Materials and Method/Results: Materials required to perform tests are the following: A microscope, inoculating loop r needle, Bunsen burner, glass slides, lens paper, bibulous paper, regular tap water, along with reagents Crystal violet, Gram’s Iodine, 95% ethyl alcohol, and seafaring. The first test I conducted was the gram stain. In preparation for the actual gram staining process I had to make a smear. Dodo this, I acquired a clean glass slide. I then placed one drop of water onto the center of the slide.
Using the flame from my Bunsen burner I sterilized my loop by passing the malleable end through the flame, making sure that the flame went up to the edge of the handle and holding it in this position until it was glowing. I then opened my unknown tube and also passed it through the flame, Just briefly. I pulled some of the specimen from my unknown tube. I then passed the tube through the flame again before placing the cap back on. I used a circular motion to place the sample of the unknown specimen onto the center of the single drop of water on the slide. After a few concentric circles with my inoculating loop, I let the slide air dry.
After approximately five minutes the slide was dry. My last step, before the actual staining process, is to heat fix my slide. This is done by making a few passes though the flame of the Bunsen burner. The next step is to complete the gram staining process. To do this I placed a few drops of my Crystal violet reagent onto the smear, being sure to completely cover the entire smear, and let it stand for about 60 seconds. This will stain all cells purple. After the 60 seconds was up I rinsed the slide with water. Then I repeated this process with the Gram’s Iodine and again let stand for another 60 seconds.
Gram’s Iodine works as a mordant which basically helps the cells take the stain better. After the 60 seconds is up, I rinsed with water. My next step was to use my decontrolling reagent. The decontrolling agent is 95% ethyl alcohol. This agent acts as a protein dehydrating agent as well as a lipid solvent, which will dissolve lipids. Tilting my slide slightly I ran one drop at a time through my smear until the drops had only a blue tinge to them. Then I again rinsed with water. At this point I countersigned using seafaring. Using this stain will stain the cells that we Just decolonize, pink.
And since only gram negative sells get decolonize, they will now absorb the countersink. For the seafaring I put about three generous drops on to my slide and let sit for about 45 seconds and the rinsed with water. I used my bibulous paper to blot my slide dry. I was then able to view my slide under my microscope using the oil immersion lens. I saw what looked like sausage links in a nice deep pink color. From this observation I had come to the conclusion that it was a gram negative stain with bacilli cells. The next step was to read the result of the Carbohydrate fermentation test.
I had one of my lab assistants inoculate the organism prior to reading the results. By viewing the tube, I determined that the Dextrose, Lactose and Sucrose test were all yellow with a bubble inside the Durham tube. This simply meant that the specimen was positive for Carbohydrate fermentation (acid) and produced gas. My next step would be the first part of the MOVIE (Indolent, methyl red, Vogues-persuade, and citrate utilization) test, the Indolent test. In order for me to complete the test I needed the Kava’s reagent and the previously inoculated tube that my lab assistant had previously set up.
I took the previously inoculated tube and added 10 drops of the Kava’s reagent. I gently swirled the tube around for a few moments. I then observed the specimen and concluded the outcome was negative, which simply meant that the substrate atrophy was not hydrolysis in that particular sample. I then moved on to the second part of the MOVIE test, the Methyl Red test and Vogues Procurers test or MRS. test. For this test I needed two clean tubes and the reagents Methyl Red indicator, Barite’s reagent A and Barite’s reagent B.
The MRS. broth is very rich in glucose so, one ml needed to be transferred to a clean tube before doing any further tests. When performing this test, we are looking for the organism’s ability to ferment glucose. For the MR. portion I took the new tube that had the one ml and deed five drops of Methyl red. I gave the tube a few swirls and the contents turned red. The reaction was positive for Methyl red. That meant the presence of acid and high pH was detected. For the second portion of this test I added 10 drops of Barite’s A reagent to the split fraction and shook the tube a bit. Then I added 10 drops of Barrister’s B reagent.
I shook the tube vigorously every three to four minutes for about fifteen minutes and waited for the reaction to occur. The UP test determines if neutral products such as action and butadiene are formed. I observed a dark, partially cloudy, yellow- brown outcome. A red ring was not present around the top of the liquid, so I concluded I had a negative reaction. The last part of the MOVIE test is the Simmons Citrate Agar. As in previous tests, my assistant had already inoculated this (sample for me, prior to my arrival). I examined the agar slant culture for the presence or absence of growth and coloration change of the medium.
My culture was a nice deep blue, which was a positive reaction. This was done by citrate and promontory blue (pH indicator) converting citrate to carbon dioxide, which makes an alkaline product. This turned the pH indicator from green to lee. Had I observed a green color in the culture, this would have shown a negative reaction. That concludes the MOVIE portion of the test series. The next test was the Areas test, also inoculated by my assistant. I observed a solid pink color throughout the culture. This indicated a positive reaction and also indicated a high PH. A solid yellow culture would have indicated a negative reaction.
The next to last test to be observed, was the Triple Sugar Iron (ITS) test. This test tests enteric for sugar utilization, gas (G) production and HAS production with the pH indicator being Phenol red. The different things that may be observed are as follows: Alkaline (K) fermentation product will turn the agar red/pink, an acid (A) fermentation product will turn the agar yellow, the presents of HAS production will turn the agar black, and last the reaction to be observed is the copious gas production which will result in the agar being cracked and possibly separated.
I observed my tube and it showed a yellow pigment, which meant there was a presence of acid (A). I also saw bubbles in the agar which suggests gas (G) was present. Finally we have come to the Gelatin test, again, that my assistant completed ahead of mime. This tube was placed in a bucket full of ice for five minutes. If I were to remove the tube from the bucket of ice and it was still solid, the reaction would be deemed negative. If I removed the tube and the contents liquefied, this will indicate a positive reaction.
Once I removed the tube from the ice bucket, I noticed that the contents were solid, which was a negative result and meant the gelatin was not hydrolysis. Data Table: Conclusion: I was able to conclude that the unknown sample was Kielbasa Pneumonia. I was able to confirm this hypothesis by using my Microbiology text book, professor signed power points, and my Person’s Custom Lab Manual. Mr.. Plough had been admitted to the hospital a few weeks prior for a severe tibia fracture in which he needed plates put in for stability.
Mr.. Slough told me that the symptoms came on suddenly and he couldn’t figure out how he had become sick. He stated that he did not come in contact with anyone who was sick. Going off of what Mr.. Slough told me about the time he spent in the hospital and his sudden onset of symptoms, I believe that the form of Kielbasa Pneumonia that he has contracted, is a form of monoclonal infection. This is an infection that is spread throughout a medical facility by way of human transport.
All employees must take note of the mode in which these infections are spreading and take action to protect themselves, as well as our patients.