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Unknown Laboratory Report - Serratia marcescens

Anastasia Dakis April 28th, 2020 Fundamentals of Microbiology Dr. Coombs

Abstract

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Serratia marcescens is a common nosocomial pathogen that was the unknown bacteria in this experiment. Serratia marcescens was identified by 5 common microbial identification methods, including the Indole test, methyl red test, voges-proskauer test, arabinose fermentation - phenol red broth and xylose fermentation - phenol red broth. The results of the 5 tests informs that Serratia marcescen cannot split the amino acid tryptophan into the indole compound, does not go through mixed acids fermentation after being given glucose, produces 2,3-butanediol as a fermentation product from glucose and cannot can ferment arabinose or xylose as a carbon source. Introduction In the field of science, experimentation is the cornerstone of changing hypotheses into theories. In the subject matter of microbiology specifically, the classification and categorization of bacteria relies on many different tests that serve as mini experiments to confirm or deny a specific trait in a bacteria. Some examples of these tests are the Indole test, methyl red test, voges-proskauer test, arabinose fermentation - phenol red broth and xylose fermentation - phenol red broth. In this exercise, there were about 16 different possible bacteria that could have been the unknown. The results of the 18 different tests were researched for each of the 16 bacteria. The results were compared to the given unknown results. Through comparing the results of 18 tests for about 16 different possible bacteria, it was confirmed that Serratia marcescens was the unknown bacteria. Serratia marcescens is a gram negative bacteria that is considered to be a nosocomial infection. The classification of S. marcescens is: domain: bacteria, phylum proteobacteria, class: gammaproteobacteria, order: enterobacterales, family: yersiniaceae and genus: serratia. S. marcescens was discovered in 1823 and is well known for its ability to produce a red pigment.

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Nosocomial infections occur when pathogenic bacteria infects any large wound in the body that compromises the skin barrier, specifically catheter infections, urinary tract infections and wound infections. Similar bacteria and fungi cause about 99,000 deaths annually from hospital acquired infections (Kouchak & Askarian. 2012). S. marcescens is also considered an opportunistic pathogen, meaning that it may not cause harm in a healthy host but if the host is immunocompromised, it will take advantage. Aside from nosocomial infections, Serratia marcescens is also the pathogen that causes Meningitis (Khanna A, Khanna M & Aggarwal 2013). Methods Indole Test: Tryptophan broth was inoculated with Serratia marcescens and incubated at 37℃ for 24-28 hours. 0.5 mL of the Kovac’s reagent was added. After the test tube was agitated, the upper layer of the liquid was observed. Serratia marcescens yielded a negative result therefore the broth was only yellow color (Department for Evaluations, Standards and Training. 2010). Methyl Red Test: 0.1 g of methyl red was dissolved in 300 mL of 95% ethanol. 200 ml of deionized water was added to the mixture. The methyl red solution was stored at 4°C. The methyl red test and Serratia marcescens was incubated at 35°C for 48 hours. MR-VP was prepared at room temperature. One tube of MR-VP broth was inoculated from a fresh pure culture of the test culture. A light inoculum was transferred to the 5 ml MR-VP broth tube. 2.5 ml of culture was transferred into a new sterile culture tube. 5 drops of the methyl red reagent was added. The yellow color of the mixture was observed, confirming a negative result for Serratia marcescens (McDevitt S. 2009). Vogues - Proskaur Test: Barritt’s reagent A (VP-A), 5% a-naphthol in absolute ethanol and

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Barritt’s reagent B, (VP-B) 40% KOH in deionized water were prepared fresh. MR-VP was prepared at room temperature. One tube of MR-VP broth was inoculated from a fresh pure culture of the test culture. A light inoculum was transferred to the 5 ml MR-VP broth tube. 0.6 mL of Barritt’s reagent A and 0.2 mL of Barritt’s reagent B was added to the remaining 2.5 mL of culture grown in MR-VP broth. The tube was agitated for 30-60 seconds. The tube was left stationary for 30 minutes. A positive result was observed for Serratia marcescens, since there was a red top layer of liquid (McDevitt S. 2009). Arabinose and Xylose Fermentation - Phenol Red Broth: The phenol red arabinose broth medium was used. The inoculating loop tool was sterilized with a bunsen burner flame. The caps of the test tubes were taken off, maintaining aseptic technique. The mouths of the test tubes were also sterilized over the flame. The sterile inoculating loop was used to pick up an inoculum from the culture tube of Serratia marcescens. The inoculum was transferred into the sterile medium. The mouths of the test tubes were reflamed and the caps were put back on. The inoculating loop was also reflamed. The inoculated tube was placed into the 35 ℃-37 ℃ incubator for 24 hours. After the results were observed, Serratia marcescens produced a magenta color, confirming a negative test for fermentation (HiMedia Laboratories. 2015).

