Yesterday I looked through my algae test tubes and I found these little things that were visible to the naked eye, they looked sort of like hair which was a little weird. The test tube that I saw it in was the control with no treatment to it just plain Rio Salado water. Matt and I identified to species of blue-green algae one was Nostoc, and the other one was Chroococcus. The picture below is Nostoc but when we first identified it, it looked some what different. This is a picture of a day old algae out of water.
 

The Bacteria Stuff

I have been playing around with various types of bacterias in the lab. I was told, after this particular project, I was going to be quite the biology expert. Starting this, I was a bit skeptical of that statement. After only doing two of the 10 test needed to be done on 9 different bacterias (to start with) I am slowly becoming a believer.

This is a project that is going to be done within two different people to ensure that the results gathered from each individual is good. The goal of the entire project is to get every bacteria we have available and run many of the test on them to ensure we still dealing with a good set of chemicals and materials.
As I had mentioned before, I have only scratched the surface with solely two of the test done, but I expect to be done by next week with the rest, hopefully. :(

On a more serious side, I can say this project has helped me a lot with doing many of these test, and doing them correctly. That is a big thing I have taken from the whole project. Well I guess that all. Ohh yes, I almost forgot, I had a bit of a set back in the beginning because the ink on the plates that had my grown bacteria had smeared off when they were put in the refrigerator. I almost cried, but I kept my cool and realized I had taken pictures of each individual plate and named them. So, can you say problem conquered? :)

Heres a picture of my Salmonella after it has been plated on TSA and incubated for a day. I felt this was the more cool looking one that just had to be shared.

I started my second set of pea plants and currently I'm waiting for their week cycle to finish so that I can record the result. Today I started on my Algae project again but it came to a stop because i didn't have any pond water to work with because they forgot to get me some so now I'm going to wait until Wednesday so that Matt or josh can get me some pond water. I have made all the solutions for the experiment already so that I can start it right away on wednesday

It's been such a long time....

Since the last time I was able to blog I was finishing up my yogurt project. I have now been involved in a different project which consist of things, in my opinion, more disgusting than rotten milk. My project is to determine whether temperature has an affect on the respiration of different insects. So far I have been able to test this hypothesis out on crickets and worms, in the temperature of cold and room temp. Here is a little bit of my results:





Graph one:  Worm's Oxygen levels at room and cold temp.



Graph two: Cricket CO2 levels at room and cold temp.



What these graphs basically show is that as temperature decreased so did the respiration of these insects. Although I did feel bad for possibly killing off half of the crickets in the Phoenix (not really, don't worry) but I did end up getting some useful results. The bars that are found on the graph are called errors bars and what these bars indicate is that the majority of my data results are found between these lines. What I was taught was that if these bars would have overlapped then my results would've been insignificant. These bars state that there is a significant difference between the two sets of data and that is a good thing. :)

The way that I tested this out is through a device that records the levels of Oxygen and CO2 for 10 minutes creating a graph and giving a slope. I ran this device 10 times on a set of 6 crickets then 6 worms in both a room temperature and cold temperature (on ice). This gave me a set of 40 different slopes, which I averaged to get four individual slopes which then became the data that helped me create four different graphs, similar to the two above. I am very happy to report that I do not have to kill anymore insects, hopefully.  

10/8/12

As I finished running the PCR experiment I have started and I'm almost finished with writing a procedure for it step by step so that one of my colleges will be able to do it in a shorter time while fixing/adding separate steps to acquire the best results possible. Today I will be blowing out eggs to collect the shell for one of the labs and its a little boring and messy but it should pass time very quickly.

During the past couple of weeks I have been working on a PCR for PV92. PV92, a human-specific alu insertion on chromosome 16. The PV92 genetic system has only two alleles indicating the presence (+) or absence (-) of the alu transposable element on each of the paired chromosomes. This results in three PV92 genotypes (++, +-, or --). The + and - alleles can be separated by size using gel electrophoresis. When I first started this kit I got no results from the primers or from my extraction which resulted in a busted so I started all over again and ran the experiment multiple times until I got some result. About 4 weeks later after multiple attempts I finally got some results but I had to change the procedures just a small bit. The procedure asked for a .9% saline wash to remove the cheek cells from your mouth but instead I did the 10ml of saline wash and 10 ml of DI water and mixed them. After that I centrifuged it multiple times until I got an average size pellet at the bottom of the pcr tube. This procedure change helped me with getting results even though they were not the best results one should expect. I'll probably have to run it again so that I acquire optimal results. Also after this PCR experiment I will continue with my previous experiment on euthrophication in a more in depth research based project.  

