Lab report for unknown bacteria (microbiology)

Anonymous
account_balance_wallet $40

Question Description

I want a lab report (not essay just lab report ) for identification of unknown bacteria though biochemical test

I have some pics to include in the report. I’ve attached a photos from the manual for what you should include in the report (worth 40 points).

My unknown is Salmonella. Type III ( my TA said it is not right, but I can’t change because it’s based on my result. there are two other choices for Salmonella ( Typi cai, Typhi) when I asked my TA if it was Typi cai she said ‘’ close’’ so I’m not sure. However, I have 13 maching test for Type III and 12 for Typi cai while only 9 for Typhi. Focus on the unknown report grading rubric for the writing direction. Turnitin must be less than %20 if more will count as ZERO. There is no limit for length (just include all parts in the grading rubric ) so feel free to make it short and informative as it is qualitative report. Don’t forget the flow chart as well (I’ve attached example *from the internet).

I’ve included an example report (from the internet ) for same class same school, just to give you an idea, however, it is too long so I want it for sure shorter.


Feel free to contact me for any question.

Unformatted Attachment Preview

Grading Rubric I had Unknown# 22 I did the following tests: YOU DON’T HAVE TO WRITE ABOUT ALL OF THEM ONLY THE ONES IN RED ARE MUST TO INCLUDE IN THE REPORD THEN CHOCE ANY OTHER 2-3 TESTS Test Methyl Red (MR) Indol VP Simmon’s Citrate KIA , H2S This test I did it twice as the first time I got a Negative result because I did it wrong while second time I got positive when I did it right. *my TA wanted me to redo it. Motility Urea Hydrleses Gas from D-Glugose Lysin Malonate Utilization Moller’s Ornithine BCP Adonitol BCP Arabinose Phenylalanin deaminase (PDA) Result + + + + + + + + - Include the wrong (-) KIA TEST ON THE LEFT, then motality, citrate, D-GLUGOSE, indole, MR -------------------------------- From the left: the green one I didn’t use it, KIA (-), D-GLUGOSE, CITRATE, MOTALITY, INDOLE ============================================ LYSINE SAME LYSINE ============================= BCP ARABINOSE =================== PHENYLALANINE DEAMINASE =================================== Identification of Enterobacteriaceae 1 Identification of Enterobacteriaceae Sarah Yoshioka San Diego State University Microbiology 350 Lab Sec: 3 November 9, 2017 Identification of Enterobacteriaceae 2 Introduction The main goal of this experiment is just as the title implies, being able to isolate, identify, and characterize bacteria in the Enterobacteriaceae family (Maloy et al., 2017). Enterobacteriaceae are Gram-negative rods that also have the ability to ferment sugars. Some of these can be pathogenic, so being able to identify which strain of bacteria it is can be very important in many industries. As the lab manual states, these Enterobacteriaceae that are being tested are heterotrophs that grow in a 35℃ environment. Enterobacteriaceae is commonly known to be a member of the intestinal flora in humans and animals and can be isolated (Guentzel, 1996). To narrow down and identify the sample unknown #6, many physiological tests were performed over the course of many lab periods. A colony of bacteria was isolated and cultured and then a Gram stain was performed to make sure that it was indeed Gram-negative and a pure culture. After this, the bacteria was put through a series of tests that included testing whether it could ferment glucose and other sugars, produce the enzyme tryptophanase, decarboxylate (catalyze the break down) certain amino acids, use arginine dihydrolase, remove amino acid groups from phenylalanine, catabolize citrate, deaminate cysteine, utilize malonate, hydrolyze urea, and have motility (Maloy et al., 2017). All of these tests were performed to narrow the bacteria down to only one strain of Enterobacteriaceae, which was Citrobacter farmeri. This bacterium followed all of the guidelines within the lab manual so by using all of the tests listed above; this is the final identification for unknown #6. Identification of Enterobacteriaceae 3 Materials and Methods Isolation and Initial Characterization of Unknown In order to obtain a pure culture of the unknown bacteria, a Trypticase-Soy agar (TSA) plate was streaked and left to incubate at 37℃ for 24-48 hours. After putting the plate in the incubator, a Gram stain was performed to confirm that it was a Gramnegative bacteria. After the 24-48 hours, a bacteria colony was divided into two halves and each half was streaked onto two different TSA slants. These slants were then incubated at 37℃ for 24-48 hrs. Physiological Tests for the Identification of Unknown Bacteria FERMENTATION TESTS Two fermentation tests were performed to see whether the bacteria could ferment glucose. These tests included the Methyl Red-Voges-Proskauer (MR-VP) test and the Dglucose test. For the MR-VP test, a tube containing the MR-VP medium was inoculated with the grown pure cultures from the TSA slants. This tube was then placed in the incubator at 35℃ for 24-48 hrs. After this time period passed, the MR test was performed when the culture was placed into a separate tube and a few drops of methyl red was added in. The VP test was performed next when the culture was placed into a separate test tube. Barritt’s reagent A (𝛼-napthol) and Barritt’s reagent B (KOH) were added to each tube and vortexed. They were then left for 1-2 hrs. The gas production from D-glucose test was performed when a tube of Bromcresol purple (BCP) broth with D-glucose and a Durham tube was inoculated with Identification of Enterobacteriaceae 4 the pure colony. This tube was then incubated at 35℃ for 24-28 hrs and examined for gas within the Durham tube. AMINO ACID CATABOLISM TESTS Four amino acid catabolism tests were performed to see whether the bacteria could catalyze certain amino acids. These tests included the Indole test, the Lysine & Ornithine decarboxylase tests, the Arginine dihydrolase test, and the Phenylalanine test. The Indole test was performed when a tube of Tryptone was inoculated with the pure colony and incubated at 35℃ for 24-48 hrs. After this time period, some of the culture was removed into a separate test tube and Kovacs’ reagent was added in to produce a color change to see whether Indole was present or not within the sample. The Lysine and Ornithine tests were both performed the same way except one tube contained Moeller’s decarboxylase broth with lysine and another contained Moeller’s decarboxylase broth with ornithine. A control was also made with just pure Moeller’s decarboxylase broth. Each tube was inoculated with the pure colony and mineral oil was placed on top of the sample to keep oxygen out. These tubes were incubated at 35℃for less than 24 hrs. Each tube was then examined to see the results of each test compared to the control. The Arginine dihydrolase test was performed when a tube of Moeller’s decarboxylase broth with arginine and a control was inoculated with the pure colony. Both of these tubes had mineral oil added to the top to not allow oxygen into the sample and were incubated at 35℃ for up to four days and then were examined for results. Lastly, the Phenylalanine deaminase test was performed when a slant of phenylalanine agar was inoculated with the pure colony and incubated at 35℃ for 24 hrs. Identification of Enterobacteriaceae 5 Whenever growth appeared, ferric chloride was added to cover the slant surface so it could either produce a color change or not indicating the result for this experiment. HYDROLYSIS OF MACROMOLECULES TESTS Four tests were performed to see whether the bacteria could catabolize citrate, deaminate cysteine, utilize malonate, and hydrolyze urea. These test included the Citrate utilization test, the Kligler’s iron agar (KIA) test, the Malonate utilization test and the urea Hydrolysis test. The Citrate test was performed when the pure bacteria colony was streaked onto a Simmon’s citrate slant. This was then incubated at 35℃ for 24-48 hrs. with a loose cap so that the oxygen could get in and then looked at later to interpret the results. The KIA test was performed when a KIA slant was inoculated with the pure colony. Two other KIA slants were inoculated with Proteus vulgaris (+ control) and Escherichia coli (- control). These slants were stabbed and then streaked with the same colony and incubated at 35℃ for 24 hrs. Directly after 24 hrs., the test was interpreted for the results. The Malonate test was performed when a medium containing high malonate, low glucose and bromothymol blue was inoculated with