Soil Collection: This particular soil sample was collected in an apartment grass area which was next to a cement sidewalk and apartment building. When I contacted the people there they did not know what pesticides were used on the grass. Soil Enrichment: The solution containing my enrichment culture has turned a slight yellow. This is great news as it is indicating that the bacteria is paraoxon degradating. Streaking for Isolation: We used a agar plate to allow for streaking for isolation. I have used the inoculation loop to gather some of the bacteria that I have isolated to then streak on the plate. When I came back to look at the plate, I had successfully been able to streak for isolation. My isolates were white in color. I will use these to see if they are pure with inoculation and then using a microscope. Inoculating Broths and Agar Deeps: I used the enrichment culture from last week to allow for my bacteria to grow in the broth. When I came back the next week, the broth had gone from a slight yellow hue to a cloudy yellow. This is indicative of having bacteria growth in the broth. I also used the broth to stab the agar deep to see the results of the oxidation requirements. Agar Deep and Oxygen Requirements: The agar deep showed growth at the top as well as throughout the column I had created. This is showing that my bacterial isolate is a facultative anaerobe. It would not require oxygen to grow but it would prefer to use oxygen as it will yield more energy from doing this. Negative Staining: I have completed a negative stain for my bacterial isolate to identify its size, grouping, shape, and purity. After the successful negative stain, I was able to see that my bacterial isolate was roughly 2.5 micrometers, singular, bacillus, and was pure. The purity is a great sign as aseptic technique is very important in completing this research accurately. Gram Staining: Next, I needed to complete gram staining to see the gram status of my bacterial isolate. Upon completing the gram staining, I used a microscope to see my results. My results show a pink coloration which is an important characteristic of being gram negative. I then used an ocular micrometer to estimate the size of the cell. It turned out to be 2.0 micrometers which was smaller than the cells found in the negative stain. This change in size is characteristic of having a capsule. KOH Test: In addition to the gram staining, I completed a KOH Test to check for sure what gram status my bacterial isolate would have. To do this, we used a specialized microcentrifuge and microcentrifuge tube of my isolate and ran for 1 minute. Once we decanted the supernatant, the solution was snotty. A snotty solution is characteristic of being didermic or gram negative. The KOH Test supports my visual of the Gram Staining. My cell now is gram negative, 2.5 micrometers, singular, bacillus, and pure. ABIS Dichotomous Key: Today, I used a dichotomous key to further isolate my bacteria. When following the map I realized that I need to complete a catalase test and an oxidase test to continue down my classification. Catalase Activity Test: I received the materials of a catalase activity test and collected a bit of my bacterial isolate and placed it on a glass slide. Then, I applied the catalase to the top of it and quickly placed a cover slip on top of it. Once placed, you could see the production of bubbles. This is showing that CO2, carbon dioxide, is being produced as a byproduct. This shows that my bacteria is carrying out catalase activity. Oxidase Activity Test: Next I used a special substrate and specialized paper with my bacterial isolate to check for oxidase activity. I placed the bacterial isolate on the paper and then placed the specialized substrate. For a positive test, the color will have to change to blue or purple within 30 seconds. My sample took 25 seconds to change to the blue color. Now my bacterial isolate is positive for oxidase activity. Hanging Drop Motility Test: Another important test to narrow down my bacterial isolate is the hanging drop motility test. This test uses a specialized slide and cover slip to have the bacterial isolate hanging and will show movement of cells through a microscope. Once the setup was completed, the drop showed that the cells within were moving in all different directions and were moving fast. This shows that my bacterial isolate is motile and it is not due to Browning’s motion. Unfortunately, motion could not be captured so I had another peer watch and consent that the cells moved. Acid and Glucose Test: After finding out the test results from the oxidase, catalase, and hanging drop motility tests, I next wanted to determine further what tests were needed to isolate my bacterial isolate. The ABIS Dichotomous Key said that an Acid from Glucose test and a Nitrate Reduction test were next to determine the genus of my bacterial isolate. The acid from glucose solution starts at a purple/red color and