John FreesackSection A24
Kim Daffer, John Chang
September 23, 2012
Introduction:
Bacteria are everywhere. Some can be seen with the naked eye and some require a microscope but how do we distinguish one kind of bacteria from another? To answer this question, we were required to complete three bacterial labs that helped us to understand what microorganisms are and how to identify and classify them. Thus, the main purpose of this project is to identify our unknown microorganisms, more specifically, our unknown bacteria.
There are many ways to distinguish and classify bacteria. You can distinguish bacteria from other microorganisms by its size. Bacteria are around one micrometer and yeast is 4-40 micrometer …show more content…
and fungi are even bigger and can be seen easily with the human eye. Besides size, you also can distinguish bacteria by its shape. There are three shapes: cocci (spherical), bacilli (rod-like), and spirochete (helical). Another way to distinguish bacteria is by looking at its colonies. The shape of the colony (circular, rhizoid, punctiform, etc.), the edges of the colonies (entire, lobate, curled, etc.), the surfaces of the colonies (smooth, rough, wrinkled, etc.), and the elevation of the colonies (flat, raised, convex, etc.) (Leicht17).
To classify bacteria, there are numerous ways. The Gram test can be used to classify bacteria as Gram-positive or Gram-negative (Leicht 24). Bacterial cells with a thick peptidoglycan would retain the color of the dye (blue/purple) after being washed with alcohol and would be classified as Gram-positive bacteria while bacterial cells with a much thinner peptidoglycan would get its dye color washed out due to its thin peptidoglycan and turns pink/red and would be classified as Gram-negative bacteria.
The KOH string test also can be used to classify bacteria as Gram-positive or Gram-negative. Potassium hydroxide is combined with cells from a concentrated bacterial culture and strings may or may not form. Gram-positive bacteria are negative (does not form string) and Gram-negative bacteria are positive (forms string) in this test (Leicht 24).
The growth on selective medium test can also be used to classify bacteria. Gram-positive bacteria can grow on PEA agar and Gram-negative bacteria can grow on EMB-lactose and Vancomycin agar. Growth on Manitol Salt Agar (MSA) can also be used to classify different types of bacteria. It contains a high concentration of sodium chloride that permits the growth of most bacteria.
Catalase and Oxidase test can also be used. The Catalase test differentiates certain types of bacteria from others, those that can produce the enzyme catalase to neutralize the bactericidal effects of hydrogen peroxide and those that cannot (Leicht 33). Oxidase test differentiates a bacterium that possesses cytochrome c oxidase and those that do not. Those that possess cytochrome c oxidase are test positive and are aerobic species and those that do not possess cytochrome c oxidase test negative and are anaerobic species.
Another way to classify bacteria, more like identify, is DNA sequencing of the 16S ribosomal RNA gene of a bacterium. By sequencing the 16S ribosomal RNA gene, we are able to identify the best matches to the sequence of the bacterium.
Materials and Methods:
For this project, the first thing that we did was we used a sterile cotton swab and swabbed it all over the surface of the keyboard in the lab and streaked the swab across the surface of our growth medium and incubated the medium to allow the bacteria to grow. After incubating, we observed our medium and recorded our observation of the bacterial growth. We then picked a colony and prepared the sample (live culture) for PCR (polymerase chain reaction) amplification.
Afterwards, we then purified our PCR products to get rid of the unwanted waste. We added several buffers at different stages to our spin column and centrifuged it and repeated the process a couple of times in order to get rid of the unwanted waste, but we kept the spin column. We then added the last buffer and centrifuged it one last time to obtain the flow through. The flow through is the 16S rRNA gene that is used later for sequencing.
Moving on, we used some of the leftover of our “live culture” and observed it under a microscope and recorded our observation.
Then we performed a KOH string test to see if our unknown bacterium is Gram-positive or Gram-negative. We added a drop of 3% KOH (potassium hydroxide) to the leftover of our colony that we chose earlier to see if the mixture becomes viscous and forms a string. We recorded our observation and moved on to incubating our live culture on the EMB-lactose and PEA. Vancomycin wasn’t available to be used. We added our live culture to the EMB-lactose and PEA and added sterile beads to spread the bacterial cells all over the surfaces of the two agars and removed the beads and incubated the two agars. While the agars were incubating, we prepared our gel and loaded our respectful samples and ran the gel. After the gel finished running, we got a picture of our gel and recorded our observation for later …show more content…
use.
After the two agars finished incubating, we recorded our observation into Table 1, which can be found in the “Results” portion of this paper. From Table 1, we concluded that our unknown bacterium is Gram-positive and so we had to do a Catalase test and a growth on mannitol salt agar (MSA) test. For the Catalase test, a drop of 3% hydrogen peroxide was added to a sample from our LB agar. The result of the Catalase test is recorded in Table 2 that can be found in the “Results” portion of this paper. For the growth on MSA test, we created another “live culture” and add it to the MSA media and added sterile beads and spread it across the surface of the media and removed the beads and incubated the medium. After incubation, we recorded our observation in Table 2.
Now with all of the tests completed, we moved on to DNA sequencing. We added two microliter of our 16S rRNA gene with six microliter of Big Dye mix and two microliter of distilled water and ran the sample in a thermocycler. Then the sample was sequenced in an automated sequencer and we received our sequenced file a few days later and used FinchTV to correct the bases that are labeled “N”. We then copied and pasted the DNA sequence to Blastn and blast our DNA sequence and the results can be found under the blast result which can be found in the “Results” portion of this paper.
Results:
Unknown bacteria description:
The colony that we chose for this project came from the swapping of the keyboard. It is a big-yellow circular colony with an entire edge. It had a smooth surface and a raised elevation. We discovered that the bacterial cells of the unknown bacterium are cocci in shape and are in clusters after examining them under a microscope.
