Bacterial unknown - BIOS 313 lab PDF

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Identification of Bacterial Unknown

Abstract The identification of microorganisms is important for a variety of reasons. For one, it is used to determine the cause of diseases. Another important use for identification is being able to identify microbial contaminants in food. This can also be important in research settings when isolating and identifying microbes with antibiotic resistance. To isolate bacteria, scientists use a variety of biochemical tests along with differential and selective media. Selective media will allow certain types of microorganisms to grow whilst inhibiting the growth of other organisms. Differential media were used to differentiate closely related organisms. For this project the fallowing biochemical tests were used to aid in the identification of an unknown bacteria: Phenol red, methyl red and Vogues-Proskauer (MR-VP), catalase test, oxidase test, citrate test, phenylalanine deaminase test, starch hydrolysis test, casein hydrolysis test, urea hydrolysis test, and the SIM medium test. A flow chart was used to map out these tests in a way that would lead to the identification of a bacteria. This method proved highly effective because it made a clear and easy path to fallow while trying to identify the bacteria. Introduction The biochemical tests performed for this project were important in the identification of the unknown bacteria. Preforming these tests, using a flow chart as a guide, it was possible to quickly identify the bacteria. These tests are also important because it allows scientists to understand and characterize the bacteria to then better understand its properties and capabilities. For mixture number nine the first bacteria to be identified was Staphylococcus aureus (S. aureus). This bacterium will have grape like clusters, be cocci, gram positive, round, had convex elevation, had entire margins, and had a golden yellow pigmentation. This bacterium is also a

facultative anaerobe, meaning that it can row in the presence or absence of oxygen. This bacterium is highly opportunistic and can cause skin infections, as well as respiratory and bone infections. It is considered to be the most dangerous of the staphylococcus bacteria. In humans, it commonly colonizes the nasal passages and the axillae. S. aureus can cause many diseases in humans such as pneumonia, osteomyelitis, phlebitis, and meningitis (Mandal, 2017). The treatments involved to alleviate and cure these illnesses are antibiotic regiments designed to kill of the infection. It is also prevalent in livestock, specifically pigs, and is found to be a greater problem in farms that carry more livestock as the bacteria is able to spread more quickly F (Guardabassia). For S. aureus the identification process took three biochemical tests. After observing that this bacterium was gram negative and cocci, the next step was to then do the urea hydrolysis test. This test had a negative result, as the medium was a yellow/orange color, which then led to the casein hydrolysis test. After observing a positive result, as it had a clearing around the bacteria line, the final test was the catalase test. The bubble formation indicated a positive result for the catalase test. This last test left the only possible identification to be S. aureus. The second bacteria identified from the mixture was Enterobacter aerogenes (E. aerogenes). This bacterium is bacilli shaped, has a single cell formation, and was gram negative. The morphology was observed to be single cell, had a shiny off-white color, had entire margins, and a convex elevation. It also has the ability to grow in the presence or absence of oxygen making it a facultative anaerobe. This is an opportunistic bacterium that is a multiresistant pathogen. It causes infections in wounds, the reparatory tract, and the urinary tract. It also causes soft tissue infections, endocarditis, and intra-abdominal infections. It is a very dangerous pathogen as it is resistant to last-line antibiotics such as carbapenems and colistin. It is also the

fifth leading cause of ICU infections in hospitals (Anne and Pages, 2015). Treatment for this pathogen varies by case, however a general antibiotic strategy is to optimize pharmacokinetic and pharmacodynamic properties of antimicrobial regimens which can lead to better clinical outcomes and less antibiotic resistance (Quinn). For E. aerogenes the identification process took two biochemical tests. After observing a bacterium that was gram negative and bacilli the first test to do was the MRVP test. After adding the a alpha naphthol and KOH reagents, the test yielded a positive result as the mixture turned red. This then led to the casein hydrolysis test which had a no clearing around the bacterial streak which then left the only possible bacteria to be E. aerogenes.

