Unknown Lab Report

Custom Student Mr. Teacher ENG 1001-04 17 December 2016

Unknown Lab Report

I. Introduction

My unknown organism #6 is Morganella morganii, which is a gram-negative bacillus rods commonly found in the environment and also in the intestinal tracts of humans, mammals, and reptiles as a normal flora. (3, 5) This bacterium Morganella morganii, was first discovered in the 1906 by a British bacteriologist named H. de R. Morgan. (2) Despite its wide distribution, it is an uncommon cause of community-acquired infection and is most often encountered inpostoperative and other nosocomial settings. (2, 3) Morganella morganii infections respond well to appropriate antibiotic therapy; however, its natural resistance to many beta-lactam antibiotics may lead to delays in proper treatment Morganella morganii was previously classified under the genus Proteus as Proteus morganii. (7)

The genus Morganella currently consists of only one species, belongs to the tribe Proteeae of the family Enterobaci Morganella morganii, with two subspecies, morganii and sibonii. (6) In the late 1930s, Morganella morganii was identified as a cause of urinary tract infections and also sepsis, pneumonia, wound infections, musculoskeletal infections, CNS infections, pericarditis, chorioamnionitis, endophthalmitis, empyema, and spontaneous bacterial peritonitis. (7, 8) Anecdotal reports of nosocomial infections began to appear in the literature in the 1950s and 1960s. (3) Tucci and Isenberg reported a cluster epidemic of Morganella morganii infections occurring over a 3-month period at a general hospital in 1977and of these infections, 61% were wound infections and 39% were urinary tract infections. (2, 4) The most common source of bacteraemia was postoperative wound infection, and most infections occurred in patients who had received recent therapy with a beta-lactam antibiotic. (6) Other important epidemiological risk factors in these studies included the presence of diabetesmellitus or other serious underlying diseases and advanced age. (3)

II. Material and Methods:

Materials used included:

Gloves, Bunsen burner, flint burner, flint strike, 3 microscope slides, inoculating loop, water bottle, staining tray, crystal-violet, test tube rack, Iodine, Gram’s decolorizer, Safranin, microscope, oil immersion, nutrient agar, potassium hydroxide (KOH), SIM media, Kovac’s reagent, inoculating needle, MRVP broth, Methy red pH indicator, VPA reagents, VPB reagents, empty test tube, mineral oil, MOI, lysine media, and motility medium with TCC.

Methods:

First, Dr. Carrington allowed us to pick our organism from a test tube tray. The number of my organism I chose was #6. After picking my organism, the first test I had to do was the gram stain. I had to label everything in order to keep my lab result organized and confusion free. I started out by gathering all the material I needed to do the gram stain test. I transferred a loop full of water on a slide, and then aseptically transferred some of my organism to the slide. I allowed the slide to be fully air dry before I heat-fixed the smears. Heat fixing the slide kills the organism while also helping it retain the dye. After it was heat-fixed, I placed my slide on a staining tray, covering the smear with Crystal-violet for 60 seconds, and then I rinsed it with water.

Next I covered the smears with Iodine and allowed that to sit for 60 seconds before rinsing it with water. Then I put about 4 drops of Gram’s decolorizer on top of my smears until all unbound dye was gone. I immediately rinsed it with water to stop the decolorization and covered it with Safranin for another 60 seconds and rinsed it again. I blotted the smear dry and it was red, therefore I concluded this organism was a gram-negative. I than began to examine it under a microscope starting at 50X and gradually moving to 1,000X. I used oil immersion so I could see the organism clearly. I was able to determine that it was a bacillus shape, rectangular shape. It had a variety of arrangement, some was short, and some was long. Next I performed a KOH test to further confirm that my organism was a Gram-negative species.

For the KOH test, I added 3 drops of 10% potassium hydroxide (KOH) to a small drop of distilled water onto a clean microscope slide, transferred a visible clump of organism to the KOH solution using my inoculating loop. I than mixed the cells into the solution using small, circular motions for 60 seconds and then lifted up the loop to look for what appears to be a “stringing” affect which means it’s confirmed that it is gram- negative species. Next, I created a streak plate using nutrient agar so that I could see pure culture of my organism.