Summer

Last month I worked on a bunch of things ranging from cleaning tables to preping labs and things of that nature. The most recent project that I worked on was a photosynthetic pigment experiment. In this experiment I had to extract pigments from different vegetables spinach, and purple cabbage. During the next couple of weeks I will be repeating this experiment but with more vegetables to test the different wave lengths that the pigments can absorb. I will also be creating a video like presentation about photosynthetic pigments. 

I'am Back!!! :)

Well haven't done this in a while but I am going tell you what I have been up this summer. I come everyday Monday - Thursday and it is always something new to explore here in the Bioscience's department. Recently I have been working on a yogurt project were I have been using different starters to see the result of the fermented milk. What basically happens when milk is fermented is the bacteria, mainly lactobacillus and streptococcus, (sorry if it is spelled wrong, they are very hard words) eats the carbohydrates (sugars) in the milk creating the end result of an organic acid, which is lactic acid. As all this is happening there are proteins in the milk that go by the name casein. These little guys like to tangle together, which coagulates the milk and solidifies. There are also times where the milk begins to curd, and the rest of the liquid in milk is separated to the top. This yellowish liquid is called whey. Whey and casein are two proteins that are found in milk, when milk coagulates the whey is left behind.Whey is very good to help with digestion.

What I had to do was pretty simple. I had to make yogurt but I had to add different types of starters to see how it affects or doesn't affect the end result. So, I started with, of course, the milk. One I heated it up to the right temperature I let it cool. Once it got to around 110 degrees ferinheight then I can add in my starters. I used yogurt along with 3 other ingredients. Those ingredients were sugar, dried milk and rennet. the different result were pretty interesting if you ask me.


This is my attempt to make chocolate yogurt with chocolate milk instead of white milk. It of course didn't work that well and I ended up with curdled milk instead. Mmmm, sounds goods doesn't it? :P
     

7/16/12

What have I been doing?

My internship here at Phoenix College for this summer so far has been fun and productive. I have done the “behind the scenes” such as washing and sanitizing the materials that were used in lab experiments. Biohazard items are disposed of in a manner of placing the materials in a biohazard bag, wrapped up, and put into an autoclave. After selecting the setting “BIOWASTE”, and as the time to laps, you will take the autoclaved bag out and put into a biohazard bin.

I have also prepared lab experiments for classes before they entered the class. I would look over the list and already be familiar with the room because prior to grabbing the items, I had to go through every practical to make sure the materials were in the room. I first place various trays on stations and set a napkin on top in case of spilling during the experiments. Then, I walk around the classroom collecting the materials and setting those on the trays. Depending on how many items are on the list, it would typically take me 30-60 minutes to make sure everything is filled, correct, and looks appropriate.

I have helped preserve the bodies in the cadaver lab. The technique to handle this is by spraying the body down with a formaldehyde solution in a spray bottle. Then place a few pieces of cloth on the body, spray, wrap in a bigger piece of cloth, spray, then zip up the body bag and move to the next one unwrapping everything and spraying.

My experiment:

I have started an experiment which involves hydrogen peroxide. My main question was, “how effective of an antiseptic is hydrogen peroxide?” and I also had a sub-question which was, “where does the foaming come from?” I will be exploring these questions and find the answers to them.

 I answered some preliminary questions before I started such as, how does hydrogen peroxide work? This is answered simply by stating, when it is applied to a surface, it reacts quickly and then breaks down into water hydrogen. At the same time free oxygen radicals are released; these create oxidation, a chemical process in which oxygen combines with another substance to break down or change the function of the molecules. Through oxidation, the bacterium decomposes, rendering it harmless.

There was also the question of, why does hydrogen peroxide foam? When catalase comes in contact with hydrogen peroxide, it turns H₂O₂ into H₂O+ O₂. Catalase does this extremely efficiently-up to 200,000 reactions per second. The bubbles you see in the foam are pure oxygen bubbles being created by the catalase. Hydrogen peroxide doesn’t foam in the bottle or on your skin because there is no catalase to help the reason to occur. (How stuff works, 2012)

Is it catalase positive or negative? When performing an experiment, you look for a reaction indicating catalase positive. If so, it is considered Staphylococci and Micrococci. There is also “Listeria, Corynebacterium diphtheria, Burkholderia cepacia, Nocardia, the family of Enterobacteriaceae, Mycobacterium tuberculosis, Aspergillus and Cryptococcus.

After exploring the back history of it, I conducted a procedure in which I included hydrogen peroxide, sterile loops, and TSA plates, samples of dirt, soil, concrete, and blood. I would seclude a section of dirt and squirt about 1TBS of hydrogen peroxide and record observations. Then, I would do the same for soil, concrete, and blood. My findings were that all of them foamed although the soil didn’t foam instantaneously. When taking a sample with the sterile loop onto a TSA plate and incubating for 24 hours at 37°C, I found multiple colonies which I then took a sample of each colony with a sterile loop and transferred to a TSB tube. After letting it settle for 24 hours in a refrigerator, I took a sterile loop and took a sample from that and spread onto a clean TSA plate. We put that into the incubator and checked the next day.

