Microbiology Lab Unknowns
Microbiology Lab Unknowns BIOL 3200
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This 39 page Bundle was uploaded by kelleedusza Notetaker on Saturday September 17, 2016. The Bundle belongs to BIOL 3200 at Auburn University taught by Dr. Elisabeth Elder in Spring 2016. Since its upload, it has received 4 views.
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Date Created: 09/17/16
Davis 1 ` Name Lab Day/ Time: Thursday 3:00 p.m. Unknown #1108 Genus: Bacillus Species: Bacillus megaterium Davis 2 Identity of the Unknown: Bacillus megaterium Gram stain: Gram stain reaction: Gram Positive (purple) Cellular shape: Bacilli (rods) Arrangement: Chains Microscope: Brightfield microscope (100X objective lens with immersion oil) Davis 3 Primary Media and Streak Plates: SBA: AlphaHemolysis (greening of the media) was observed and incubated for 24 hours at 37 degrees Celsius. Isolated colonies were round, raised, green, and 1cm in diameter. TSA: Incubated for 24 hours at 37 degrees Celsius. Bacteria maintained at 5 degrees Celsius for 72 hours before making a new streak plate in order to run biochemical tests. Large, round colonies were observed; they were semiraised and seemed to be an opaque color. The isolated colonies were approximately 2 cm in diameter. Davis 4 Test Media/ Reagents Enzyme/ end Interpretatio Photo products n of the Test indicated by test Oxidase Swab, Oxidase Enzyme tested Negative reagent (N, N, for: N’,N’tetramethyl Cytochrome C p Oxidase phenylenediamine End Products: ) None KOH Slide, 3% KOH Enzyme tested Gram for: None Positive End Products: Cell wall lysis and release of DNA Davis 5 Catalase Slide, 3% H 2 2 Enzyme tested Positive for: Catalase, End Products: H 2 2H O2+ O 2 O/F 2 glucose tubes, Enzyme tested Oxidation of Glucose sterile mineral oil for: None glucose: End Products: negative Fermentation acid accumulation(a of glucose: drop in the pH) negative Nitrate Nitrate broth tube, Enzyme tested Negative Reduction 23 drops of for: Nitrate nitrate reagent A Reductase and 23 drops of End Products: nitrate reagent B, Nitrite or could Zinc Dust be released as Molecular Nitrogen (Nitrogen Ammonia) Davis 6 Voges MRVP broth tube, Enzyme tested Negative Proskauer serological tube, for: None pipette, VP A and End Products: VP B reagents Neutral end product, 2,3 butanediol; acetoin Discussion: The unknown bacteria #1108 was found to be Bacillus megaterium. Upon completion of the original gram stain, it was obvious that the bacteria was gram positive with a rod morphology. The KOH test was performed, and the bacteria did not lyse. Therefore, the gram stain was confirmed to be gram positive. Next, I performed the catalase test to see if the organism broke down hydrogen peroxide through the production of the enzyme, catalase. The results were negative. The oxidase test was the fourth test I performed on the first day of unknown testing; upon testing for the enzyme cytochrome C oxidase, the organism’s results were negative. At this point, I discovered the family of the organism was indeed the Bacillacae family. However, after viewing the gram stain in detail, I found that the organism grew in chains, indicating it could not be Bacillus Subtilis. The next day of testing consisted of running a hemolysis test on a sheep’s blood agar plate. The bacteria was specifically tested for Betahemolysis, and due to the greening of the agar after incubating it at 37 degrees Celsius, it was evident that the organism was negative for Betahemolysis. Two days later, I ran a Vogues Proskauer test on the organism to help determine whether the unknown organism was Bacillus Cereus or B. megaterium. The results for the VP test were negative, leading me to believe that it was B. megaterium. I ran a Davis 7 nitrate reduction test as well; the results for the reduction of nitrate to nitrite were indeed negative. Lastly, an oxidation/fermentation glucose test was used to confirm the results thus far. My unknown organism, B. megaterium, was, in fact, negative for both oxidation and the fermentation of glucose. Luckily, I had no trouble with conflicting results throughout the testing process, so I never doubted that my organism was B.megaterium. After being tested for its