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Unknown Lab Report-
Microbiology (Micro 321)
The University of Tennessee
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Microbiology: Unknown Lab Report
Introduction
Our class completed several tests over approximately a two week period. These tests were used to determine which organism we had by eliminating all other possible organisms (among the ones we could possibly have) and by using a confirmatory test. Assuming all the tests were performed correctly, and that the specific strains our group had were largely consistent, these several tests should independently point to one organism. With more tests confirming the identity of an organism we can more reliably confirm the identity of the organism in question. Before starting the unknowns project our group performed a knowns project, which means we knew which organism we had (each individual had their own organism) and we performed several tests on them and recorded the results as well as write a description if it was necessary. The reason for this is by the end of all our tests our instructor collected the data online so that we could make a flow chart to differentiate the several organisms at hand by way of several tests. This was necessary for our group because, rather than looking up the results through a source (in retrospect, saving time), the strains our particular group have may be different than a text book definition. This may be the case because microbes evolve at such an incredible rate that no one description of an organism (given that it’s specific enough) is going to be completely correct. Using the data from our specific strains will help us create a more accurate and reliable flow chart (particularly for our strains).
The tests we use to identify our organism tell us how a specific bacterium reacts biochemically in a given environment (its surrounding nutrients and how it utilizes it, i. its biochemical reaction to an environment). For example, I had Serratia marcescens for my
knowns project and one test our group had to do was the DNA hydrolysis test. This test is supposed to distinguish between different organisms by testing for the enzyme deoxytibonuclease (DNase). This is accomplished by inoculating the microbe onto a DNase Test Agar plate, which contains “an emulsion of DNA, peptides as a nutrient source, and methyl green dye.” The organism is positive when, after incubating for a given time (in this case, 24 hours), the organism should have a clearing around it. Conversely, a negative result would be no clearing. This is because the enzyme being tested for (DNase) hydrolyzes the DNA surrounding the bacteria into small fragments which aren’t bound to the dye. The result should be a clearing around the bacteria if it’s positive. S. marcescens was positive which tells us something about its biochemical makeup (a negative result would as well), in my case I learned that S. marcescens contains the enzyme DNase. This is the case with our other tests and is the basis for making a flow chart to identify our unknown organism. For a flow chart to be successful, it is essential that the individual find a difference between all the organisms, and then find a difference between the organisms that diverged on the left as well as the right, etc. until enough differences have been found to leave only one possible organism to be the culprit. Then follow this with a confirmatory test (a test that further confirms this is indeed the organism in question) for a reliable conclusion.
These tests have significant importance in the real world outside the lab. Bacteria can cause a variety of diseases ranging from something serious to just an inconvenience. But to combat these infections it’s important to know how to differentiate between different genuses and species. Knowing the morphology, biochemistry, and ecology of all the different microbes can be useful in diagnosis when the proper tests have been performed to determine the organism. Further, when we know the biochemistry, morphology, etc. of pathogens then we can better
second colony, which appeared on both the nutrient agar and PEA was a round colony and had a yellow appearance to it. I confirmed this was the Gram-positive bacteria with the gram stain. The next test I performed, which I mentioned several times already, was the gram stain. This is a differential stain which separates the Gram-positive bacteria from the Gram-negative ones. This is done through several steps, first by adding crystal violet then when the iodine is added it creates crystals which get stuck in the cell wall of Gram-positive bacteria. Ethanol is added to get rid of the outer membrane of Gram-negative bacteria (which makes them colorless at this point) then safranin is added as a counterstain which makes the Gram-negative bacteria pink and the Gram-positive bacteria stay purple. The result of this test has already been mentioned.
Following the Gram-negative trail (refer to Table 1), I then performed the capsule stain (see Figure 1). In the capsule stain the background of the slide is stained with an acidic stain (Congo red in this case) and the cell is colorized with a basic stain (Maneval’s stain). The capsule of bacteria isn’t stained that easily, so in this particular stain everything should be colorized except the capsule. A white halo should appear around the bacteria, which it did for the Gram-negative bacteria. Getting a positive result from the capsule stain, I next performed the mannitol salt agar (MSA) test. This test is supposed to favor staphylococci growth and inhibit growth of all other organisms. This test had two possible results (in my flow chart), which were poor growth (P) and growth with a yellow halo surrounding the organism (G/Y). The bacteria didn’t grow on MSA, so the result was poor growth. Moving right along, my next test for the Gram-negative bacteria was the citrate test. The citrate test is a utilization media which tests for the organism if it can utilize citrate as its only carbon source, meaning that it contains the enzyme citrate-permease which can bring citrate into the cell and turn it into pyruvate. And pyruvate can be used for all sorts of bacterial cell shenanigans. The media changes color as the
pH changes, blue being a positive result and my result was positive. My next test was the bile esculin test, which is a test that detects the presence of hydrolytic enzymes. It’s both a selective and a differential media, selecting for Gram-negative bacteria and, esculin being easy to hydrolyze under specific conditions, detects hydrolytic enzymes. Any darkening of the media is a positive result, and mine turned out negative. With this test I reached the end of my flow chart with Pseudomonas aeruginosa (Figure 4), and confirmed this with phenol red sucrose fermentation test as my confirmatory test. In order to be correct, the phenol red sucrose needed to turn pink with no bubbles in the tube (K), which is exactly what happened. This means that peptone was deaminated, which left alkaline end products (no gas end products). With a confirmatory test and a confirmation from my instructor, I was sure that P. aeruginosa and I were going to become very close with my upcoming paper.