will turn yellow upon a positive test. After allowing for my test to sit for the week, the tube turned a bright yellow color showing a positive test for acid by glucose. This test shows that fermentation can occur in the bacterial isolate. Nitrate Reduction Test: After the results of the acid from glucose test were positive, I needed to use the nitrate reduction test to further identifymy bacterial isolate. The nitrate reduction test needed some of the bacterial isolateto be deposited into a new tube with the nitrate broth. Once suspended, 6-8 drops of nitrate reagent were needed to test if the bacterial isolate would use a pathway with nitrates. The directions said that the color change from yellow to pink, a positive test, should happen almost instantly. After roughly three minutes, my culture had changed to the pink coloration which shows a positive test for nitrate reduction. DNA Isolation: First, I made a glycerol mix to have my isolate preserved if something unforeseen were to happen. Next, I took some of my bacterial isolate and microcentrifuged it to get a liquid version of a store for cell materials. I allowed this to sit in the hot water bath to allow for more cells to lyse so that I could retrieve the bacterial DNA. After the lab, I put my DNA microcentrifuge tube into a cryocontainer to allow for ice to form in the cells. This is in the hopes that the cells will lyse more and next week I can retrieve more DNA for PCR. I returned the next week to see that more supernatant was present at the top of the tube. I set the tube into a hot water bath for another five minutes to lyse the remaining cells. I then decanted the supernatant and prepared it to go into the PCR mix and other materials to get a PCR product. PCR for Isolated Bacteria: I grabbed the DNA from the previous week and set it aside to put it into the PCR Master Mix, a forward and reverse primer for the 16S gene in question, and water. I put all of the materials together and mixed. After, I took the mixture to Dr. Gold for it to be placed into the thermocycler in position 59. This ran until after lab was over and it was sent to be sequenced by an outside company. Gel Electrophoresis: In this lab, we made a agarose gel that was to be used for gel electrophoresis. We obtained the setup for the electrophoresis by Dr. Gold and were told to get our DNA samples from sequencing to be placed on a paper and made into a bead with the loading dye. The loading dye will be important for when the gel is placed under the transilluminator so we can see how far the DNA fragments moved. The beads were made and pipetted into the wells with glycerol to allow for them to float to the bottom and not move. Then we let the gel run on 90V until it reached 2/3 the length of the gel. After, we took the gel to the transilluminator and took a picture of the location of our DNA. My DNA was very clean and in a clean line. The fragment size, based off of the DNA ladder provided, was roughly 1400bp which is the desired length of the SSU rRNA 16S gene we will identify using a BLAST search and FinchTV. BLAST search for Bacterial Isolate: At this point, I know that my bacteria is gram negative, has a capsule, is bacillus, singular, pure, is 2.5 micrometers, positive for catalase activity, positive for oxidase activity, positive for acid from glucose, and positive for nitrate reduction. All of these factors are important and necessary for helping to identify a bacterial isolate but DNA sequencing is the most important factor. Using the ab1 file given back to me from Dr. Gold, I imported it into FinchTV to see what the DNA code was. Once it was imported, I went through and made sure the code written was the same represented by the color points at each nucleotide. I also checked for spacing to ensure no nucleotides were missed. After, I selected the clean sequence and did a BLAST search for the highest similarities. The BLAST search told me that there were three species that were very close to 100% but none of them were of the genusPasteurellaceae that I was expecting from the ABIS Dichotomous Key I had followed. The only genus showing was Pseudomonas. The top three species were Pseudomonas putida,Pseudomonas plecoglossida, and Pseudomonas hunanesis. The top identity % was at 100%, the top query cover % was at 99%, and the top max score was 1164. These scores all belonged to Pseudomonas putida. After further analysis of where to find this bacteria, I found that is predominantly found in soil and close to the top top so it is high in the rhizosphere. I also found that it passed all of the same exams I tested earlier in the semester. Lastly, I found that it is a common bacteria used in many pesticides as it has a very good biodegradation of aromatics such as styrene. After this BLAST search, I learned that the ABIS Dichotomous Key was not the perfect route to find my bacteria but I was able to successfully identify my bacterial isolate as Pseudomonas putida.
