January 26, 2012

Looking for Bacteria

                

                       

 (Pictures of TSB + Rid-X bacteria colony)

I originally was supposed to gram stain several of the samples that I streaked on petri dishes, but when I saw the result of the previous streak that I did (TSB + Rid-X), it came out thick and perhaps difficult to gram stain because there was no isolate bacteria colony. I was told to do another streak, but this time with a different method which Matt and Josh demonstrated to me. There are three steps to streak, and this is to better identify the bacteria colony rather than having it all jumbled into one.

Streaking instructions

• 1) Strike once (horizontal/vertical) 
• 2) Use a different inoculation loop and streak from the previous streak (about the tip of) to another vertical/horizontal or slanted streak. 
• 3) Use a different inoculation loop  and repeat step 2. 

With new TSA petri dishes and my sample of TSB and Rid-X, I used the method of streaks. (I was also told for future purposes that when labeling petri dishes, it's best to write at the perimeter of the dish for better viewing instead of the text covering the results). I then placed the two petri dishes in the incubator so I can gram stain them next week. 

I also had my soil + TSB samples from Tuesday and also applied the new streaking technique I learned. I also placed those in the incubator for next week's gram stain. 

First Official Gram Stain

I was eager to learn how to gram stain (seeing as it was the last or close to last step to identify bacteria) and remember how to apply it for future projects. I looked up different ways how to gram stain, but ultimately Josh helped me out and the visual presentation was more helpful than reading off from text. I thought it was going to be very difficult and have many steps (which it did), but it became simple once I got the hang of it. 

Procedures

• Gather materials (lighter, candle-like object*, clean slides, China marker, Crystal Violet, Distilled water, Gram Iodide, Decoloizer, Safromin, petri dishes with samples, inoculated loops, clothespin) 
• Make a circle on the center of the slide using the China marker for a target. 
• Apply about a drop or two of distilled water in the target. 
• Next, use an inoculated loop to swab the sample from the petri dish (TSB+Rid-X) and add it to the distilled water. 
• I clipped the slide to the clothespin, and used lighter to light fire on the candle-like object*. 
• With the fire present, I hovered my slide on top of the fire with a few back and forth movements until the water evaporated. 
• I removed the fire, and started the staining process. ***By the way, the method is called Fixing before staining. 

Gram Staining

• With slide, I added a Crystal Violet on the target and waited for 30 seconds. (Was done over sink with gloves) 
• Next, I washed the slide with Distilled Water for 5 seconds. 
• I then added Gram Iodide for 60 seconds.
• I washed the slide with Distilled Water and for 5 seconds.
• I then added Decolorizer for 1-5 seconds. 
• I washed the slide with Distilled Water for 1-5 seconds. 
• I then added Safromin for about 10-20 seconds. 
• Lastly, I washed it off with Distilled Water. 

Letting the slide dry off, it became maroon-purple like color and was ready to be seen under the microscope. The results were tricky as I saw a combination of Cocci and Bascilus bacteria. This is why I redid the streaking to get a better view of what bacteria is in the Rid-X. I will be doing this next Tuesday for a new sample + Soil and TSB samples.

*I will find out what the object is called. 

1/26 Mushroom Garden Instructions

Today Matt informed me that he had the materials for the mycelium project and that we would begin. Here's a summary of the procedure on the box...

Step 1 says to place the box indoors and to keep it from direct sunlight.

Step 2 says to open the front panel (it has a perforated rectangle for easy opening) and cut a 3x5 inch "+" (plus sign) into the bag.

Step 3 instructs to mist the box and its contents with water 2x/ day.

And then voilà! (it doesn't say that).
The mushrooms can be harvested in as little as 10 days. The process can be done a second time for a second harvest by repeating the above steps.

~I opened the box only to find more instructions that said to take the bag of soil out of the box and submerge it into a bucket of cold tap water. It took a 1000mL beaker filled to the top with water to hold the bag of soil down, along

1/22-23/12

These days I was getting to know and learn about stoma or stomata. I was working on how to look at it and what stomata is. * it's a pore found in the leaf and stem epidermis that is used for gas exchange.*

1/24

Today I worked on the paper electrophoresis of saliva and basically what you do is get saliva put it in a pcr tude and centrifuge it for about a minute or so. Get 2 microscope slides and a piece of starch paper the size of the slides. Wet the starch paper with the buffer and put it flat on one of the slide. Then get 20 microliters of saliva and put it directly in the center of the starch paper and cover it up with the second microscope slide. Put the slide into the cell chamber and fill it up with buffer until the top of the slide is covered. After that run 100 volts through it for about 1 hour. Thats all I did today.

Bioremediation Practice Updates

 


Today I went to check the cultured tubes to see the differences from the first day that I started my experiment to now. A quick recap, I had four tubes and there were two groups to track observations.

• The first tube had 0.02% tetrazolium, corn oil, and distilled water. 
• The second tube had 0.02% tetrazolium, oil, and microbial suspension (Rid-X). 
• The third tube had distilled water, corn oil, and distilled water added again. 
• The last, fourth tube, had distilled water, corn oil, and Rid-X. 