I aseptically obtained a loop full of my organism and gently inoculated one quarter of the nutrient agar plate by running the loop back and forth across the surface. I then flame sterilized the inoculating loop, allowing it to cool for 10 seconds, and then streaked the organism from quadrant I into quadrant II using a zigzag motion technique. I repeated those steps streaking from quadrant II to quadrant III and then streaking from quadrant III to quadrant IV. Once completed, I put the streak plate in the incubator at 37° for 24-48 hours. 48 hours later, I check my streak plate and it had a lot of growth on it. I was able to determine that the organism was definitely an off white color, opaque. The IV quadrant was the quadrant that best represented the colony. The whole colony was round, having irregular margins. Next, I figured out that my unknown organism was a gram- negative bacillus so I had to start off by doing the IMIV series test.

I started off by labeling my SIM media for the Indole test, methyl red-voges proskauer (MRVP), and the Simmon;s citrate agar for the Citrate test. Using an inoculating needle, I transferred my unknown organism to SIM media stabbing it straight to the bottom of the SIM media and then incubating it at 37° for 24-48 hours. I aseptically obtained a loop full of my organism and inoculated the MRVP broth with my unknown organism incubating it at 37° for 24-48 hours. After flame sterilizing the inoculating needle, I used the Simmon’s citrate agar to transfer my organism into tube using the stabbing and streaking technique, also incubating it at 37° for 24-48 hours.

After 48 hours, I took out my tubes from the incubator and continue to finish my IMIV test. For the SIM test, I added 5 to 10 drops of Kovac’s reagent to my SIM tube. After a couple of seconds, I concluded that the indole was presented because it appeared to have a cherry red layer on top of the SIM tube which indicate indole positive. Also, it appeared that hydrogen sulfide gas production (H2S) is not presented in my organism because it did not form a black ferric sulfide in the tube. But motility is presented in my organism because growth and turbidity occurred away from the stab line. (1) Following incubation for the red-voges proskauer (MRVP) test, I took the MRVP broth and carefully transferred about 2 mL of the broth into an empty test tube for the VP test.

I took my original broth and used that for the methyl red test, adding 2-5 drops of methyl red indicator to it. After a few minutes of moving and mixing the broth and the MR around, it appeared to have a reddish color. Which indicate it’s methyl red positive which means it’s Postive for acid pH 4. Next, I took the other broth that I’ve placed aside and used that broth for the VP test. I added about 10 drops of VPA and 10 drops of VPB reagents to the broth. After about 20 minutes of shaking the broth and the VPA and VPB around, it appeared to have no red color developed but a cloudy, yellow color, which indicate the organism is negative for VP. The last test for the IMIV test is the Citrate test.

Following incubation, it appeared that the Simmon’s citrate agar seem to have a greenish color with no growth on it, indicating that it’s negative for citrate utilization. (1). After the IMVI test, I concluded that the flow chart I was going to use to find my unknown organism is positive + (SIM test), positive + (MR test), negative – (MRVP), negative – (citrate test), in the back of my laboratory manual. The next test on the flow chart was the lysine decarboxylase test. Using the inoculating needle, I transfer an invisible amount of my organism into the lysine media, stabbing it straight to the bottom. I than added several drops of mineral oil on top of the lysine media until I could see a visible layer forms on top, incubating it at 37° for 24-48 hours. Following incubation, I concluded that it was negative for ecarboxylase enzyme. (1) because it formed a purple color on top and a yellow color on the bottom of the tube.

Next on the flow chart was the motility test, I figured out that my organism was motile already when I was doing the IMVIC test with the SIM tube because growth and turbidity occurred away from the stab line. But I wanted to double check my result so I did a motility test with TCC to better figure out if my unknown was motile or non-motile. I aseptically transferred my unknown organism to the motility medium by carefully stabbing a straight line, about two thirds of the way into the agar. I than incubate the tube for 37° for 24-48 hours.

