggests that the (10-3 +T) could be resistant to Tetracycline, because it was the diluted section that more bacteria's had and even though, was the agar plate with more bacterial development. In the other side, the four agar plates at least once showed bacterial growth in the agar cultivation plates without tetracycline. (10-3 -T) showed more than a hundred bacterial colonies, the (10-5 -T) plates had 73 colonies, (10-7 -T) presented 12 colonies and (10-9 -T) just had one colony. This scenario establishes the previous expectations because of the environmental conditions that the cultivation's where on. Finally, is very important to add that all colonies were measured in the colonies/mL interval to comprehend the development capacity they occupied in the agar plate.

b) Antibiotic Resistance:

Antibiotic Resistance is the ability that a bacterial organism has to avoid the action mechanism that an antibiotic could have against it. This action could be against the bacterial cell wall, the ribosomes or the DNA material through the use of the efflux pumps. In the studied bacterial colonies, it was seen that overwhelmingly the bacterial presence was less in those that had tetracycline in comparison with those that do not had tetracycline. However, the (10-3 +T), the cultivation with the most bacterial presence because it was not diluted, had the bigger bacterial presence with tetracycline. This projects the idea that these cells could maybe be resistant to tetracycline. The effect of tetracycline in the bacterial cultivation's were it was present had a 73% of effectivity, however it did not acted against the fungi presence. Going along with this, over 65 tetracycline resistant genes had been identify, which makes infer that maybe one of the obtained bacterias from the hay infusion could be resistant to this antibiotic that inhibits the protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site (Chopra et. al a). The most common resistant bacteria's to tetracycline are a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites (Chopra et. al b).

c) Bacteria Cell Morphology Observations:

To accomplish the goal of get a better approach to our cells for the precise description and identification of the studied organisms, a wet mount and a gram stain procedure was performed. For the wet mount procedure, a portion of the cell cultivation's from the chosen four agar plates was mixed on a water drop over a slide. Then, it was proceed to identify at 10x and 40x any presence of motility, morphology or bacterial shape in the slide. For the gram stain procedure, the same four bacterial colonies were used and prepared according to the established protocol and placed below the microscope to identify the same characteristics that were searched in the wet mount procedure too.

d) PCR for 16s Amplification:

In this procedure two strains were chosen for the amplification of the 16s rRNA gene with the given procedure in the lab manual. Each sample will use primers and PCR to expand the 16s rRNA gene and identify with certainty the bacteria that we are studying.

3)Raw Data:

Image 1. This image shows the evaluation of all the eight obtained agar plate cultivation's in the first step of all this methods.

Image 2. This image is a table that collects data from the wet mount procedure and the gram stain procedure.

4) Conclusion:

In conclusion, it can be stated that bacteria strains identification process is a very complicated process that range from the organism morphology through rRNA 16s gene amplification to let identify it with a database that with certainty will let you know who is the specie that you had been working with. Bacterias, are one of the most diverse organism in the world and this diversity is also marked by their ability to develop resistance to antibiotics through mutations in the DNA sequence. Bacterias are also the only prokaryotes that have the ability of make ill humans and because of their mutations as result of the high exposition to antibiotics, humans should wash their hands with non-anti bacterial soap to avoid the non-resistance development. Finally, stains like the gram stain one are very important to identify with precision if the organism below the microscope is a bacteria or no through the peptidoglycan expression in their cell wall.

5) References:

Chopra, I., Roberts, M. (26 November 2014). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Elsevier, 175, 485-493. 4 February 2016,from Google Scholar Data Base.

J.E.M.G.

Title: Identifying Algae and Protists Date: 1/28/2016

A) Hay Infusion Culture Observations

1-Purpose: The purpose of this section of the lab was to identify different living organisms organized in niches trough different zones of hay infusion culture jar. This leads to have representative samples of different populations that that populates this new formed ecosystem. This will permits identify and measure the algae's and protists that have been developing in this new environment.