the pure colony. This medium was then incubated at 35℃for 4 days then looked at for the results after that time had passed. The urea hydrolysis tests was performed when a slant of urea agar was streaked with the pure unknown colony and incubated at 35℃ for 24-48 hrs. This was then taken out and examined for the results. Identification of Enterobacteriaceae 6 CELL BIOLOGY TEST One test was performed for this and that was the motility test to see whether the bacteria can produce flagella and move through the medium or not. After a tube of motility agar was received, a needle was touched to the pure colony and then was stabbed into the agar almost to the bottom. This then was incubated at 35℃for 24-48 hrs. It was then examined for motility after this time period. Results Before the first sets of physiological tests were performed, a Gram stain was performed. The Gram stain was Gram-negative and the original color of the unknown #6 liquid was yellowish. The first set of tests that were conducted in the lab were the MR-VP, Indole, gas production from D-glucose, Citrate, Motility, and KIA tests. Table 1 shows all these first set of tests including the rest of the tests for the experiment. The MR test was performed and after adding 4 drops of methyl red, the solution turned a red color indicating a positive test. The VP test was performed and after both reagents were added, the tube still remained black/brown so this was a negative result. The Indole test was performed next and after the Kovacs’ reagent was added, the tube turned a bright pink color, which indicated a positive test. The gas production from D-glucose test was performed after and a gas bubble was found inside of the Durham tube along with a turbid broth culture, which means a positive test. The Citrate test was negative because the tube stayed green and did not turn blue. The Motility test was next and this tested positive because the bacteria had moved through the motility agar turning it pinkish throughout the whole Identification of Enterobacteriaceae 7 tube. The last test included with these ones was the KIA test, which tested negative because no black ring was formed in the tube and it just turned a reddish brown color. The second set of tests that were performed included the Malonate and Urea tests. The Malonate test was negative because the tube stayed a green color and exhibited no color change. The Urea test was performed and the agar still remained its original yellowish color, which means that the test was negative. The third and final set of tests that were performed were the Lysine, Ornithine, Arginine, and Phenylalanine tests. The Lysine test was performed and the broth still stayed the same purple color so this was a negative test. Next the Ornithine test was performed and the broth turned a yellow purple color so this was a positive test. The broth of the Arginine test also turned a yellow purple color indicating a positive test. Finally, the Phenylalanine test was performed and growth was observed, but after ferric chloride was added, the agar stayed yellow and did not exhibit a color change, so this was a negative test. Identification of Enterobacteriaceae 8 Tables and Figures Table 1: Physiological Test Results for Unknown Bacteria #6 Test Medium Incubation time 1-2 days Incubati Observation on temp. Turned 35℃ red/orange Interpretation MR MR-VP broth VP MR-VP broth 1-2 days plus 5 days to retest 35℃ Indole Tryptone broth 1-2 days 35℃ Dglucose BCP broth w/Dglucose and Durham tube Simmon’ s citrate slant Motility agar 1-2 days 35℃ 1-2 days 35℃ Stayed green Bacteria couldn’t catabolize citrate Negative 1-2 days 35℃ Pink color spread throughout agar Turned reddish brown color but no black ring Stayed green color Bacteria could produce flagella and move through medium Bacteria could not produce H2S. Positive Bacteria could not degrade malonate. Negative Bacteria could not hydrolyze urea Bacteria could not Negative Citrate Motilit y Bacteria can ferment glucose to acidic end products. Stayed Bacteria can’t brownish/black ferment glucose to non-acidic end products. Turn bright Bacteria could pink produce tryptophanase to degrade tryptophan Has air bubble Bacteria could in Durham ferment glucose and tube produce gas Positive/Nega tive Positive KIA KIA slant 1 day exactly 35℃ Malona te 35℃ Urea Medium 4 