antibacterial capabilities, acetic acid appears to be the best chemical food preservative against the bacteria, B. megaterium. (R.K. Pundir and P. Jain, 2011). This research finding was interesting to me because I am a prepharmacy major, and B. megaterium is a prevalent bacteria found in contaminated or spoiled foods. Pharmacists and chemists have researched together to fight against food spoilage and its contamination (the presence of my bacteria, B. megaterium). Dichotomous Key: Davis 8 Bibliography: Davis 9 1. Pundir R.K., Jain P. 2011. Evaluation of five chemical food preservatives for their antibacterial activity against bacterial isolates from bakery products and mango pickles. J. Chem. Pharm, Res 3(1): 2431. Name Thursday 3:00 Unknown #1021 Klebisella pneumoniae Identity of the Unknown: Klebisella pneumoniae Gram Stain: I performed a gram stain on my unknown organism and observed it using a brightfield microscope. After using the 10x, 40x, and 100x oil immersion lenses, I was able to view my stain. Under the 100x resolution it produces a total magnification of 1000x. My unknown appeared to be pink cylinder shapes. These observations led me to conclude my organism was a Gramnegative rods that are in singles, pairs and some small chains. The pink color indicated my unknown has a thin peptidoglycan wall and does not retain crystal violet but will retain safranin. Primary Media and Steak Plates: I streaked my unknown organism onto two separate TSA plates. I incubated both of them at 37°C for 24 hours. After the 24 hours, I moved one of the plates to the 5°C incubator to keep as a reserve plate. My isolated colonies configurations were round, slightly elevated and smooth. There was no hemolysis after incubation. I had heavy growth in the first and second quadrants, and was able to measure isolated colonies in the fourth quadrant. They were about 4 mm in size and were a white/yellow color. Biochemical Tests (Table) Guidelines Test: Media/Reagents Enzyme and Observation Photo: : end products and indicated: interpretation of test: KOH Slide, 1 drop of DNA “strings” Positive 3% KOH form if the strings present Gramnegative (indicates cell wall has Gram negative lysed. bacteria) Catalase Slide, 1 drop of The catalase Positive H 2 2 enzyme breaks immediately down hydrogen produced peroxide to bubbles water and oxygen (produces bubbles). Oxidase Cotton swab, 23 Cytochrome C Negative drops of Oxidase oxidase reagent didn’t change reagent makes the swab colors (N,N,N’,N’ turns blue. tetramethylp phenyl enediamine) Hydrogen SIM Tube Hydrogen Negative; there Sulfide Sulfide is no production; blackening blackening around the stab around stab line line (no color indicates change). positive test, no Organism does color change not produce indicates Hydrogen negative test. Sulfide. Indole SIM Tube, add 5 Indole Negative; no drops of Indole production; red color change. (Kovac’s) reagent color change Organism is after 48 hours of indicates does not incubation. positive test; no produce Indole. color change indicates negative test. Motility SIM Tube Motility; Negative; organism organism grew diffused growth closer to stab outward from line. Organism the stab line is nonmotile. indicates positive test, organism’s growth is only along the stab line indicates negative test. Arabinos Arabinose, This test Negative; Red e pH indicator differentiates color indicates phenol red organisms based no on their ability fermentation of to ferment Arabinose Arabinose. Color change to yellow indicates fermentation. Lactose Lactose, This test Positive; pH indicator differentiates Yellow color phenol red organisms based indicates on their ability fermentation of to ferment Lactose Lactose. Color change to yellow indicates fermentation. Mannitol Mannitol, This test Positive; pH indicator differentiates Yellow color phenol red organisms based indicates on their ability fermentation of to ferment Mannitol Mannitol. Color change to yellow indicates fermentation. Sucrose Sucrose, This test Positive; pH indicator differentiates Yellow color phenol red organisms based indicates on their ability fermentation of to ferment Sucrose Sucrose. Color change to yellow indicates fermentation. Discussion: When I received my unknown on Thursday