The dirt, soil, blood, and concrete grew fairly well. What I found was all the dirt colonies and soil colonies foamed. 1 of the 3 colonies for concrete did not foam.

Dirt Bacteria Isolated	Catalase (+,-)
Colony 1	Negative
Colony 2	Positive
Colony 3	Negative
Colony 4	Negative

Soil Bacteria Isolated	Catalase (+,-)
Colony 1	Positive
Colony 2	Positive
Colony 3	Negative

Concrete Bacteria Isolated	Catalase (+,-)
Colony 1	Negative
Colony 2	Negative
Colony 3	Positive

Blood Bacteria Isolated	Catalase (+,-)
Colony 1	Positive

My conclusion is that the foaming comes from the blood and cells which contains an enzyme called catalase. Since a cut or scrape contains both blood and damaged cells, there is lots of catalase floating around.

Presentation

I was prepared to present for the Student Conference 2012 at Estrella Mountain Community College. It was a great opportunity to present my findings to the community of professors, visitors, and other college students. In the beginning, I was nervous, but the nerves quickly went away and I was prepared to share my journey at Phoenix College. It was slow in the beginning as people were coming in and looking at other posters. Eventually, people began to come up to my board and gave me positive feedback which was great. Everyone there was very nice and surprised that a high school student was involved in a college setting. Overall, I felt great and relieved that I presented even though it was just an hour.

Hello again

Well, busy, busy, busy bees we have been. Today was a Fantastic day! We went to Estrella Mountain Community College for our poster presentations. It was so much fun and when the judges came one after another, I was a little nervous. One of the judges said that everyone from Phoenix College was impressive and phenomenal! All the judges and guests said that they were impressed with my work and my mentors should be proud of us. :)

I haven't posted in awhile because of me having loads of work from my school and putting the finishing touches on my poster which turned out great were withholding my brain. Overall, I enjoyed myself at EMCC and proud of my fellow peers as well! :)

Poster Changes

I made sure that I scanned my PowerPoint poster to make sure it was error free and made the changes after having Matt look over it and provided feedback. Some tips he gave me regarding my poster is that it is best to keep the writing in third person rather than first person, which I included plenty of 'I's. Next, he told me that I would need to include a conclusion in my abstract (about 1-2 sentences) which basically was the results I had with my experiments. After the changes, I uploaded my poster in Dropbox.

(Zoomed out image; my poster in regards to bioremediation)

Cleaning Up

Today I had time to finish my internship poster at school and did last minute editing. I made sure that I highlighted the important parts of my project rather than having the whole Powerpoint that I made on there. It took me a while to finish because I wanted to edit the layout and make it more appealing and also difficult to include what I wanted while keeping spacing in mind. However, I now have a draft version of  my poster and uploaded it to Dropbox. All I need is for Matt and/or Josh to look over my poster and give me any feedback.

I also cleaned up all the current and past experiments that I have saved. There were things that I kept from last year which I had no idea I had. If I were to get a penny for each test tube rack placed away, I would be a millionaire. I probably had about 100 test tubes that I kept throughout the months. There were some petri dishes that needed to be disposed, but I finally finished up cleaning.

Lastly, I helped Josh making cadaver preservative with simple steps (because the instructions are labeled). 

It has been a while

It has been a while since I last posted on the blog and I apoligize for that but i have been extremely busy with all the school work and also all the project work that i had on my hands. This whole period that I didn't post was mainly focused on my project which had a title of algae utopia and basically what I was looking for was algal and bacterial growth and that took a good amount of my time because I had to search under a microscope and I dentify the different species that were found in the sample. Coming towards the end I had to create a presentation to show my school what I was doing and also how eutrophiocationp was impacting phoenix. After going through my presentation I had a sense of relief because I don't have to present again and I think that i did a pretty great job in my presentation. Now we are cleaning up our project posters so that we can display them in the Estrella student conference.

Repairing Damages

After a periodic time of presenting my project, I now have some time to make some corrections and include additional information. I am planning to attend the Student 2012 Conference on May 3, and this will be my third time having my project displayed/presented to an audience of people. After two presentations, I now have a good idea on ways to present my findings. I also worked on making a poster from all the information I gathered. This is my first time creating a PowerPoint poster, especially a large size, and I need to figure out what are the highlights of my project because of limited space. Once I am finished, I will upload a digital copy of the poster to Dropbox and have Matt and/or Josh give me feedback.

3/28-29-30/12

All these days were dedicated to wrapping up our projects and presenting to Mrs. Chapman, Josh, and Matt as well as my fellow interns. I made adjustments and printed out my poster for tomorrow's presentation at school.