*Tubes 1 and 2 were Experiment A, and tubes 3 and 4 were Experiment 4. 

The observations changed today as I took a look at the tubes and realized the major difference. 

• Tubes 1 and 3 had no change, same liquid,clear appearance. 
• Tube 2 drastically changed as the oil layer turned into a maroon color, and very little to no fluids present. Underneath the maroon layer, the appearance looked like sand that was wet. The reason why the color changed was because the microbes present began to degrade the oil. This caused the chemical indicator to change structure and turn pink. 
• Lastly, Tube 4 had more of a "blob" texture like appearance, dark brown (like sand that was wet) and little fluid. This is to show that the microbes changed the composition of oil over time. 

I may more than likely will need to repeat this procedure and alter the amount of materials added in the tubes to equate the number of days for results. For example, I started the experiment last Thursday, and now Tuesday the results are dramatically different but it was supposed to be a day-to-day experience. I will need to find out how to make it even or close to even to record accordingly. 

Streaking

I also streaked the TSB + Rid-X substance in order to find out what kind of bacteria is in the Rid-X. I will more than likely gram-stain them on Thursday and figure out the results. In addition, I will most likely rework on my soil samples (with LB Nutrient Broth) to see what bacteria is residing in soil. 

Last Week

So last week I had started writing up my project. I also got to speak with Mrs. Chapman about my project and she gave me some really useful tips and ideas. Now I just need to get fimiliar with the equipment I am going to be using and begin to understand the data that is going to be giving to me after all the tests. Oh yeah, today Josh decides to tell me there is an easier piece of equipment that can tell blood pressure, which is good but not so good at the same time because I spent A LOT of frustrating time working with the other one, that I completely didn't understand. But all in all I think things are going good and I am working at a nice pace at the moment. =)

Yesterday, Wednesday 1/18

I did research on bioremediation for a possible mushroom project. The species Pleurotus Ostreatus aka the oyster mushroom is the type I may be using because it is one of few mushroom species that grows at temperatures above 75 degrees F.

A Day of Test Tubes

Bioremediation Kit Practice + TSB 

   

• 1st Picture: Me gathering materials from bioremediation kit from Carolina company. 
• 2nd Picture: Four different test tubes with corn oil, distilled water, and Rid-X 
• 3rd Picture: TSB test tube w/ a pinch of Rid-X 

Yesterday I research a Bioremediation Protocol and began the experiment today. I started off by gathering my materials, made a checklist of what I needed, and began work. 

The procedure I followed was (summarized):                                                                                    

• Labeling four different test tubes with a number and my name and date. 
• Next, I added 2 mL of 0.02% tetrazolium indicator to Tube 1 and Tube 2. 
• I added 2 mL of distilled water in Tube 3 and Tube 4. 
• I added 10 drops of Corn Oil to all four tubes. 
• I then added 2 mL of distilled water to Tube 1 and Tube 3. 
• I added 2 mL of microbial suspension (Rid-X) to Tube 2 and Tube 4. 
• I listed in my notebook what componenets each test tube had. 
• I then mixed the liquid in all test tubes by finger vortexing one test tube at a time. 
• Lastly, I recorded observations for each test tube for color, fluidity, and any other characteristics. 
• I will be seeing the difference(s) if any next Tuesday. 

TSB + Rid-X:

• Josh told me to take two TSB test tubes and add Rid-X (about a pinch or so) and next Tuesday come and see what is growing. Should be exciting to see what the results are! 

Conclusion:

I feel that by being involved in this experiment, it gives me practice with regards to microorganisms, oil, and water. I also get to see some interpretation of the real world events such as oil spills, and how it could be possible for bioremediation to come handy. The more practice I get, the better I will understand the concept and find unique ways to approach oil spill situations. 

1/19/12

So I finally figured out what my projects are going to be. The first is a Geno sensor PCR lab and since there isn't a PCR machine I can't start it yet. Currently I'm researching Paper Electrophoresis and this technique is used for the separation of small charged molecules such as amino acids and small proteins. A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes. I'm also researching Electrophoresis of saliva.

1/18/12

Happy New Year to all!!!

This year we'll be paying more attention to our projects and mine is going around Stomata and looking at it from many plants at PC. I was thinking I would use 1 from a tree, 1 from a fruit plant, and maybe 1 from a bush.

Project to focus on

Last semester, I researched about Bioremediation and gained some background information about the topic. I am someone that is passionate about saving Mother Nature, which is rather exciting to continue to research the topic and be involved this semester to find ways to save our Earth. Aquaponics/Hydroponics is a pending option, but for now it is most likely that I will put my focus on Bioremediation. Coming from a school that deals with sciences and real life situations, it would be great to introduce this topic to my school and inform students and my teachers about the different methods scientists are coming up with to clean oil spills. Time to see microbes eat oil!

What I will be doing...

Well last semester we were given a project that we needed to work on for the rest of the year. In this project we are required to test people, which is always fun, and what Josh and I have came up with is the affect of reaction time and blood pressure when an energy drink is consumed.