2-Methods/Materials:

a) Hay Infusion Collection (by zone) using the Dichotomous Key: The top zone and the bottom zone were chosen to get some drops. First, the top zone showed at 4x different microorganisms that were randomly rapidly moving. However, at 10x three organisms were identified using the dichotomous key in the top layer: Colpidium, Euglena and Volvox. Also, a fungus piece was also founded surrounded by different organisms. Colpidium is a protozoa that produces its own energy through photosynthesis. The identified one in the sample had a size of 49µm and among its physical traits distinguish its motility. On the other hand, Euglena is an algae that produces its own energy too through photosynthesis. The sighted one in the studied sample had a size of 39µm and among its physical traits distinguish its motility. Finally, the Volvox algae was also seen in the hay infusion top layer. Interestingly, Volvox is a photosynthesis energy producer algae, that moves through the use of its motility and the one that was seen had a size of 3µm because it was founded individually.

The bottom zone just showed up one organism in the sample. The Pandorina is a motile-algae that produces its own energy through photosynthesis and the identified one in the studied sample measured 125µm.

b)Preparing and Plating Serial Dilutions: To develop bacterial cultivation's for the microbiology lab of the next week, four tubes (10-2, 10-4, 10-6, 10-8) of 10 mLs sterile broth were labeled to carry out a serial dilution. Then four nutrient agar and four nutrient agar plus tetracycline plates, were labeled for the cultivation's. Later, the Hay Infusion Culture was mixed and 100µl were added to the the first (10-2) tube. Then, 100µl from the 10-2 tube is well mixed and so forth until reach the last tube (10-8). Finally, all the tubes after being diluted were spread throughout the plate and set on a rack incubated at room temperature.

3-Raw Data: Serial Dilutions Procedure

Fungus Image below the microscope

Pandorina Image below the microscope

Volvox Image below the microscope

4-Conclusions and Future Experiments:

In conclusion, it can be stated that biodiversity and the development of life is something that can be found at all levels as long and the abiotic and biotics components set the bases. With the use the bacterial growths in the agar plates, it will be seen their reproduction efficiency regardless of being diluted, but also how strong are this organisms against the presence of antibiotics. This experiment also let's better comprehend how diverse can an isolated ecosystem be. On the other hand, and taking the founded Volvox presence in the top layer, it can be stated that its presence is because it fulfills with all the life requirements to be alive; it produces energy, is a cell, it has genetic information, can replicate and throughout the time it had evolved in different species. Finally, it is considered that if the hay infusion culture is evaluated in two more months, the development of fungus will be higher. And because of the anaerobic environment, some organisms will not be able to keep alive in this scenario leading to the extinction of a huge portion of niches. This explains why do the obtained hay infusion culture has a repulsive odor like muck.

J.E.M.G.

Title: Biotic and Abiotic Components Collection from AU campus Date: 1/15/2016

1)Purpose: The purpose of the biotic and abiotic components collection through AU campus was to identify different ways of life in our campus. This will later let us know, with greater certainty, what are the different protists, bacteria’s and other ways of life in the assays we found.

2) Methods/Materials:

a) Material Collection Process: For the soil collection process, the group went to the American University’s garden and collected soil and biotic and abiotic components from throughout the all garden for a period of 25 minutes. In total, approximately 1 lb. of soil and other materials was collected. During the process, pictures were take it to identify the spaces from where the materials where collected.

b) Hay Infusion Culture: To create a hay infusion culture, we need an empty glass container. Then, we proceed to measure 12 g of the collected soil and biotic and abiotic materials in the weight scale and added to the container. Later, 500 mLs of water are measured with a graduated cylinder and added to the glass container. 0.1 g of dried milk are weighted in the weight scale and added too to the glass container. Finally, everything is mixed by manually lateral movements.

3) Raw Data:

4) Conclusions and Future Experiments

In conclusion, it can be stated that nature is composed of abiotic and biotic material that conform our living spaces and our environment. The contact of these conditions with artificial materials, such the mix of dried milk with them, can produce organisms that surround us and conform all ways of life. In the future, instead of use water to mix it with the collected material, the use of acid juice (like lemon) could let us understand the anti-bacterial resistance that maybe this substance carry.

J.E.M.G