3/27/12

Today was not the greatest. I had to measure the width of the stomata for all of my plants and finishing up the density. My powerpoint is almost complete and also a work in progress. (Mostly done)

3/21-22/12

These days were focused and dedicated on working on my powerpoint and paper for our presentation. Not much exciting details for here but my presentation will look great as it's coming together nicely!

Back from Spring Break

Return from spring break. It's been a while since I have been in the lab because of a week off. However, as soon I returned I immediately continued on an experiment that I left off on, and I also started a new experiment today. 

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Bioremediation Tests 

Before the break, I had set up 13 test tubes with 13 different species of bacteria and Crisco oil. I didn't need to include the color indicator tetrazolium because the point of the test was to see if bacteria truly did do something with the oil, and also see if there would be some kind of cloudy appearance. I noticed small spots at the bottom of the test tubes that had the bacteria:

• E. coli
• Pseudomonos fluoroscens
• Salmonella
• Serratia marcescens
• Proteus vulgaris

I wondered why these specific species of bacteria had those results but the other results didn't. I showed Matt and Josh my results and I was told to repeat the experiment but this time add 1 drop of the bacteria rather than a loop, and also try a new oil -- motor oil. I may have to continue with the additional procedures another day because I needed more material like test tubes, and the room I usually work on my experiments was busy. 

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Bacteria Chart
Today I also worked on a chart, and the data that I used for the chart was a bioremediation experiment weeks ago using different species of bacteria, corn oil, and tetrazolium to see which ones turned red/dark pink. The chart consisted of four things:

1. The specie of the bacteria.
2. If there was color change or not. 
3. Gram positive or negative. 
4. Cell shape. 

(Click for better view)

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2/29-3/1-3/7-3/8

Wow, first of all, I apologize for this very late post. I did my work but forgot to post. These days were all about working on my abstract for the Estrella Mountain Community College Student Conference. I had Matt revise it, I re-worded again then worked through it with Matt and submitted it.

I also started working on a table for my project on stomata. I listed all the species of plants by common name and scientific name. I have to add the different densities.

Caffeinated

Today went by pretty fast. I was surprised how quickly time went by today and I did not even get to the second part of my newest experiment yet.

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First, 

I checked the incubator today to see how the DI water and seawater with Crisco oil and motor oil resulted with the bacteria. As of now, there are no obvious change and I recorded "no change". Change probably did occur, but if it did then it was not visible because I did not include tetrazolium (color indicator that determines if bacteria degrades oil). The only new observation that I did notice were very small bubbles (about 4-5 in each plate), but other than that nothing. I may need to wait longer than a day to see what the results were. I returned everything in an incubator and plan to take observations within a lengthy period to compare a day to about a week (more of less). 

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More soda and exercising!

Esperanza asked if she could run a test and I would be a subject which I agreed, but what I had to do was drink a can of soda in about 5 minutes and then wait 20 minutes after drinking it. I couldn't drink it down in 5 minutes, but I did my best to make sure that I finished it and then set the timer of 20 minutes. After, I went to Esperanza and what I had to do was click a button (w/ headphones on) every time I hear a sound. After, I was told to run up to the highest floor of the building using the stairs, and then run back down. I then needed to repeat the hearing test and the results came better than my first attempt. 

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Working with more bacteria and oil

Matt gave me a new experiment which was to apply 2 mL of Crisco oil in 13 tubes, and then apply 13 different species of bacteria. I was curious to see how this would work, or how I would know if the bacteria is working without the addition of tetrazolium (the color indicator). After inoculating the species of bacter to the tubes with oil, I let it set in a test rack at room temperature. 

Petri Dish Experiment

On Tuesday (yesterday) and today, Josh introduced me to a new experiment. For weeks I have been testing to see how bacteria reacted, if possible, to corn oil. After several studies, I found that for the most part all the species of bacteria that I chose ranging from E. coli to Bacillus subtilis. I immediately noticed a sudden change of pink color in the tubes compared to Enterobacter aerogenes. However, it becomes strange that a particular bacteria took days or even a week to show results while these other species of bacteria had five-second effects.

This is when a new experiment arose. This time, I would use six blank petri dishes and then add certain key ingredients.The purpose of this experiment was to apply two different species of bacteria to four petri dishes with distilled water or salt water and oil applied. I had to do some background research to find how to make seawater (my original task), but we did not have the necessary ingredients to make it at the moment. Instead, I found how to make 'seawater'.

