Unknown Project

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Feb 20, 2024

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Identifying an Unknown Bacterial Organism through Biochemical Testing Project Overview: In this unknown organism identification project, you will learn the process of identifying an unknown bacterial organism through a series of steps and biochemical tests. This project aims to develop critical thinking skills while reinforcing the principles of microbiology. The project is worth 100 points - 10 points per question. Answer the following questions using the lecture textbook and credible online resources. Make sure to cite your sources where needed. 1. You are given a test tube that contains an unknown broth culture. What is the first technique that should be performed to obtain valuable information about the broth culture and why? (Hint: this step doesn’t involve staining.) The technique used when dealing with a mixed culture is known as the t-streak plate technique which is applied to different types of plates allowing for various microbial growth, selective, or differential appearance microorganisms to grow or express through direct dilution onto plate through loop transfer. Through the use of the Tryptic Soy Agar (TSA) plate the mixed broth culture will be separated into sections that will separate the types of bacteria contained within the culture. Then using special types of microorganismal agars like the Eosin Methylene Blue (EMB) agar will allow for the growth of only a selective group of the negative bacteria, while also depicting fermentation productive ability. This will allow for the separation of larger groups of the gram- negative bacteria and depicting the abilities of the certain families or groups apart of the larger gram-negative bacteria group. After allowing these agar mediums to incubate will allow the depiction of these differences. 2 . Now that you have the valuable information from question 1, you perform a differential stain. What differential stain should be done and why? The differential staining that should be performed is a Gram stain procedure, this will allow for the distinguishment of the bacterial cells shape morphology and the type of bacteria and its bacterial cell structure which will either be a gram-positive configured with a simple one-layer cell wall with multiple types of protein connected or a gram-negative possessing a second layer in addition to its one layer which will possess a toxic lipid that is harmful to the human body. 3. After performing the differential stain, you have obtained a microscope image that shows the unknown organism has an outer membrane. Based on this information, you know the unknown organism is Gram-Negative and rod shaped. 4. The unknown organism has been narrowed down to the following organisms: Escherichia coli Proteus vulgaris Enterobacter aerogenes Pseudomonas aeruginosa Klebsiella pneumonia Which of these organisms belong to the Enterobacteriaceae family? The organisms that can be found within the Enterobacteriaceae family include Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, and Klebsiella pneumonia.

Now that you have some preliminary information, it is time to further explore how to determine the unknown bacterium. The image below is a dichotomous taxonomic key, which is also known as a bacterial identification flowchart. The flowchart serves as a systematic and efficient tool for the identification of unknown bacteria. It simplifies the complex task of classifying bacteria by assessing their metabolic characteristics and reactions, ultimately helping microbiologists determine their identity and attributes. The next few questions pertain to biochemical tests that should help you identify the unknown bacterium in this project. 5. The first biochemical test you conduct is a catalase test. Based on the bacteria that you listed as members of the Enterobacteriaceae family, which bacteria are catalase positive (+) and which bacteria are catalase negative (-)?

Catalase Activity Escherichia coli Enterobacter aerogenes Klebsiella pneumoniae Proteus vulgaris 6. The second biochemical test you conduct is an isolation streak on Eosin Methylene Blue agar. From the bacteria that you listed as catalase positive, which bacteria are positive for growth (+/-) and which bacteria are positive for growth and pigment production (+/+)? Eosin Methylene Blue Agar Proteus vulgaris Escherichia coli (metallic Green) Klebsiella pneumoniae Enterobacter aerogenes 7. The third biochemical test you conduct is on motility. What medium did we use earlier in the semester that would be a great biochemical test to use now? The biochemical test conducted to differentiate between the motile and non-motile Enterobacteriaceae family species uses the medium Sulfide Indole Motility (Semi-soft) (SIM) Agar, which allows for the possibility of movement if possessed by the organism. + - +/+ +/-

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8. Based on the organisms that were positive for growth and negative for pigment production, which bacterium is positive for motility (+) and which bacterium is negative for motility (-)? Motility Proteus Vulgaris 9. What flagella arrangement does the motile organism possess? Proteus Vulgaris is an organisms that like most types of organisms of the Enterobacteriaceae family poses the motile ability through the possession of flagella which are arranged in a peritrichous arrangement. 10. The motile organism is your unknown bacterial organism. What is the name of your unknown bacterial organism? The unknown bacterial organism found within the bacterial broth culture is Proteus Vulgaris. + -