I was able to complete a gram stain, KOH test, oxidase test, and catalase test. First, I performed my Gram stain and used a brightfield microscope to observe my unknown with 100x magnification, where I observed pink cylinder shape organisms. This observation led me to conclude my unknown was Gram negative rods. My next test I performed was KOH, and observed strings indicating a positive test. A positive test confirms a Gram negative bacteria in which the cells are able to lyse in KOH solution. Third, I performed oxidase and after having my GTA add the oxidase reagent, my swab exhibited no color change. With a negative oxidase test, I concluded there was no presence of cytochrome oxidase. Fourth, I performed a catalase test adding hydrogen peroxide to my unknown on a microscope slide. It bubbled immediately indicating a positive test, and that my microorganism produces catalase which allows it to break down hydrogen peroxide. These four test led me to my family, Enterobacteriaceae. I came back the next day, Friday, to perform other tests including; citrate, urea hydrolysis and SIM tube. I came back the following Monday to observe and analyze my results. After inoculating a citrate tube I allowed it to incubate at 37 C over the weekend, I observed my tube turned from green to a deep blue indicating a positive test. The positive test allows me to conclude that my unknown bacteria uses citrate as its sole carbon source. My next test, urea hydrolysis, was also a positive test. I inoculated this tube on Friday and also let it incubate over the weekend at 37 C. The tube changed colors to an intense pink color caused by urease, produced by my unknown, resulting in ammonia to be released and changes the pH to become alkaline. My last test, SIM tube, allowed for me to perform three tests in one: Hydrogen Sulfide production, Indole production, and Motility. My SIM tube was negative for Hydrogen Sulfide production because there was no blackening around the stab line. I also had a negative Indole test, which was observed after adding Kovac’s reagent and observing no color change. When I observed my SIM tube for motility, I ran into difficulty determining whether it was positive or negative. I could still see bacteria around the stab line, but the rest of the medium appeared cloudy. I decided to run the test again. I streaked a plate and came back on Tuesday to inoculate another SIM tube. Along with this SIM tube, I decided to run Phenol Red Mediums, Arabinose, Lactose, Mannitol and Sucrose, to confirm my bacteria incase I had more conflicting results with observing motility. After incubating these tubes for 48 hours at 37 C I was able to observe my results. For my second SIM tube, I had the same results for Hydrogen Sulfide production and Indole production. I observed my bacteria was centered around the stab line which indicated it was negative for motility. At this point I was able to determine my unknown bacteria was Klebisella pneumoniae. Even though, I was sure about my unknown bacteria I decided to analyze my Phenol Red Mediums to ensure I had the correct bacteria. For Arabinose my tube did not change colors indicating a negative test, which allowed me to conclude my unknown is not able to ferment Arabinose. For my Lactose, Mannitol, Sucrose my tubes turned colors from red to yellow, which indicated my microorganism is able ferment those carbohydrates. I was also able to conclude my unknown was a obligate anaerobe. After observing these results, I was extremely happy because these results point towards Klebisella pneumoniae. Although I only had one conflicting result on my SIM tube, I encountered many students struggling with conflicting results. After seeing this, I decided to run extra tests, Phenol Red Mediums, to ensure I had the correct bacteria. This unknown process taught me that its important to follow all directions, and that you can never be too carful with running unknown tests. It is very likely that I will encounter Klebisella pneumoniae with in the hospital setting. I have recently switched from majoring in Biomedical Sciences Prepharmacy, to Information Systems Management. I have always had a love for technology and I plan to use this degree to work on the systems that are in