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. 

2/8-9/12

What did I do? I went around campus and had Matt help me pick specific species of plants to look under the microscope and after they were picked, I painted the leaves with nail polish the rest of the day. There were 11 types of plants including Criosote, Yucca, Aloe Vera, Brittle Bush and Mesquite. Today I looked under the microscope expecting to find (for most) a lot of stomata, however, I couldn't see much, close to nothing, and didn't understand why. Hopefully, I will take pictures through the microscope next week.

National Gram Staining Day

A New Holiday

Today was a day dedicated to fixing and gram staining. I gram stained so much today that I have all the steps memorized. I actually liked the excess in practicing gram staining because I become more familiar with the process, and I also do not need to rely on looking at the instructions (which is time consuming) anymore. Now I know what to do and how to do it. 

Redo

Matt told me last Thursday to try gram staining different bacterial colonies from the petri dish because there was a variety of colors. I decided that I wanted to choose three different colonies from both petri dishes (same soil samples) and see any differences. First, I chose one petri dish with the bacteria and did the fixing process. I then gram stained and labeled those three slides (1) along with the petri dish. Those would be group 1. Next, I repeated the same process with a duplicate petri dish and labeled it (2). 

Top: Group 1, Bottom Group 2 

After gram staining, I organized my slides and kept them safe in a box, labeled. 

Next, I went to check on the Fermentation Test with the Mannitol, Lactose, and Glucose with the garden soil and TSB sample. I concluded that it was positive because the tubes had changed color to yellow, and there was gas trapped. The next step was the Tryptone Broth and Indole Reagent Test. I would need to set the tube with soil sample in incubation at  37 degrees C for 24 hours. 

(Results from soil sample for Fermentation Test)

(10 grams soil sample w/ dilution blank)

(1 gram soil sample w/ dilution blank)

Lastly, I took a look at the room temperature soil samples with 10 grams and 1 gram. I gram stained the samples and will be checking all slides on Thursday under the microscopes. I may make permanents of them as well. 

Broth and Exercise!

Fermentation Test Results + A Bit of Exercise 

[Pictures above are results of TSB + Rid-X after a 24 hour incubation of 37 degrees Celcius. Left picture is Glucose, middle is Lactose, and right is Mannitol]

Recap

Today I looked at the Rid-X and TSB sample from my petri dish that was in the incubator for 37 degrees for 24 hours.According to the fermentation test, if the color of the glucose changed from red (original color) to yellow, there is acid and the result is positive. If there is a gas bubble trapped in the Durham tube, then the result is still positive. Lastly, if there is not change then the result is negative. Seeing as there was change and a bubble trapped in the Durham tube, I concluded the the result is positive. I need to redo my soil sample experiment, which I redid the Fermentation Broth test and incubated the tubes (lactose, glucose and mannitol) in an incubator. 


Focusing on the Rid-X and TSB results with the Fermentation Broth, I then proceeded to the next step which was a Tryptone Broth and Indole Reagent. I needed to get a Tryptone Broth tube and used an inoculation loop to dip the bacteria to the tube and incubate at 37 degrees Celcius. I will then need to check on the tube Tuesday and work on part 2 of the test, which is to add Indole Reagent. 

Lastly...

Matt took a look at my permanent slides that I made about two weeks ago, and he suggested that I take individualized bacteria colonies rather than a group of them to get a better image when I gram stain and look under the microscope. He recommended for me to do that on Tuesday, but for now try another approach. He gave me Dilution Blanks (2) and he wanted me to add 10 grams of Garden Soil sample that I had in one, and 1 gram of soil in another. After shaking it for 10 minutes, I would need to make streak on two different plates and leave it incubated at room temperature. 

[Left, 10 grams of soil, and right 1 gram of soil]