Examples of journal article citations. MLA Citation information  MLA format - the author's last name is followed by their first name. The title of the article is written in sentence case and is enclosed in quotation marks. The title of the journal is italicized or underlined, and the volume number is specified, followed by the issue number in parentheses. The publication date is listed, and the page range of the article is indicated with "pp." preceding the page numbers  Author: Johnson, Lisa M.  Title of Article: "The Impact of Technology on Workplace Productivity."  Title of Journal: Journal of Business and Management Volume Number: 45 Issue Number: 2  Publication Date: 2023 Page Numbers: 127-142.  MLA Citation: Johnson, Lisa M. "The Impact of Technology on Workplace Productivity." Journal of Business and Management, vol. 45, no. 2, 2023, pp. 127-14 APA Citation information  APA format - the author's last name is followed by their initials. The publication date is included in parentheses. The title of the article is written in sentence case and is not enclosed in quotation marks. The title of the journal is written in title case and italicized. The volume number is specified in italics, followed by the issue number in parentheses (not italicized). The page range of the article is indicated.  Author: Johnson, L. M.  Publication Date: 2023.  Title of Article: The Impact of Technology on Workplace Productivity.  Title of Journal: Journal of Business and Management. Volume Number: 45(2). Page Range: 127-142.  APA Citation: Johnson, L. M. (2023). The Impact of Technology on Workplace Productivity. Journal of Business and Management , 45 (2), 127-142.

MLA Citations Abram, Dinah, et al. “Basal Structure and Attachment of Flagella in Cells of Proteus Vulgaris.” Journal of Bacteriology, vol. 90, no. 5, American Society for Microbiology, Nov. 1965, pp. 1337–54. https://doi.org/10.1128/jb.90.5.1337-1354.1965 . Ahmed, Asma, et al. “CHARACTERIZATION AND IDENTIFICATION OF TYROSINASE PRODUCING BACTERIA FROM SOIL SAMPLES.” ResearchGate, Mar. 2023, https://doi.org/10.17605/OSF.IO/6ASBR . Bauman, Robert W., et al. Microbiology : With Diseases by Body System. Pearson Education, 2012, pp. 58, 59, 107, 149. Budin, Ghyslain, et al. “A Magnetic Gram Stain for Bacterial Detection.” Angewandte Chemie (International Ed.), International ed. in English, vol. 51, no. 31, 2012, pp. 7752–55, https://doi.org/10.1002/anie.201202982 . Enterobacteriaceae - MeSH - NCBI. www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Term= %22Enterobacteriaceae%22[MeSH%20Terms . Ewing, William H., et al. “Roundtable: How Far to Go with Enterobacteriaceae?” The Journal of Infectious Diseases, vol. 119, no. 2, 1969, pp. 197–213. JSTOR, http://www.jstor.org/stable/30102297. Accessed 25 Oct. 2023. Gram Negative Cocci. medicine.osu.edu/-/media/files/medicine/departments/pharmacy/asp/clinical- microbiology/gramnegorganisms.pdf?la=en&hash=910F30C209BBB7E2E857CF1034E240F65428E922. “---.” Iheartpathology, www.iheartpathology.net/micro-gramnegrods#:~:text=Many%20gram%20neg%20rods %20are%20considered%20enteric%20or,yourself%20with%20the%20overall%20categorization%20of%20these %20bacteria.%20%20https://www.ncbi.nlm.nih.gov/books/NBK8035 . Klaif, S., et al. “The Genetic Relationship for Klebsiella Pneumoniae Isolated from Human Urinary Tract and Beef.” Iraqi Journal of Veterinary Sciences, vol. 33, no. 1, 2019, pp. 75–80, www.iasj.net/iasj/download/67b1ed49c4c748ae. Accessed 27 Oct. 2023. Leininger, Dagny Jayne, et al. “Use of Eosin Methylene Blue Agar to Differentiate Escherichia Coli From Other Gram-Negative Mastitis Pathogens.” Journal of Veterinary Diagnostic Investigation, vol. 13, no. 3, SAGE Publishing, May 2001, pp. 273–75. https://doi.org/10.1177/104063870101300319 . Unknown Identification - Microbiology Resource Center - Truckee Meadows Community College. www.tmcc.edu/microbiology-resource-center/lab-protocols/unknown-identification .

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