Table 1:
This table contains the information from the incubation of live culture on EMB-lactose and PEA. Vancomycin wasn’t available to be used. The EMB-lactose had no growth and so is considered Gram-positive. The PEA had a large amount of growth with colonies looking like a beige-ish yellow color and is considered Gram-positive. There was no string formation during KOH string test so it is considered Gram-positive.
Table 2:
This table contains the information on the Catalase test and the growth on MSA test. The Catalase test showed an immediate formation of bubbles and thus is considered a strong result. For the growth on MSA, there was growth but there weren’t any colored zones around the colonies. This meant that the unknown bacterium is a non-fermenter.
Gel Electrophoresis:
5715000114300000
B1 and C1 are mine and B2 and C2 belongs to the other group. SS is between C1 and B2.
Edited DNA Sequence:
GCCCACGNCNNGNGNGNGGCNNCGGCTCCGGGCCTAAACN CNTTCNGTAGGANGCACGAAGNANGGNCCCGCAGAAGAAG CACCGGCTAACTACGTGCCAGCAGCCGCGGTAAACGTAGG GTGCGAGCGTTATCCGGAATNTTGGGCGTAAAGAGCTCGT AGGCGGTTTGTCGCGTCTGTCGTNNNNNTCCGGGGCTTAA CCCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGCAGT AGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCA GATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGG GCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAA CAGGATTAGATAC
Blast result:
Discussion:
With a 97% certainty of what my unknown bacterium is, according to the top blast result in the “Results” portion of this paper, I cannot affirmatively say what my unknown bacterium is because of the other 3%. I have an idea of what it could be but the other 3% means that there may have been a contamination somewhere. Either in the process of swapping the keyboard to a false negative in the KOH string test due to not having enough of a high concentration of bacterial DNA to something as simple as correcting the wrong “N” base on FinchTV, which may have affected the blasting of the DNA sequence and thus having only 97% certainty of what the unknown bacterium is. I think that an uncertainty of 3% is accurate because our gel electrophoresis picture showed no bands in our C1, which is expected. If there were bands in C1, then I think that our uncertainty percentage would be much higher.
A 97% certainty is still very high and according to the blast result in the “Result” portion, I believe that the unknown bacterium is a Micrococcaceae, more specifically, Micrococcus luteus. Here are my reasoning’s. First off, after the unknown bacterium was collected and incubated, the colony was observed to be yellow and had a cocci (spherical) shape. Citizendium’s website confirms that Micrococcus luteus is indeed yellow and has a spherical shape. This supports my theory.
Table 1 shows that there was a large amount of growth on the PEA and no growth on EMB-lactose. We know that Gram-positive bacteria can only grow on PEA and only Gram-negative bacteria can grow on EMB-lactose and Vancomycin. Since we didn’t have the Vancomycin, we can’t fully prove that the unknown bacterium is 100% Gram-positive but the growth on PEA is suggesting so. Citizendium’s website also states that Micrococcus luteus is a Gram-positive bacteria. This supports my theory even more.
Then we have the KOH string test. Again, Gram-positive bacteria do not form string and Gram-negative bacteria do. There was no string formation so this points the unknown bacterium further towards being a Gram-positive bacterium. But since there may have been a contamination, we have to move on to the Catalase test. As you can see in Table 2, our unknown bacterium had a strong catalase positive reaction. This means that it can be one of the following: Bacillus, Staphylococcus, or Micrococcaceae.
Narrowing down to three types of bacteria, this is where the growth on MSA test comes in.
MSA contains a very high concentration of salt that allows only two Gram-positive species to grow: Staphylococcus and Micrococcaceae. Now we are down to two suspects. We know that according to Table 2, our unknown bacterium did grow on MSA but there weren’t any colored zones around the colonies. This means that our unknown bacterium is a non-fermenter. This was confusing at first but Superfarmer’s Weblog confirms it. On their website, they’ve confirmed that Micrococcus luteus (Micrococcaceae) do indeed grow on MSA and it is a non-fermenter thus backing up my believe that Micrococcus luteus is our unknown bacterium. But I also know that you’re saying “Staphylococcus can also grow on MSA too!” and I totally agree but as you can see under the blast result in the “Result” portion of this paper, none of the top blast result had Staphylococcus but they did have Micrococcus luteus which backs up my belief even more that Micrococcus luteus is our unknown
bacterium.
References:
Berenbaum, May, Craig Heller, David Hillis, and David Sadava. Life The Science of Biology. 9 ed. Sunderland, MA: Sinauer Associations, Inc., 2011. Print.
FinchTV 1.4.0 (Geospiza, Inc.; Seattle, WA, USA; http://www.geospiza.com)
Knisely, Karin. A student handbook for writing in biology. 3rd ed. Sunderland, MA: Sinauer ;, 2009. Print.
Leicht, Brenda, and Mark Holbrook. Diversity of Form and Function Biology 1412. Southlake, TX: Fountainhead Press, 2014. Print.
"Mannitol Salt Agar (MSA) Test." Superfarmers Weblog. N.p., 26 May 2008. Web. 20 Sept. 2014. < http://superfarmer.wordpress.com/2008/05/26/mannitol-salt-agar-msa-test/ >.
"Micrococcus Luteus." Citizendium. N.p., 03 Dec. 2010. Web. 20 Sept. 2014. < http://en.citizendium.org/wiki/Micrococcus_luteus >.
Morgulis, Aleksandr, George Coulouris, Yan Raytselis, Thomas L. Madden, Richa Agarwala, Alejandro A. Schäffer (2008), "Database Indexing for Production MegaBLAST Searches", Bioinformatics 24:1757-1764.
Zhang, Zheng, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14.