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Unknown Report Brynn Allen Bacteria: Micrococcus Luteus and Enterobacter Aerogenes My Steps: GR+ & GR- One: Gram Stain Both

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Unknown Report Brynn Allen Bacteria: Micrococcus Luteus and Enterobacter Aerogenes My Steps: GR+ & GR- One: Gram Stain Both Unknown Report Brynn Allen Bacteria: Micrococcus Luteus and Enterobacter Aerogenes My Steps: GR+ & GR- One: Gram stain both bacterias labeled “6” and “F.” Purpose: Observe what they may be under oil immersion lens after gram staining. ​ Observations: Gram positive cocci appear to be tetrads in 6 plate, and Gram negative bacillus ​ with long rods in F plate. GR+ & GR- Two: Inoculate the two fresh bacteria’s given to me from the tubes onto Nutrient Agar plates. Incubate for 24 hours. Purpose: Detect growth from my two plated organisms and see if any colors or distinct colonies ​ have revealed themselves. Observations: 6 Plate with Gram positive bacteria reveals vivid yellow streak isolation on it’s ​ plate. F plate reveals a light pink, almost transparent color with high motility and a filamentous colony appearance. GR+ & GR- Three: Performed catalase test on GR+ bacteria from 6 plate and GR- from F plate. Purpose: To see if the bacteria neutralizes toxic forms of oxygen metabolites (Hydrogen ​ Peroxide h2O2) Observations: Bacteria bubbled once catalase solution was applied to both bacteria’s. This ​ indicates a Catalase positive result. GR+ Four: Performed streak isolation on Gr+ bacteria onto an MSA plate. Incubated for 24-48 hours. Purpose: To see if this bacteria can survive in high salt concentration, if it can — the pH will ​ decrease to 6.8 and will change from red to yellow, therefore indicating it uses mannitol. If the pH goes up to 7.4, it uses amino acids and frees ammonia, it will change from red to pink. Observations: Gr+ bacteria had no change once checked after incubation. I have knowledge of ​ Gr+ non-mannitol fermenters appearing clear, colorless or white — with the exception of Micrococcus Luteus. This may be my unknown bacteria for my 6 Gr+ tube. GR+ & GR- Five: Performed Thioglycolate test on 6 organism and F organism. Purpose: To determine the oxygen requirements of my bacterias. ​ Observations: Gr+ 6 organism indicated obligate aerobes. Gr- F organism indicated obligate ​ aerobes with high motility along with many dispersed bubbles. GR+ Six: Performed Phenol Red testing for Gr+ with a lactose, glucose, and sucrose Durham tube, along with a control tube for each. Purpose: To determine if my organism can utilize any of these enzymes. ​ Observations: After a 24 hour incubation period, I concluded that there was a negative result ​ with each of my tubes.There was a slight change with the sucrose tube, but overall indicted a 76-89% negative result. Therefore, my unknown organism does not break down any of these sugars. This narrows down my search to Micrococcus Luteus and Sporosarcina Ureae. Due to the vibrant yellow color on my 1st, 2nd, and 3rd generations of inoculated nutrient agar plates, along with the tetrads and clusters seen once observed under oil immersion lens — I have figured out that my unknown Gr+ “6” bacteria is Micrococcus Luteus. In addition, M. Luteus ​ ​ has obligate aerobes. GR- Seven: Performed phenol red testing with GR- bacteria with a lactose, glucose and sucrose along with a control tube for each. Purpose: To determine if my bacilli bacteria will utilize either enzyme. ​ Results: positive reaction with lactose, glucose, and sucrose. ​ GR- Eight: SIM testing up next. Stabbed the SIM medium with my gram negative bacteria using a sterilized loop. Incubated for 24 hours. Purpose: used this differential medium to see if my organism will reduce sulfur, produce indole ​ or have motility throughout the agar. This test is a common one to use in order to differentiate members of enterobacteriaceae. ​ ​ Observations: after returning 24 hours later, there was no change. This means my bacteria ​ does not reduce sulfur. Although, there was much growth radiating outward from the stab line — therefore indicating high motility. I have knowledge of Enterobacter aerogenes being capable of motility via peritrichous flagella. After observing my negative result of sulfur reduction, I added Kovac Reagent to see if the top of the medium will turn red — therefore determining my bacteria produced tryptophanase and tryptophan is not hydrolyzed — though once adding the Kovac Reagent, no reaction occurred. Enterobacter aerogenes is known to be indole negative. GR- Nine: Methyl red testing up next. Inoculated a freshly grown sample from my GR- bacteria on nutrient agar into methyl red testing tube. Incubated for 48 hours. Purpose: to determine if my bacteria will produce stable acids by mechanisms of mixed acid ​ fermentation of glucose. The methyl red additive will give me a better idea if it produces stable acids during glucose fermentation, and the Voges Prosakeaur additive will be the indicator of whether my bacteria is capable of 2,3 butanediol fermentation following mixed acid fermentation. Observations: obtained two sterile tubes to perform my testing. Then, added 1mL of my methyl ​ red medium incubated