days with high malonate, low glucose and bromothy mol blue Urea agar 1-2 days 35℃ Stayed yellow Lysine Moeller’s 1 day 35℃ Purple color Negative Positive Positive Negative Negative Identification of Enterobacteriaceae decarbox ylase broth with lysine Ornithi Moeller’s 1 day ne decarbox ylase broth with ornithine Arginin Moeller’s 1 day e decarbox ylase broth with arginine Phenyla Phenylala 1 day lanine nine agar 9 produce lysine decarboxylase 35℃ Yellow purple color Bacteria did produce ornithine decarboxylase Positive 35℃ Yellow purple color Bacteria could use arginine dihydrolase Positive 35℃ Growth occurred but did not color change Bacteria could not remove the amino group from phenylalanine Negative Table 2: Escherichia coli Tests vs Citrobacter farmeri Tests Test Escherichia coli Citrobacter farmeri Indole + + Methyl Red + + Voges-Proskauer - - Simmon’s Citrate - V KIA - - Urea - D Motility V + Lysine Decarboxylase V - Arginine Decarboxylase V + Identification of Enterobacteriaceae 10 Ornithine Decarboxylase V + Phenylalanine Deaminase - - Malonate Utilization - V Gas from D-Glucose + + Lactose + V Sucrose V + D-Mannitol + + Dulcitol V - Salicin V V Adonitol - - D-Sorbitol V + L=Arabinose + + Raffinose V + L-Rphamnose V + Gelatin (no longer performed in lab) - - Identification of Enterobacteriaceae 11 Figure 1: Flow Chart of Tests Performed. This figure describes the tests that were performed on the unknown #6 and shows which bacteria are eliminated and which are left. This flowchart was made in Google Docs (by Sarah Yoshioka) and was then copied into this document. Identification of Enterobacteriaceae 12 Discussion For the first Gram stain that was performed, this resulted in being Gram-negative which means that the bacteria was correctly isolated and also was within the Enterobacteriaceae family because Enterobacteriaceae are Gram-negative, short rods (Tankeshwar, 2013). Since the MR test turned out to be positive shown in Table 1, this means that the bacteria was able to ferment glucose into acidic endproducts with a pH less than 4.4. This eliminated the bacteria Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter aerogenes. The eliminated bacteria for each test can also be viewed in the flowchart Figure 1. The VP test turned out negative, which means that the bacteria is not able to ferment glucose into (non-acidic) endproducts that are ethanol or 2,3-butanediol. Because this bacteria cannot ferment glucose into non-acidic endproducts, this then eliminates the bacteria Klebsiella oxytoca, Enterobacter sakazakii (3), Enterobacter gergoviae, Serratia marcescens, Serratia rubidaea, Yersinia enterocolitiea, Pantoea agglomerans, Pantoea disperse, Rahnella aquatilis, and Ewingella americana. The Indole test was a positive test, which means that the bacteria is able to produce tryptophanase that can degrade tryptophan into “yielding indole, pyruvate, and NH3” (Maloy et al., 2017). This positive test then ruled out the bacteria Shigella sonnei, Salmonella Typical, Salmonella typhi, Salmonella Type III, Citrobacter freundii, Citrobacter youngae, Citrobacter werkmanii, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Hafnia alvei, Serratia liquifaciens, Proteus mirabilis, Yersinia Pseudotu-berculosis, and Yersinia Pestis. Identification of Enterobacteriaceae 13 The gas production from D-glucose test was positive, which means that the bacteria are able to ferment glucose and produce the gases H2+CO2 or just CO2 by itself. This positive test eliminates the bacteria Shigella other and Providencia stuartii. The Citrate test was negative so this means that the bacteria was not able to catabolize citrate and could not grow on the Simmon’s citrate medium because they were not able to obtain an energy source. This negative test rules out the bacteria Citrobacter braakii and Providencia rettgeri. The Motility test was positive which means that the bacteria can produce flagella, which allows the bacteria move through the medium and spread out. This positive test then eliminates the bacteria that have already been eliminated shown in Figure 1. The KIA test was negative, which meant that the bacteria could not deaminate cysteine and produce H2S, which would then react with iron to produce a black ring. At first, I thought this test was positive because the agar had changed