Materials and Methods The materials needed for this project are the fallowing: inoculating loop, nutrient agar plates, Bunsen burner, incubator, refrigerator, microscope, glass slides, crystal violate, iodine, safranin, ethanol, methyl red, alpha naphthol, KOH, hydrogen peroxide, citrate tube, casein hydrolysis plate, urea hydrolysis tube, and SIM medium tube. The methods used for beginning portion of this lab were gram staining, sterile inoculation, and quadrant streaks. The gram staining was done by first transferring the bacterial mixture to a glass plate and was then heat fixed. Next, crystal violate was used to cover the sample and was allowed one minute to remain on the sample and then rinsed off with water. Next, iodine was placed over the sample and was allowed to stay there for one minute and then rinsed with ethanol fallowed immediately by water. The final step was to cover the sample with safranin for one minute and then wash off with water. The slide was then dried off and observed under the microscope to identify cell morphology and gram staining results. Sterile inoculation

was very important for this project. If this was not done properly the results of this experiment could have been greatly skewed. To do this, use a Bunsen burner to sterilize the inoculating loop. It is sterile when the metal is glowing. After each transfer the loop should be inoculated as a precaution. The quadrant steak was used to separate the bacterial colonies from one another. After the quadrant streak was performed on the nutrient agar plate, the plate was then stored in the incubator at 37 degrees Celsius for 48 hours. The methods used for the biochemical tests fallow the procedures of the urea hydrolysis, casein hydrolysis, catalase, MRVP, and the glucose tests. For the urea hydrolysis test, a test tube of urea was obtained and inoculated with the bacteria. Then, the test tube was stored in an incubator at 37 degrees Celsius for 48 hours. Upon return, if there was a color change from the yellow/orange color to red/pink, then the test is positive. If it remained the yellow/orange color, then the test was considered negative. For the casein hydrolysis test, a plate of casein was obtained, and a single streak of bacteria was applied to the plate. It was then stored in the incubator at 37 degrees Celsius for 48 hours. Upon return if there was a clearing around the streak then the test was positive. If there was no clearing, then the test was negative. For the catalase test, use a wooden stick to transfer bacteria onto a glass slide. Then drop hydrogen peroxide onto the sample. If there is bubble formation, then the test is positive and if there is no bubble formation then the test is negative. For the MRVP test, a MRVP tube was inoculated with the bacteria and then stored in the incubator at 37 degrees for 48 hours. Upon return, a 1ml sample was placed into two separate tubes. In one tube, three drops of methyl red was added to the mixture. If the mixture turned red then it was positive, if not then it was negative. In the other tube, 15 drops of alpha naphthol and five drops of KOH was added. The results were read for 30 minutes at 10 minute intervals. If during that time the mixture turned red, then the test was

positive. If it stayed the original copper color, then it was negative. For the glucose test, a tube with peptone, a carbohydrate, and a pH indicator (phenol red) was inoculated with the bacteria and placed in an incubator for 48 hours at 37 degrees Celsius.

Results S. aureus was observed to be cocci in shape, have grape like clusters, and held the crystal violate color indicating that it was gram negative. After isolation, the bacteria was streaked onto a nutrient agar plate. After incubation the colonies were observed to be round, had convex elevation, had entire margins, and had a golden yellow pigmentation. The order of tests performed to identify this bacterium started with the urea hydrolysis test, then the with casein hydrolysis test, and finally the catalase test. The urea test was negative as it remained an orange yellow color. This indicates that the bacteria did not convert urea to ammonia to be used a nitrogen source. This led to the casein hydrolysis test which was positive as seen by the clearing around the bacterial streak. This result indicates that this bacterium did not secrete casease to break down casein as a source of amino acids. The final test to preform was the catalase test and it was positive as indicated by the production of bubbles after dropping hydrogen peroxide onto a sample of the bacteria. This indicates that the bacteria produces the catalase enzyme that breaks down hydrogen peroxide. Figure 1: Flow chart for S. aureus identification

Figure 2: Morphology

Figure3: Urea Hydrolysis test: negative

Figure 4: Casein test: negative

Figure 5: Catalase test: positive

E. aerogenes was observed to have a bacilli shape, single cell formation, and was a pinkish red color which indicated that it was gram negative. After streaking it onto a nutrient agar plate and let incubate for 48 hours at 37 degrees Celsius the morphology was observed to be single cell, had a shiny off-white color, had entire margins, and a convex elevation. The order of tests performed to identify this bacterium started with the glucose test, then the MRPV test, and ended with the casein hydrolysis test. For the glucose test, a tube with peptone, a carbohydrate,