Preparation

1. To make table 'seawater', have 35 grams of Kosher salt and apply it to a contained 965g of distilled water.
2. Stir the water and the salt until there are no salt particles present. 
3. Next, apply distilled water into three petri dishes, enough so that there are no open gaps. 
4. Repeat step 3 with seawater. 
5. Apply 5 microlitre of Crisco oil to petri dishes (2 labeled with seawater and 2 labeled distilled). [One petri dish will only contain DI H20 and Seawater to be used as control plates.]
6. Wait for 20 minutes for any spreading of the oil.
7. With a thin ruler, measure the diameter of the round oil shape for each petri dish. 
8. Apply one drop of designated bacteria to the designated plates. 
9. Carefully placed the plates in an incubator and wait for 24 hours. 

*I also replaced Cisco oil with motor oil, but did the exact same steps. 

Results

The ones with Cisco oil: I noticed that the plates with distilled water formed a small circle of only .5 centimeter while the plates containing seawater had more of a spread with the oil and the measure of the diameter was 1 centimeter.

The ones with motor oil: I saw immediate dispersion of the oil into the water. I waited for 20 minutes to see how it would form and then apply results.Since the oil was still segregated, I measured the default length of the diameter of the petri dish which is 14 cm. 

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It has been two days with this experiment. There were some flaws and little success, but I will see tomorrow if the newly added plates containing a couple of bacteria have shown results.

*I also submitted my abstract, but before that I had to continuously check for errors and make sure that it was solid. I was grateful for the feedback and help that I received to make it turn out as it is now.

Today's Color: Pink

Recap: Yesterday I incubated a total of 24 petri dishes. There was a total of 12 different species of bacteria (and a duplicate for each bacteria). I needed to wait 24 hours for the bacteria to incubate to begin the bioremedation experiments. One experiment (experiment 1) would consist of a control tube (just 2 mL of tetrazoilium and 10 drops of corn oil), and other tubes with the same substances but the unique bacteria added in each tube. Another experiment (experiment 2) would consist of just the 2 mL of tetrazolium, and tetrazolium with the unique species of bacteria. 

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Results from incubation of each bacteria

Bacillus Subtilis that has been streaked and incubated for 24 hours. It looks like thick circles that form some kind of worm-like appearance. 

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Experiment 1 and 2

Bacteria: Salmonella enterica 

There was immediate result when adding Salmonella to the tetrazolium and corn oil. Since there are obvious signs of a pink color, it shows that Salmonella is capable of degrading the oil. 

With experiment 1, I tested 10 tubes and one control tube which was just 2mL of tetrazolium and 10 drops of corn oil. 


The types of bacteria used:

• Bacillus subtilis
• E. coli 
• Serratia marcescens 
• Salmonella enterica
• Proteus vulgaris
• Enterococcus faecalis
• Pseudomonas aeroginosa
• Staphylococcus epidermidis 
• Proteus mirabilis  

For the most part, the results showed many of the species of bacteria to take immediate or gradual, but quick effect of changing into a pinkish to dark pink color. Usually, it takes a day or two to take effect (Enterobacter aerogenes), but many of the bacteria tested were much quicker than that. Experiment 2 also was the same, but I excluded corn oil and left it only with tetrazolium and added the same species of bacteria. All same results except P. aeroginosa, E. faecalis and S. epidermidis showed no effect.

[Bacillus subtilis (gram positive) takes immediate effect on just tetrazolium]

Not infected yet! A day with bacteria

Matt and Josh came up with an exciting experiment to work on which was to test all the stock of bacteria solution available and see which bacteria would degrade corn oil and which ones did not. While I could not get the official [bioremediation] test running today , I had to first take a loop-full of each bacterial solution to a TSA plate (two samples for each bacteria). I then placed all the petri dishes in an incubator and would need to leave it for 24 hours.

[Left: Tubes of different types of bacteria. Right: Different types of bacteria streaked in TSA plates]

Some bacteria included:

• Salmonella 
• E. coli
• Pseudomonas aeruginosa 
• Staphylococcus epidermidis 
• Staphylococcus aureus 

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Final results of E. aerogenes and its effect on corn oil
An end was put to the E aerogenes bioremediation test. After three different trials of the same bacteria, not only was it to confirm that Enterobacter aerogenes was Rid-X, but also to confirm that E. aerogenes degrades oil. This is evident based on the color indicator of the tetrazolium which was if the liquid changed to a pinkish color, then there are signs of the microbes degrading the oil. After of a week of the initial experimentation, the results came out just as expected. Enterobacter indeed helps degrade oil and is likely to be in Rid-X.

[Left: control tube with only 2 mL of tetrazolium and 10 drops of corn oil. No signs of a pink color because there are no microbes that were added. Right: test tube of 2 mL of tetrazolium, 10 drops of corn oil and a large inoculated loop of Enterobacter aerogenes. There are definite signs of color alteration which represents E. aerogenes taking affect and consuming the oil.]

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So what's next?