color, but according to the Bergey’s Manual of Systematic Bacteriology, the agar can change color but a positive test must show a black ring within it (Krieg, 2010). This negative test eliminates the bacteria Edwardsiella tarda, and Proteus vulgaris. The Malonate experiment tested negative, which means that the bacteria was not able to degrade malonate so then the high concentration of malonate inhibited the catabolism of the bacteria’s TCA cycle so then the glucose was fermented into acidic endproducts. This negative test ruled out the bacteria Citrobacter sedlakii. The Urea test also tested negative meaning that the bacteria couldn’t produce the enzyme urease, which can hydrolyze urea into ammonia and CO2. This also possibly Identification of Enterobacteriaceae 14 means that this bacteria does not contain a high virulence factor. This test eliminates the bacteria Morganella morganii. The Lysine test was negative, which shows that the bacteria were not able to produce lysine decarboxylase. This test then eliminates the bacteria that has been already ruled out within Figure 1. The Ornithine test was positive, which meant that the bacteria could produce ornithine decarboxylase causing an alkaline reaction within the tube. This eliminates the bacteria Providencia alcalifaciens leaving only two bacteria, Escherichia coli and Citrobacter farmeri. The Arginine test was conducted and turned out to be positive also, which means that the bacteria could use arginine dihydrolase in catabolizing arginine to end products. This eliminated bacteria that had already been eliminated. Finally, the Phenylalanine test was performed and turned out to be negative which means that the bacteria could not remove the specific amino acid group off of phenylalanine in order to deaminate phenylalanine. After all of the tests were completed the two bacteria that were left to choose from were Escherichia coli and Citrobacter farmeri. There was only problem that was encountered and it was that these two bacteria had no remaining tests to differentiate which one it would be. All of the BCP sugar tests either turned out as the same result (+/+ or -/-) for both bacteria or one would be variable (+/V; -/V, V/V, etc.), which is not a reliable source. Table 2 clearly shows this problem with the tests and results. Later, a slide with the unknown bacteria was made and looked at under the microscope. It was then compared to pictures online of both the bacteria. It was very similar to both Identification of Enterobacteriaceae 15 Escherichia coli and Citrobacter farmeri. In the end, the unknown rods were shorter and more clumped together which resembled Citrobacter farmeri, which is one reason that this bacteria was chosen to be the final unknown. According to Dr. Wang and Dr. Chang, citrobacter is a Gram-negative bacilli that is for the most part motile (Wang & Chang, 2017). Furthermore, another reason why the unknown is Citrobacter farmeri, is because that type of bacteria had less variables in the test results as compared the Escherichia coli, so this most likely means that it is a more accurate strain of bacteria for the unknown #6. Identification of Enterobacteriaceae 16 References Guentzel, M.N. (1996). Ch. 26 Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter, and Proteus, 4th edition. https://www.ncbi.nlm.nih.gov/books /NBK8035 Krieg, N.R. (2010). Bergey’s Manual of Systematic Bacteriology. Springer. Maloy, et al. (2017). Biology 350: General Microbiology Laboratory Manual. 137159. Tankeshwar, A. (2013). Enterobacteriaceae F ...

This question has not been answered.

Create a free account to get help with this and any other question!

Brown University





1271 Tutors

California Institute of Technology




2131 Tutors

Carnegie Mellon University




982 Tutors

Columbia University





1256 Tutors

Dartmouth University





2113 Tutors

Emory University





2279 Tutors

Harvard University





599 Tutors

Massachusetts Institute of Technology



2319 Tutors

New York University





1645 Tutors

Notre Dam University





1911 Tutors

Oklahoma University





2122 Tutors

Pennsylvania State University





932 Tutors

Princeton University





1211 Tutors

Stanford University





983 Tutors

University of California





1282 Tutors

Oxford University





123 Tutors

Yale University





2325 Tutors