and a pH indicator (phenol red) was inoculated with the bacteria and placed in an incubator for 48 hours at 37 degrees Celsius. The result was a yellow color indicating it had acid and gas production. The next test was the MRVP test , a MRVP tube was inoculated with the bacteria and then stored in the incubator at 37 degrees for 48 hours. Upon return, a 1ml sample was placed into two separate tubes. In one tube, three drops of methyl red was added to the mixture. The mixture turned red which indicated it was positive. For the VP tube, 15 drops of alpha naphthol and five drops of KOH was added, and it turned a red color indicating it was positive. The positive MR test indicates that this bacterium preforms mixed acid fermentation. The positive VP test indicates that this bacterium converted its acidic fermentation products to acetoin and 2,3butanediol. This led to the final test which was the casein hydrolysis test. For this, a plate of casein was obtained, and a single streak of bacteria was applied to the plate. It was then stored in the incubator at 37 degrees Celsius for 48 hours. Upon return there was no clearing around the streak which indicated the test was negative. This series of tests led to the bacteria being identified as E. aerogenes. Figure 6: Flow chart for E. aerogenes identification.

Figure 7: Glucose test: A/G (acid gas)

Figure 8: MRVP test: positive

Figure 9: Casein hydrolysis test: negative

Discussion Mixture number nine was composed of two bacteria mixed together. To isolate them and identify them, gram staining, quadrant streaking, and various biochemical tests were used. The first isolated bacteria was gram positive and cocci in shape. This led to the first test being the urea hydrolysis test. The urea test was negative as it remained an orange yellow color. This indicates that the bacteria did not convert urea to ammonia to be used a nitrogen source. This led to the casein hydrolysis test which was positive as seen by the clearing around the bacterial streak. This result indicates that this bacterium did not secrete casease to break down casein as a

source of amino acids. The final test to preform was the catalase test and it was positive as indicated by the production of bubbles after dropping hydrogen peroxide onto a sample of the bacteria. This indicates that the bacteria produced the catalase enzyme that breaks down hydrogen peroxide. Through these tests the bacteria was identified as S. aureus. The second isolated bacteria was gram negative and bacilli in shape. Fallowing the flow chart, this led to the glucose test. Here, a tube with peptone, a carbohydrate, and a pH indicator was inoculated with the bacteria and placed in an incubator for 48 hours at 37 degrees Celsius. The result was a yellow color indicating it had acid and gas production. The next test was the MRVP test , a MRVP tube was inoculated with the bacteria and then stored in the incubator at 37 degrees for 48 hours. Upon return, a 1ml sample was placed into two separate tubes. In one tube, three drops of methyl red were added to the mixture. The mixture turned red which indicated it was positive. For the VP tube, 15 drops of alpha naphthol and five drops of KOH was added, and it turned a red color indicating it was positive. The positive MR test indicates that this bacterium preforms mixed acid fermentation. The positive VP test indicates that this bacterium converted its acidic fermentation products to acetoin and 2,3-butanediol. This led to the final test which was the casein hydrolysis test. For this, a plate of casein was obtained, and a single streak of bacteria was applied to the plate. It was then stored in the incubator at 37 degrees Celsius for 48 hours. Upon return there was no clearing around the streak which indicated the test was negative. This series of tests led to the bacteria being identified as E. aerogenes.

Conclusion The result of this unknown bacterial identification project demonstrated that the first isolate was gram positive S. aureus and the second isolate was gram negative E. aerogenes. References Davin-Regli, Anne, and Jean-Marie Pagès. “Enterobacter Aerogenes and Enterobacter Cloacae; Versatile Bacterial Pathogens Confronting Antibiotic Treatment.” Frontiers in Microbiology, Frontiers Media S.A., 18 May 2015, www.ncbi.nlm.nih.gov/pmc/articles/PMC4435039/. Guardabassia, Luca, et al. Individual Predisposition to Staphylococcus Aureus Colonization in Pigs on the Basis of Quantification, Carriage Dynamics, and Serological Profiles. Danish Council for Independent Research, Technology, and Production Science, aem.asm.org/content/aem/81/4/1251.full.pdf. Quinn, John. “Enterobacter Species.” Enterobacter Species - Infectious Disease and Antimicrobial Agents, Antimicrobe, www.antimicrobe.org/b97.asp....