Next, I will wait a day for the newly added TSA streaked plates to be incubated. After I get some bacteria growth in the plates, I will begin the bioremediation test using the Carolina Bioremediation Kit. I also have to look online to see where I can get more tetrazolium since I am running short of the product. Lastly, I will need to finish my abstract for the Estrella Mountain Community College and I need to find credible resources to support my findings with Bioremediation.

2/21-23/12

Arizona Ash Tree
Golden BarrelCactus

All three days were dedicated to capturing pictures of the stomata I have collected since the beginning. I captured 29 different species of plants on the SPOT Advanced system. I put all of the slides on 40x on the microscope to get a better picture.

Gram Staning Continuation + Bacteria Findings

It was another day full of tasks and I tried to tackle as many as I could. I came today (Wednesday) so that I can see immediate and accurate results from my bioremediation experiment and jot down observations. I also gram stained a set of 10 slides from TSB and soil isolated bacteria.
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Pictures above are a 24 hour incubated(12 set) isolated bacteria colonies from TSB tubes. After incubation, it was clearer to see that in each TSA plate (petri dish) there was pure bacteria rather than having multiple bacterial colonies all at once. As I was observing them, the majority of the plates had bacteria growing in them. However, there were two petri dishes that did not show any visible presence of bacteria which I excluded when I was fixing and gram staining. 

10 different slides from gram staining (yet to be seen under a microscope). It took quite some time to get all of them gram stained, but after the process I was excited to see what I would see under the microscope on Thursday. The petri dishes did not need any special placement (such as in the refrigerator or incubator). I stored them at room temperature. After numerous amounts of gram staining, I would say that today was the best improvement. I was very proud of how the stains was well structured in the middle and nothing messy. This should help when looking under the microscope for accurate results.

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Bioremediation Experiment Updates

Below are pictures of what is currently present in the contents in the tube.

• All tubes consists of:
• 0.02% Tetrazolium (acts as a color indicator; if contents in tube change red/maroon/pink, then that indicates that the microbes are degrading the oil)
• 10 drops of corn oil 
• Choice of microbes (Rid-X, Enterobacter aerogenes, E. coli, and soil)

Left: This tube represents the microbe Rid-X. As you can see, there maroon/red color present. The Tetrazolium worked as it is evident that color change from a dark/sandy color to a maroon/red color. This applies to the first bioremediation that I worked on and to the newest one. There is no change but the color being present.

 Right: To the right, that is the bacteria Enterobacter aerogenes. After two attempts to confirm that E. aerogenes did "eat" oil, it is starting to come possible and promising that Rid-X indeed contains this certain bacteria. Fun fact: Enterobacter aerogenes is found in fecal matter, dairy, soil, and water.



Left: To the left, the bacteria applied is E. coli. With two attempts to see if E. coli was indeed found in the garden soil at P.C., there seems to be some possibility that it is indeed that bacteria. There is a hint of pink color present in the liquid. Unlike the soil sample (where very little to no color change present), this shows some signs. Interesting observation.

  

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Lastly, I made a control tube with only tetrazolium and corn oil, and another tube with Enterobacter aerogenes. Since I had incubated a loop full of E. aerogenes solution, I had more of a "spread" to add in the tube. It will be interesting to see the difference from a drop of E. aerogenes to a large amount. I will find out the results on Thursday.

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My collection of findings and experiments thus far. It's getting "packed", but I have plenty to observe and it is fun to partake in different experiments and still being able to manage and focus on them individually.

Continuation of Streaking + Confirmation

I started out with working on fixing and gram staining the culture samples from Thursday. On Thursday, I streaked 12 individual bacterial colonies on 12 TSA plates. There was a better result today because I could see some pure colonies, but for the most part there were still some that had a mixture of different colonies. Nonetheless, I gram stained all 12 for comparison. 

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With the same bacterial colonies, I had TSB tubes (as an alternative to TSA plates) and streaked them to 12 different TSA plates. I want to see what choice gives better results, and I was told by Matt that I may have better results with the tubes. 

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Lastly, I checked to see the first experiment I did with Bioremediation and my second experiment for any differences and comparisons. It looks like both are relatively the same. The things that I noticed was:

• Enterobacter aerogenes (on both) had some pink color. 
• E. coli (on both) showed some presence of pink color.
• Soil from first experiment had very, very little pink color while the new experiment didn't show anything. 
• Rid-X on both showed obvious change of color (maroon-red). 

*Color change of red/pink indicates the microbes degrading the oil. Tetrazolium (0.02%) acts as a color indicator. 

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Lastly, I streaked a loop-full of Enterobacter aerogenes to a TSA plate and left it to incubate for 24 hours. I will return tomorrow (Wednesday) to record observations and continue on fixing and gram staining. 

