Material and Methods: Materials used included: Gloves, Bunsen burner, flint burner, flint strike, 3 microscope slides, inoculating loop, beater bottle, staining tray, crystal-violet, test tube rack, Iodine, Gram’s decolonize, Seafaring, microscope, oil immersion, nutrient agar, potassium hydroxide (KOCH), SIMI media, Kava’s reagent, inoculating needle, MRS. broth, Methyl red pH indicator, VIA reagents, BP reagents, empty test tube, mineral oil, MOM, Fusing media, and motility medium With ETC. El 3 Methods: First, Dry.
Carination allowed us to pick our organism from a test tube tray. The number of my organism chose was #6. After picking my organism, the first test 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. Allowed the slide o 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 rinsed it with water. Next covered the smears with Iodine and allowed that to sit for 60 seconds before rinsing it with water. Then put about 4 drops of Cram’s decolonize on top of my smears until all unbound dye was gone, immediately rinsed it with water to stop the decentralization and covered it with Seafaring for another 60 seconds and rinsed it again. Toted 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 SOX and gradually moving to I ,OX. Used oil El 4 immersion so I could see the organism clearly. 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 KOCH test to further confirm that my organism was a Gram-negative species.
For the KOCH test, I added 3 drops of potassium hydroxide (KOCH) to a small drop of distilled water onto a clean gyroscope slide, transferred a visible clump of organism to the KOCH solution using my inoculating loop. 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 RSI confirmed that it is gram- negative species. Next, created a streak plate using nutrient agar so that could see pure culture of my organism.
I aseptically obtained a loop tulle 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 into quadrant II using a zigzag motion technique. Repeated those steps streaking from quadrant II to quadrant Ill and then streaking from quadrant Ill to quadrant IV. Once completed, I put the streak plate in the incubator at 370 for 2448 hours. 48 hours later, 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 Vass the quadrant that best Less represented the colony. The whole colony was round, having irregular margins. Next, figured out that my unknown organism was a gram- negative bacillus so had to start off by doing the NIMBI series test. Started off by labeling my SIMI media for the Indolence test, methyl red-vogues persuade (MRS.), and the Simmons;s citrate agar for the Citrate test. Using an inoculating needle, transferred my unknown organism to SIMI media stabbing it straight to the bottom of the SIMI media and then incubating at 370 for 24-48 hours. Aseptically obtained a loop full of my organism and inoculated the MRS. broth with my unknown organism incubating it at 370 for 24-48 hours. After flame sterilizing the inoculating needle, seed the Simmons citrate agar to transfer my organism into tube using the stabbing and streaking technique, also incubating it at 370 for 24-B hours. After 48 hours, I took out my tubes from the incubator and continue to finish my NIMBI test, For the SIMI test, added S to 10 drops of Kava’s reagent to my SIMI tube.
After a couple of seconds, concluded that the indolence was presented because it appeared to have a cherry red layer on top of the SIMI tube which indicate indolence positive. Also, it appeared that hydrogen sulfide gas production (HAS) is not presented in my organism because it did not form a black ferric sulfide in he tube. But motility is presented in my organism because growth and turbidity occurred away from the stab line (I) Pillowing incubation for the red-vogues persuade (MRS.) test, I took the MRS. broth and carefully transferred about 2 ml Of the broth into an empty test tube for the UP test.
El 6 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 Positive for acid pH 4 Next, took the there broth that I’ve placed aside and used that broth for the UP test. Added about 10 drops of VIA and 10 drops poppa reagents to the broth.
After about 20 minutes of shaking the broth and the VIA and BP around, it appeared to have no red color developed but a cloudy, yellow color, which indicate the organism is negative for UP. The last test for the NIMBI test is the Citrate test. Following incubation, it appeared that the Simmons citrate agar seem to have a greenish color with no growth on it, indicating that its negative for citrate utilization. (1). After the NEVI test, concluded that the flow chart I was going to use to find my known organism is positive 4 (SIMI test), positive (MR. test), negative – (MRS.), negative – (citrate test), in the back tot my laboratory manual.
The next test on the flow chart was the lysine destroyable test, using the inoculating needle, transfer an invisible Amoco 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 could see a visible layer forms on top, incubating it at 37′ for 2448 hours. Following incubation, concluded that it was negative for carboxylic enzyme. (1) because it armed 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 was doing the IAMBIC test with the SIMI tube because growth and turbidity occurred away from the stab line. But wanted to double check my result so I did a motility test with ETC 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 bono thirds of the way into the agar. I than incubate the tube for 370 for 2448 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 HAS and I have already figured this out while performing my IAMBIC test using the SIMI media. The result appeared that no hydrogen sulfide gas production (HAS) 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 Marginally inorganic. Leg Ill. Results: Test Observation Results I
Gram Stain I Red, bacillus shape, rectangular shape I Gram- Negative Bacillus I Streak Plate-Nutrient Agar Representing best collaborations IV I Round, white color opaque ,irregular margins, flat I KOCH “Stringing” Gram- negative IAMBIC SIMI-elementariness I Red color layer on top Blackening Growth and turbidity Red color Yellowstone With no growth I – -Indolence Positivists not present Motility Positive for acid pH a Negative Negative Lysine Destroyable I Purple color on top, yellow color below I Negative for carboxylic enzyme Motility with ETC I Tube turned red Motile I IV. Conclusion:
After several different tests my results yielded that my unknown organism #6 was Marginally inorganic, The gram stain was red, which proved that is was a Gram-negative bacterium but wanted to confirm that my organism was a Gram-negative species so performed the KOCH test, which results came out to be “stringing”, which confirmed it was a gram-negative. By looking through the microscope 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 needed to do the NIMBI test in order to determine which flow chart was going to use. My IAMBIC result came out to be + Indolence positive, methyl red positive, UP negative, and citrate negative. While doing the IAMBIC test I figured out that my organism has no hydrogen sulfide gas production (HAS) 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 Destroyable test, which concluded that it was negative for carboxylic enzyme, because a purple color was on top, and a yellow color on the bottom.
After that, the low chart directed me to do the motility test Which I have already figured this out by doing the NIMBI test which was motile but wanted to double check my confirmation by using doing a motility test With ETC The result confirmed my previous result, the tube turned red, which was an El 10 indicator for motile. The flow chart than directed me to do a hydrogen sulfide production test (HAS), which have already figured this out also by doing the IAMBIC test, which I concluded that it was negative.