a hospital. Klebisella pneumoniae is known to be found in ventilators or in intravenous catheters in patients. It can also spread through persontoperson contact of patients or healthcare professionals (Woldu, 2015). With working in a hospital, it is still likely I could come into contact with this bacterium if I am in contact with someone that is contaminated or I am in a contaminated environment. Unknown week has been very helpful in using what we have learned in lab and in lecture, giving us a hands on experience! Works Cited Woldu MA. 2015. Klebsiella pneumoniae and Its Growing Concern in Healthcare Settings. Clinical and Experimental Pharmacology Clin Exp Pharmacol 06. Name Tuesday 1pm Unknown #339 Staphylococcus epidermidis Gram Stain: I performed a gram stain and observed my unknown using a brightfield microscope. After focusing on the 10x and 40x, I used immersion oil to view my stain at 100x resolution. This produced a total magnification of 1000x. My unknown appeared to be purple clusters of little spheres. The purple color indicated that the cells were able to retain the crystal violet, and that the alcohol had no effect on the bacteria’s peptidoglycan walls. I concluded that my microorganism was a Gram-positive cocci. Primary Media and Streak Plates: I streaked my unknown organism onto two separate TSA plates. I incubated both of them at 37°C for 24 hours. After the 24 hours, I moved one of the plates to the 5°C incubator to keep as a reserve plate. After examining my plates, I noticed that there was heavy growth in the first quadrant, and isolated growth in the fourth quadrant. The colonies in the fourth quadrant had a round configuration, smooth margins, and a flat surface. They were very small (1 mm) and were yellow/white in color. Biochemical Tests (Table) Guidelines Test: Media/reagen Enzyme Observatio Photo: ts: and end n and products interpretati indicated: on of test: KOH test Slide DNA Negative - “strings” No strings 1 drop of 3% form if the present KOH Gram- (indicates negative Gram cell wall positive has lysed. cell) Catalase Slide The Bubbled catalase immediatel 1 drop of enzyme y - positive H2O2 breaks down hydrogen peroxide to water and oxygen (produces bubbles). Oxidase Cotton swab Cytochrom No color e C change - 2-3 drops of oxidase negative Oxidase makes the reagent swab turns (N,N,N’,N’- blue. tetramethyl- p-phenyl- enediamine) Coagulase Rabbit Coagulase Negative - plasma tube causes clot no clot (coagulase formation. formation tube) (fluid liquid; not viscous) Nitrate Nitrate broth Nitrate is Positive - reduction reduced to color 3 drops of nitrite or changed to both nitrate nitrogen red after reagent A gas; Red reagents A and nitrate color after and B were reagent B; A and B added zinc powder indicates if necessary positive test, and red color after zinc indicates negative test. Novobiocin TSA plate Zone of Susceptible Susceptibil inhibition ; Zone of ity Novobiocin (<= 16; inhibition disk (C) resistant) > 17 mm Bacitracin TSA plate Any zone Resistant; Susceptibil of No zone of ity Bacitracin inhibition inhibition disk (A) indicates Susceptibl e test result. Growth Mannitol Salt This test Growth (+) and Acid Agar Plate differentiat Acid (-) Production (MSA) es bacteria on based on (The yellow Mannitol their in the first Salt Agar ability to quadrant grow at can be 7.5% NaCl ruled out, and because ferment there were Mannitol no isolated (yellow yellow indicates colonies in acid any other production quadrant.) ). Mannitol; Mannitol - This test Negative; Acid from Phenol Red differentiat Red color Utilization Medium es indicates of organisms no Carbohydra based on fermentatio tes(Phenol their n of Red ability to Mannitol Medium) ferment Mannitol. Color change to yellow indicates fermentati on. Trehalose; Trehalose – This test Negative; Acid from Phenol Red differentiat Red color Utilization Medium es indicates of organisms on Carbohydra based on fermentatio tes(Phenol their n of Red ability to Trehalose Medium) ferment Trehalose. Color change to yellow indicates fermentati Gram Stain Negative Positive Enterobacteriacea Streptococcaceae Staphylococcaceae e Bacillaceae Pseudomonadace Morphology Morphology Rods Cocci Rods Cocci Enterobacteriaceae Bacillaceae