2/21/12

Today I continued working on the fresh water biology project which is mainly trying to see what kind of algae grows in pond water when we add nutrients to enrich the water. The first set of test tubes contained pond water and a plant nutrient NPK. The second pair of test tubes that were done today held a Nitrogen stock solution and another one with a phosphorus but since we I had help from robert we decided that he would do phosphorus and I would do nitrogen. The project takes about 2 weeks for the algae to grow in the incubator and when they are finished I will take some samples and look at it under a microscope to determine what type of algae it is and also anything that we can identify in the sample.

Feb. 16, 2012

Today I was able to get through most of the second part of my test. So for most of my "subjects" they no longer have to suffer the exercise of my test. There are still a few people that I need to test so I plan on having that all done by next week, hopefully. The results were actually quite interesting. It doesn't really seem like my hypothesis is has a fighting chance but we shall see. =) And that is basically it. The test took FOORRREVVER... but it feels good to be closer to the end.

What the "subjects" had to do:

-Drink a 12oz can of Sierra Mist Natural (in 5minutes)
-Wait 20minutes
-Then preform the test (both resting and post exercise) again.

That was my day.

Identity:

Updates on Bacteria Identification and Bacterial Isolation

[Left, 12 TSB tubes with soil, Center are the TBS tubes w/12 TSA plates, Right are the two TSA source plates of soil]

Today, Matt suggested that I isolate the bacterial colonies in the two TSA plates (one with dilution blank of 1 gram of soil, and another one with 10 grams of soil) and either inoculate in TSB tubes or streak the isolated bacteria in TSA plates. I decided to choose both to see which method worked best, and I streaked a total of 12 TSA plates (6 with 1g of soil and 6 with 10g of soil), and inoculated 12 TSB tubes (6 with 1g of soil and 6 with 10g of soil). I was also told that I should leave the TSA plates and TSB tubes at room temperature. Next Tuesday, I will try a new technique involving oil.

[ The source for the bacterial colonies was from my previous weeks worth experiment where I used dilution blanks to apply 10 grams of soil in one blank, and 1 gram of soil in another. Then I had to streak the samples in two different TSA plates. ]

Necessity of Isolation
Despite the fact that I had to isolate each bacteria colony various times throughout this year, I think that it is important to isolate the bacteria (once present in the TSA plates) to see if there are different types of bacteria present in whatever substance is being tested on. For example, when I saw the result of bacteria growing on a TSA plate for Rid-X, there weren't any different shapes or colors of bacteria and everything looked the same. Ideally, the bacteria in Rid-X should be in unison and not have multiple bacteria. Therefore, it would make sense that in natural sources like soil, there would be a mismatch of bacteria identification because of cross contamination and natural forces. When I saw the different shapes and colors of bacteria growing in the TSA plates for soil, there were just too many to see the sample itself all at once. Isolating each bacterial colony individually was necessary for better and accurate results.

 

[Left, 4 different experiment tubes. Middle, Rid-X (control tube), Right garden soil tube]

 

[Left, Enterobacter aerogenes tube, Right Escherichia coli] 


Results
Since I added 0.02% tetrazolium when I conducted the experiment, if in the following days the experiment changed color to red/pink, then that was an indicator to show that the microbes present began to degrade oil. This caused the chemical indicator to change structure and turn to a red/pink.

Which tube experiments were the ones that turned into red/pink?

Rid-X, because it was already tested to show that it worked, was a tube that changed color (maroon). The Enterobacter aerogenes tube also turned into a light pink color, which is possibility that Rid-X is that bacteria (I concluded that E. aerogenes was in Rid-X). The garden soil tube did not change at all, and the E. coli tube had no change at all. Could this mean that E. coli, the bacteria that I thought was in the soil, is not the correct one? I will see what happens in the next few days and update a last observation to see if the effect of E. Coli and microbes in the garden soil worked or not. 

Today February 15, 2012 :0

So today I got to look deeper in how I am going to set up the data in project.

One word - S T A T S

Matt showed me how to notice One Way ANOVA (One Way Analysis of Variances). Being that I am a Calculus student and not a Statistics student, it was quite complicated for me. He sat me down with Josh and we discussed what this is going to look like for me.
So this is how its looking at the moment:

My variable - Reaction time
Treatment - Resting state, post-exercise, sugar - resting, sugar post - exercise.
So what I got out of ANOVA is that it is the mean of all four treatments and it's average difference from mean and values (+/-).
ANOVA is going to give me the F-Score that turns into --> P-Value (percent value) and that determines whether you have a significant difference in your data or a not so significant difference. So tomorrow, when I get to do the second part of the test on everyone I can start organizing my stuff.