with my bacteria to each of these tubes. Next, added 15 drops of VP reagent A and 5 drops of VP reagent B to one of my sterile tubes with the incubated medium, and I stirred it gently to allow for adequate oxygen access. After observing this tube for one hour, there was no change at each 10 minute interval. Next, I added the methyl red solution to my second sterile tube with methyl red medium along with my bacteria. I observed results immediately and found no change, therefore indicating a negative result. GR- Ten: Performed urease testing and gelatinase testing next. Inoculated my GR- bacteria into a clean urea medium, along with a control tube, incubated for 24 hours. Obtained one control gelatinase tube and one with my bacteria, stabbed the medium and incubated for 24 hours. Purpose: The intention of my gelatinase test was to determine if my bacteria has the ability to ​ produce gelatinases. Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus. The test can also be used to differentiate genera of gelatinase-producing bacteria such as Serratia and Proteus from other members of the family Enterobacteriaceae. The purpose for my urease testing was to determine if I had a bacteria capable of hydrolyzing urea with the enzyme urease. This test may indicate a Protus bacterium, or potentially H. Pylori bacteria — which are both associated with urinary tract pathogens and gastric ulcers. Observations: After returning 24 hours later, and then 48 hours later, my gelatinase and ​ urease testing both came back negative. There was no color change or solidification in either of my tubes with my GR- bacteria. GR- Eleven: Last two tests — Citrate and oxidase. Acquired an oxidase chromogenic reducing agent q-tip and swabbed my GR- bacteria directly from a fresh nutrient agar plate. Read results after 45 seconds. Acquired two citrate tubes, inoculated one with my bacteria and had one control. Incubated for 48 hours. Purpose: My citrate testing was undergone to see if my bacteria utilizes citrate as a sole carbon ​ source. This test was one part of the series referred to as the IMViC (Indole, Methyl Red, Voges-Proskauer, and Citrate tests) that distinguishes between members of the Enterobacteriaceae. My oxidase test was undergone to determine if my bacteria contains the respiratory enzyme cytochrome c oxidase. This test can be helpful in differentiating oxidase-negative Enterobacteriaceae. Observations: After 48 hours, my citrate testing indicated a positive result. There was a distinct ​ blue color within the medium that entirely differentiated from the control (green) medium. Enterobacter aerogenes is known to be citrate positive. After 45 seconds, my oxidase testing indicated a negative result by not appearing blue/purple within 20 seconds. This tells me that my bacteria does not contain cytochrome c complex. Enterobacter aerogenes is known to be oxidase negative. As a result of my final two tests, I have determined that I have Enterobacter ​ Aerogenes. ​ SUMMARY: During this lab project, I was given two unknown bacterias labeled “6” and “F.” After gram staining each of them, I concluded that I had a Gram positive cocci in 6 and a Gram negative bacillus in F. After doing multiple plating generations for my Gram positive bacteria, I noticed a bright yellow isolation streak each time I checked the plate post incubation. I’m convinced that my 6 is Micrococcus luteus due to it’s positive reaction with catalase, negative reaction with MSA plating, negative reaction with glucose, lactose and sucrose within phenol red testing, and it’s obligate aerobe determination within thioglycolate testing. Furthermore, my 6 bacteria revealed circular, pinhead colonies which were convex with entire margins once plated. Regarding my F, I’m almost certain it is Enterobacter Aerogenes. I think this due to it’s Gram negative long rods found once gram staining. Along with the characteristics of shiny colonies with entire margins and convex elevation once plated for 3 separate generations, spaced about one week apart. In addition, the extremely high motility indicated in the SIM testing and thioglycolate testing further proved that my bacteria most likely belongs to an Enterobacteriaceae genus. My catalase positive reaction, oxidase negative reaction, inability to reduce sulfur, citrate positive reaction, urease negative reaction, gelatinase negative reaction, along with positive reactions to glucose, sucrose, and lactose within my phenol red testing all indicate the characteristics and results that Enterobacter Aerogenes fall under. MICROCOCCUS LUTEUS OBSERVED IN THE CLINICAL SETTING - potential infections it may cause Although Micrococcus luteus is not a known organism to cause disease, there are skin infections in immune system compromised patients that may appear — especially in those affected with HIV virus. Micrococci itself has occasionally been identified as the cause of pneumonia, meningitis associated with ventricular shunts, septic arthritis, bacteremia, peritonitis, endophthalmitis, CR-BSI and endocarditis. Micrococcus luteus can be transferred just through contact with contaminated people or objects, especially in the hospital setting.
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