Following incubation, the tube turned red all around, indicating that it’s motile. The next test on the flow chart was to perform the hydrogen sulfide production test to see if my organism produces any H2S and I have already figured this out while performing my IMVIC test using the SIM media. The result appeared that no hydrogen sulfide gas production (H2S) is presented in my organism because there was no visible black ferric sulfide in the tube. The result from the hydrogen sulfide production test was negative. Therefore leading to my unknown was Morganella morganii.

IV. Conclusion:

After several different tests my results yielded that my unknown organism #6 was Morganella morganii. The gram stain was red, which proved that is was a Gram-negative bacterium but I wanted to confirm that my organism was a Gram-negative species so I performed the KOH test, which results came out to be “stringing”, which confirmed it was a gram-negative. By looking through the microscope I was able to tell that it was bacillus-shaped. By doing the streak plate using nutrient agar I could easily describe that it was a round, white color opaque, flat, and it has a irregular margins colony. I had a gram-negative bacillus which means I needed to do the IMIV test in order to determine which flow chart I was going to use. My IMVIC result came out to be + + – -, Indole positive, methyl red positive, VP negative, and citrate negative.

While doing the IMVIC test I figured out that my organism has no hydrogen sulfide gas production (H2S) is present and also it was motile because growth and turbidity occurred away from the stab line. The flow chart than directed me to do the Lysine Decarboxylase test, which concluded that it was negative for ecarboxylase enzyme, because a purple color was on top, and a yellow color on the bottom. After that, the flow chart directed me to do the motility test which I have already figured this out by doing the IMIV test which was motile but I wanted to double check my confirmation by using doing a motility test with TCC. The result confirmed my previous result, the tube turned red, which was an indicator for motile. The flow chart than directed me to do a hydrogen sulfide production test (H2S), which I have already figured this out also by doing the IMVIC test, which I concluded that it was negative. There were no H2S presented because the tube did not blacken. After thoroughly testing this species, the unknown organism #6 is Morganella morganii.

References:

1. Carrington, Elizabeth, Ph.D. Microbiology Laboratory Manual. Second Edition. Dubuque: Kendall Hunt Publishing Company, 2006, 2009. 2. E Almaz, et al. “First case of CAPD-related peritonitis caused by Morganella morganii.” Peritoneal Dialysis International: Journal Of The International Society For Peritoneal Dialysis 30.1 (2010): 119-121. MEDLINE. EBSCO. Web. 1 May 2011>. 3. Farmer, J. J., III, B. R. Davis, F. W. Hickman-Brenner, A. McWhorter, G. P. Huntley-Carter, M. A. Asbury, C. Riddle, H. G. Wathen-Grady, C. Elias, G. R. Fanning, A. G. Steigerwalt, C. M. O’Hara, G. K. Morris, P. B. Smith, and D. J. Brenner. 1985. Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microbiol. 21:46-76 4. Fulton, M. 1943. The identity of Bacterium columbensis Castellani. J. Bacteriol. 46:79-82 5. Hu, L. T., E. B. Nicholson, B. D. Jones, M. J. Lynch, and H. L. Mobley. “Journal of Bacteriology.” Morganella Morganii Urease: Purification, Characterization, and Isolation of Gene Sequences 176.6 (1990): 3073-080. Department of Medicine, University of Maryland School of Medicine. Web. 1 May 2011. <http://Journals.ASM.org>.

6. Katz, L. M., R. J. Lewis, and D. G. Borenstein. 1987. Successful joint arthroplasty following Proteus morganii (Morganella morganii) septic arthritis: a four-year study. Arth. Rheum. 30:583-585. 7. McDermott, Carolyn, and Joseph M. Mylotte. “Morganella Morganii: Epidemiology of Bacteremic Disease.” Infection Control 5.3 (1984): 131+. The University of Chicago Press on Behalf of The Society for Healthcare Epidemiology of America. Web. 1 May 2011. <http://www.jstor.org/stable/30142630>. 8. Winslow, C.-E. A., I. J. Kligler, and W. Rothberg. 1919. Studies on the classification of the colon-typhoid group of bacteria with special reference to their fermentative reactions. J. Bacteriol. 4:429-503

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  • University/College: University of Arkansas System

  • Type of paper: Thesis/Dissertation Chapter

  • Date: 17 December 2016

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