Streptococcaceae Pseudomonadaceae Staphylococcacea Confirmation Tests: e Gram Stain = Gram positive 3% KOH = No strings gram positive Catalase Catalase Catalase = (+) Oxidase = (-) Positive Negativse = (-) Pseudomonadac Negative Positive eitrate reduction = (+) Streptococcaceae Staphylococcacea Novobiocin Susceptibility = (S) e Bacitracin Susceptibility = (R)riac Negative Growth and Acid Production on Reosiitvet Staphylococcus MaNegative OxidasePositive StaphiResistantCoagulaseSuscpepbiele Salt Agar = (+) growth and (-) S. saaureusticus S. ei.lrtedsis Eacidobacteriac Pseudomonadace S. saprophyBoacirocinaphylococcus Discussion: When I first received my unknown on Tuesday, I was able to complete a gram stain, KOH test, oxidase test, and catalase test. First, I performed a gram stain, and used a brightfield microscope to observe my microorganism with 1000x magnification. I saw purple, spherical bacteria, and I concluded that my microorganism was a Gram-positive cocci. Second, I completed the KOH test. When I lifted up my loop, there were no “strings” attached. This confirmed that my microorganism was Gram positive. Third, I performed the oxidase test. My swab exhibited no change in color; therefore, the test was negative. Fourth, I performed a catalase test. Once I added hydrogen peroxide, the contents on my slide bubbled immediately. This indicated a positive test result, because the catalase broke down the hydrogen peroxide to water and oxygen (bubbles). This led me to the family Staphylococcaceae. I ran out of time during my normal lab time, so I came back to the lab on Thursday in order to complete my streak tests. After streaking two TSA plates, I incubated them for 24 hours in 37°C. On Friday, I performed Mannitol Phenol Red Medium, Trehalose Phenol Red Medium, and a nitrate reduction test. Since all of these tests are safe to do over the weekend, I was able to come back into lab on Monday to check my results. Both of my Phenol Red tests came back negative, and my nitrate reduction test came back positive. This led me to Staphylococcus epidermidis. During that week, I performed a series of tests in order to confirm my microorganism. I found out that my microorganism was negative for coagulase, as well as susceptible for Novobiocin and resistant to Bacitracin. All of these tests confirmed Staphylococcus epidermidis. To be 100% positive, I performed a MSA test in order to look for growth and acid production. The biggest challenge I faced was the MSA test. Initially, I saw yellow coloring in the first quadrant, and I began to worry. The microorganism that I thought I had was supposed to be negative for acid production! To make sure I did not do the test wrong, I performed the MSA test again to double- check myself. I got the exact same results as before: slight yellow coloring in the first quadrant. To figure out this issue, I compared my plate with the pictures in the back of the lab as well as the pictures in the lab manual. Finally, I asked Dr. Miller for advice. Through all of these venues, I figured out that isolated, yellow colonies in the fourth quadrant were what I was looking for. This was great news, because that meant that my test result pointed towards Staphylococcus epidermidis. This process taught me how to check and double-check my results, compare with information from academic sources, and finally, ask a reliable source. It is very likely that I will encounter Staphylococcus epidermidis in my career as a nurse. This microorganism is part of our skin’s flora, but it can cause many problems with indwelling medical devices. Staphylococcus epidermidis is the number 1 cause of nosocomial infections, and it commonly occurs in catheters and prosthetic joints. As a nurse, I will be treating many patients who already have this infection, or I will be insuring that they do not contract it. Unknown week has been extremely helpful in taking what I have learned over the semester and teaching me how to apply it in a hands-on manner. Works Cited: Otto, Michael. "Staphylococcus Epidermidis — the 'accidental' Pathogen." Nature Reviews Microbiology 7.8 (2009): 555-67. Web. 26 Apr. 2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/>.
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