Oh yeah, I almost forgot...
My product is going to be a bar graph =)

Guess Who: Bacteria Style

Today I learned how to view my slides under a microscope that was connected to a computer and watch it live. I took several pictures, but I will continue to take pictures on Thursday.

Today was oriented on determining if the bacteria that I found in Rid-X and Soil were correct. A recap of last week's findings.

• In Rid-X, I found that the bacteria is Enterboacter aerogenes. This type of bacteria is found in soil, water, dairy products, and in the intestines of animals and humans. 
• In soil from the garden, I found that the bacteria is Escherichia coli. This type of bacteria is found in animal feces and lower intestines of animals. 

Bioremediation Experiment 2.0 

Knowing that Rid-X worked in decreasing the amount of corn oil in a tube, it became my control tube. I had three other tubes that I was going to test; In the replacement of Rid-X, I would add soil to one tube see if it would clean the oil, another tube with Enterobacter aerogenes, and another with Escherichia coli. This would help me see if the bacteria that I chose for garden soil and Rid-X was correct. (Hoping that it was!) 

Procedure

1. Label four tubes with each testing material, your name, and date. 
2. Starting with the Rid-X tube, add 2 mL of 0.02% tetrazolium with a measuring pipet. Then apply with the other three tubes. 
3. Add 10 drops of corn oil to each test tube. 
4. Add 1 gram of Rid-X in the Rid-X tube. 
5. Add 1 gram of soil in the soil tube. 
6. Add 1 drop of Enterobacter aerogenes to the proper tube. 
7. Add 1 drop of Escherichia coli to the E. coli tube. 
8. Finger vortex all test tubes. 

Observations

I jotted down observations for each tube to see how they compare in a couple of days. 

• Rid-X = Tan color (Rid-X) at the bottom of the tube, yellow layer and clear liquid. 
• Soil = Dark brown soil at the bottom of tube, clear liquid w/ yellow layer. 
• Enterobacter aerogenes = Clear liquid w/ yellow layer. 
• Eschericia coli = Clear liquid, yellow layer. 

I will then need to wait for Thursday to see the results and write down new observations. 

2/10/12

The slides I prepared and observed

The leaves I used for the slides


I came in today and applied more nail polish on the leaves but I brought some from home. The ones I brought consisted of Lemon tree, Grapefruit, Jalapeno, Habenero, Ash, Ajo, and Hibiscus. All of the bottom sides of the leaves were perfect. However, the top sides of all of the leaves didn't have any which I found odd. So I'm going to say that with only a little bit of practice, I would expect a couple mishaps. But I'm confident that the results will be great.

Bacteria In You

Today was another hardworking day, and I performed a variety of tasks. The first thing that I did was to get the Tryptone Broth from the incubator that I left on Tuesday, and added indole (I would need to add some drops to the side of the tube and let it flow down), and then gently rotate the tube for a minimum of 10 minutes.) I had to make sure that I left it undisturbed to get accurate results.

 

(Because the picture shows a red top layer, it is positive.Left is before the indole reagent was added, and right  is when the indole reagent was added.)

Results
After vigorous gram staining, streaking, and a variety of tests, I finally had results to what bacteria was in Rid-X and garden soil. The bacteria in Rid-X is Enterobacter aerogenes.

• "Enterobacter genus originates primarily in the intestinal tracts of most warn-blooded animals. They are smaller rod-shaped cells that are motile and encapsulated compared to others in the same family of Enterobacteriaceae. 
• It is known to be resistant to antibiotics.
• Found in soil, water, dairy products, and the intestines of animals and humans.
• Causes diseases in humans 
• Are opportunistic and only infect those who already have suppressed host immunity defenses. 
• Infants, the elderly, and those who are in the terminal stages of other diseases are prime candidates for such infections."

Information from:

microbewiki.kenyon.edu/index.php/Enterobacter_aerogenes 

The garden soil sample contained Escherichia Coli (E. Coli). 

• "It is an aerobic, gram negative, rod-shaped bacteria that can be commonly found in animal feces, lower intestines of mammals, and even on the edge of hot springs. They prefer to live at a higher temperature rather than the cooler temperature. It does not sporulate, and it is easy to eradicate by simple boiling or basic sterilization.
• Can help the digestion breakdown and absorption, and vitamin K production in intestines. 
• Most strains are not harmful. 
• Can cause severe infections to mammals and animals."

Information from: 

microbewiki.kenyon.edu/index.php/escherichi_coli

I found these findings to be very interesting and surprising. The bacteria that I found could be harmful, but there are benefits such as helping digestion and removing toxic substances from crude oil. Bacteria is a helpful and harmful organism!

 

Lastly, I gram-stained 10 slides, they were 10g of soil sample (6 different bacterial colonies) and 1g of soil sample (4 different bacterial colonies). It took me some time to do, but finally finished and will be looking under the microscope on Tuesday.