Results When performing the indole test, Serratia marcescens bacteria gave a negative result. Indole test evaluates if an organism can split the amino acid tryptophan into the indole compound. Tryptophanase is the enzyme present that hydrolyzes

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https://microbiologyinfo.com/ind ole-test-principle-reagentsprocedure-result-interpretationand-limitations/

Figure 1. Serratia tryptophan to produce indole. When Kovac’s reagent is added, if any indole is present the Marcescens Indole Test Result. compound will turn red. In the case of Serratia marcescens, when conducting the indole test, the resulting compound remained yellow. This confirms a negativ

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marcescens cannot split Tryptophan into the indole compound. Thu

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to react with the Kovac’s reagent and produce the red color. After conducting the methyl red test, Serratia marcescens

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methyl red test evaluates if a bacterium went through mixed acids

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glucose. The products a bacteria produces by the anaerobic f

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scientists identify and categorize different bacteria. When testing Se

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result is produced. The culture remained yellow and experienced n that less acid is produced from the fermentation of glucose.

https://microbiologyinfo.co m/wpcontent/uploads/2015/10/R esult-Interpretation-of-MRtest.jpg

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Figure 2. Serratia When performing the Voges Proskauer test, it was Methyl determined Marcescens Red that Test Result. Serratia marcescens yields a positive result. The Voges Proskauer test measures if a specific bacteria produces 2,3-butanediol as a fermentation product from glucose. However, a positive test result doesn't outright mean 2,3-butanediol is present. The reason for this is that 2,3-butanediol is the product of a long fermentation pathway, making it difficult to detect. To get around this obstacle, the Voges Proskauer test instead, tests for the presence of acetoin, which is one of http://image3.slideserve.com/660 1347/voges-proskauer-test-n.jpg

Figure 3. Serratia Marcescens Voges Proskauer Test Result.

the intermediates of the fermentation pathway. In the case of Serratia marcescens, a positive result is observed. This means that the acetoin reacted with the reagents present to create a red layer. Serratia

marcescens

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demonstrates a negative result for both the arabinose fermentatio

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xylose fermentation - phenol red broth test. The purpose of the a determine if the bacteria can ferment arabinose as a carbon sour

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Figure 4. Serratia the pH would drop. Since Serratia marcescens had a negative result, there was no pH drop Marcescens Arabinose and Xylose Fermentation therefore no color change. The same mechanism is seen for the carbohydrate xylose. If xylose Phenol Red Broth Test Result. was fermented, the pH would drop. The negative result shows that there was no pH change therefore xylose is not fermented in Serratia marcescens. Discussion This experiment characterizes how different tests can help distinguish bacteria. There were about 18 different tests and 16 possible bacteria to choose from. Using the positive or negative test results, the unknown bacteria was identified as Serratia marcescens. In this report, the focus was on five tests: Indole test, methyl red test, voges-proskauer test, arabinose fermentation - phenol red broth and xylose fermentation - phenol red broth. Serratia marcescens had a negative result for the indole test, a negative result for the methyl red test, a positive result for the voges-proskauer test, a negative result for the arabinose fermentation - phenol red broth and a negative result for the xylose fermentation - phenol red broth. This series of results gives a lot of vital information about Serratia marcescens. It informs that Serratia marcescens, cannot split the amino acid tryptophan into the indole compound, does not go through mixed acids fermentation after being given glucose, produces 2,3-butanediol as a fermentation product from glucose and cannot can ferment arabinose or xylose as a carbon source. Overall, this emphasizes the importance of different tests in microbiology that aid the classification of bacteria.

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References HiMedia Laboratories. 2015. Phenol Red Arabinose Broth. 1: 1-2. Khanna A, Khanna M and Aggarwal A. 2013. Serratia Marcescens- A Rare Opportunistic Nosocomial Pathogen and Measures to Limit its Spread in Hospitalized Patients. Journal of Clinical and Diagnostic Research 7(2): 243-246. Kouchak, F., & Askarian, M. 2012. Nosocomial infections: the definition criteria. Iranian journal of medical sciences, 37(2), 72–73. McDevitt S. 2009. Methyl Red and Voges-Proskauer Test Protocols. American Society for Microbiology 1: 1-9. Standards Unit, Department for Evaluations, Standards and Training. 2010. Indole Test, National Standard Method. Centre for Infections 2: 1-10....