Coming back to the trail of my Gram-positive organism (refer to Table 2), the first test on my flow chart was the capsule stain. The organism did not have a halo surrounding it, which means it was a negative result (refer above to when I explain how the capsule stain works)(See Figure 2). The next test on my list to be conquered was the acid fast stain. This is a differential stain that tests for mycolic acid in the cell wall. Acid-fast cells, due to the mycolic acid, bind with the primary stain much more easily and resist decolorization much more effectively. A positive result (for the particular test our group performed) would be reddish-pink and a negative result would be blue. The test for my organism turned out blue, so my result was negative (meaning my organism didn’t have mycolic acid in its cell wall, so it couldn’t resist decolorization). Next, I did the gelatin hydrolysis test which tests for the enzyme gelatinase. If the organism has the enzyme, the gelatin will liquefy and stay liquefied after it’s cooled down. If not, then it will remain solid and become solid when cooled down (if it liquefied due to
what I could of those onto the negative side of the capsule stain. In the end I ended up having to perform only one test, which was the acid fast stain. The other tests, the gelatin hydrolysis (used for a confirmatory between B. subtilis and B. megaterium) and the nitrate reduction test were used instead. My second time asking my instructor if I had chosen the correct organism I got an emphatic “yes,” that it was Micrococcus luteus. Some of the tests listed above could possibly be different in other text books, which can be expected. The reason for this difference is because each strain of organisms is a little different from a “textbook” example. The reason for this is because bacteria mutate, or evolve at a fast rate. These mutations differ from species to species which may show different results depending on the test used.
Both of these organisms are important in one respect or another. I will describe each of the organisms in respect to where they can be found, how they’re important clinically, etc. starting with the Gram-negative bacteria. The genus Pseudomonas can be found in many places, including the soil, water, and plant and animal tissues and is known to produce a soluble blue- green pigmentation. The species, aeruginosa, is specifically an opportunistic pathogen which doesn’t miss an opportunity to infect and cause a disease in someone who is immunocompromised (burn victims and cystic fibrosis being examples). It’s very common for someone who has been hospitalized for a while to develop a disease associated with P. aeruginosa. Such diseases include pneumonia, urinary tract infections, and bacteremia (bacteria in the blood), abscesses and meningitis. The enzyme extracellular protease is important in the virulence of these bacteria because it helps it adhere and invade its victims. Fighting this bacterium has also proven difficult because it has even been known to not only resist several antibiotics, but also has been known to grow in some antiseptics like quaternary ammonium compounds. This genus also has a very efficient efflux system, which makes it very resistant to
antibiotics (because they just pump the antibiotics out before they can do any harm). The genus is also a culprit in spoiling milk and is also responsible for defects in the taste of milk after it has been processed. This is probably due to certain enzymes (lipase, lecithinase, extracellular protease) present with the genus. Pseudomonas, with its large genetic capacity, can produce a lot of different enzymes that help it find several niches in the environment for it to utilize the nutrients for its advantage. Another environmental effect it has is some pseudomonads can use nitrate as a terminal electron acceptor, which means they are responsible for the loss of valuable nitrogen in the soil. They convert this nitrate into gas (N 2 ) which goes into the atmosphere.
Micrococcus luteus can be found in areas such as the skin, dust, water, and soil. It can also be found in beer and dairy products. This species is also responsible for the bad odor someone gets from sweating (and not wearing deodorant) because M. luteus breaks those compounds down and produces the odor as a waste product. They can also grow well in a nitrogen environment and is halophilic. For the most part Micrococcus is harmless, but there have been instances where this genus has caused problems with people who are immunocompromised, which would make it opportunistic. This is especially the case with people who are HIV+ and can be a problem because this bacterium isn’t well known to cause any diseases and because it’s one of the many normal floras of bacteria that live on our skin. This bacterium is beginning to be recognized to cause major problems as an opportunistic bacterium has been noted to cause several infections, which include “bacteremia, septic shock, septic arthritis, endocarditis, meningitis, intracranial suppuration, and cavitating pneumonia” all of these being in people who have a compromised immune system. It also takes a while to diagnose a problem (whatever the problem is) as belonging to Micrococcus because all other
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Table 1 Pseudomonas aeruginosa Test Name Resul t Descriptio n Gram Stain - Capsule Stain + Mannitol Salt Agar P PEAPhenol Red - Sucrose K Citrate + Bile Esculin -
Table 2 Micrococcus luteus Gram Stain + Acid-Fast Stain - Capsule Stain - PEA + Nitrate Reduction - Gelatin Hydrolysis -
Unknown Lab Report-
Course: Microbiology (Micro 321)
University: The University of Tennessee
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