MCB 2610 L Lab Report - Grade: 100 PDF

Preview

Summary

This is the cumulative final lab report for the lab section of the MCB2610 course with Dr. Rossi....

Description

MCB 2610 Sec 52L: Isolation and Identification of Bacteria Lab Report Introduction Bacteria are found in almost all natural environments. In the lab they can be found on many surfaces, as well as within the human body. Within the human body bacteria can be beneficial or pathogenic. Pathogenic bacteria may cause many symptoms when they enter the body through the eyes, nose, mouth, urogenital openings, or an open wound, sometimes leading to fatality. For this reason, bacteriostatic agents are used to control bacterial growth and bactericidal agents are used to kill bacteria. The identification of bacteria is important due to their unique structural and internal features that may help determine how they will respond to treatments. Bacteria must be grown in a pure culture to observe their morphology and physiology. In this lab, two species of bacteria were isolated and grown in pure cultures to first observe their morphology and their gram stain reactions, and second how they respond to a series of fermentation and enzymatic tests. An unknown mixed culture was given to each student containing two of the following species bacteria: Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Klebsiella aerogenes, Salmonella enterica, Pseudomonas aeruginosa. To isolate the two species, a T-streak was performed on a Tryptic Soy agar plate using aseptic technique. In a T-streak the liquid culture is progressively diluted between three sections of the agar plate by streaking each new section from the previous section. After incubation, two isolated, morphologically distinct colonies can be observed (The Molecular and Cell Biology Department 23). These distinct colonies are then T-streaked on their own plates to form a pure culture of each species. Once two pure colonies were achieved, a gram stain was performed on each. A gram stain

differentiates bacteria based on their cell wall composition, specifically their peptidoglycan content. Gram negative bacteria produce a pink/red stain due to a thinner peptidoglycan layer in their cell walls, while gram positive bacteria produce a purple/blue stain due to retention of the crystal violet stain after being washed with ethanol by their thick layer of peptidoglycan. After the gram stain and morphology of each isolated species was known, a flowchart of diagnostic fermentation and enzymatic tests was made to determine what tests to perform after each previous tests’ positive or negative result. Fermentation tests are used to determine which sugars the bacteria are able to use for anaerobic respiration. Products of fermentation are often acids, producing a color change in the media, and/or gases, producing bubbles in the media. Enzymatic tests are used to determine what enzymes the unknown bacteria can or cannot produce based on their ability to break down a specific substance. Subsequent tests were performed until the identity of each bacteria was definitive, thus allowing students to combine knowledge gained throughout the microbiology lab course to achieve results in the “Isolating and Identifying of Bacteria” lab.

Figure 1. This flowchart displays my experimental plan, including gram stain reaction results, followed by tests conducted to identify Isolate A and B respectively.

Discussion

The purpose of this lab was to isolate pure cultures of two unknown bacterial species in order to observe their morphology, followed by several tests, including a gram stain, fermentation tests, and enzymatics tests, which lead to a conclusive identification of each species. An experimental plan was made in the form of a flow chart in order to visualize what tests needed to be performed based on the characteristics of each bacterial unknown, determined by the result of each test. With each subsequent test, the possible identity of each bacterial species was narrowed from the original eight species until only one species was possible for that series of results. This organization of tests was important to ensure accuracy of final results in that only one species of the original eight could have the set of morphological and physiological characteristics determined by all tests performed. After isolation of unknown species A and B in pure culture was achieved through Tstreaks, unknown A’s shape was observed to be sphere/coccus, and unknown B’s shape was observed to be rod/bacillus. Next a gram stain was performed on each unknown, resulting in a purple gram positive reaction for isolate A and a pink gram negative reaction for isolate B. This reveals that isolate A has a thick porous layer of peptidoglycan in its cell wall. In contrast, isolate B has a thin layer of peptidoglycan in its cell wall, allowing the ethanol wash to decolorize the crystal violet stain, followed by isolate B’s retention of safranin counterstain to produce the pink color observed. Because isolate A showed a positive gram stain and coccus cell shape, it’s identity was narrowed to Enterococcus faecalis or Staphylococcus aureus. Next, a catalase test was performed to differentiate between these two bacterial species. Catalase is an enzyme that breaks down hydrogen peroxide into water and oxygen byproduct. A catalase test is performed by adding a drop of 3% hydrogen peroxide to a bacteria colony. A positive catalase result indicates that the

organism has the catalase enzyme, and is indicated by the observance of quickly forming oxygen bubbles. Catalase positive organisms are usually aerobes or facultative anaerobes, while lactic acid bacteria usually lack catalase (The Molecular and Cell Biology Department 63-64). Staphylococcus aureus is a facultative aerobe that contains catalase and is not a member of the lactic acid bacteria group, while Enterococcus faecalis is a facultative anaerobe of the lactic acid bacteria group and does not produce catalase. The catalase test of isolate A showed a positive result with quickly forming bubbles, allowing the conclusion that isolate A is Staphylococcus aureus. Isolate B produced a negative gram stain and has a rod shape. Because all gram negative bacteria in this lab are rod shaped, morphology was not a diagnostic factor in determining the identity of isolate B. The gram negative species in the “Isolating and Identifying Bacteria” lab include Salmonella enterica, Escherichia coli, Enterobacter aerogenes (Klebsiella aerogenes), and Pseudomonas aeruginosa. The next test performed on isolate B was an oxidase test. A positive oxidase test indicates the presence of cytochrome c. Bacteria possessing cytochrome c can oxidize the compounds N,N-tetramethyl-p-phenylene-diamine and/or dichlorophenol indophenol, which are both colorless in their reduced forms. When oxidized, these compounds change in color to a dark blue or red, therefore a color change in the oxidase test indicates a positive result for the oxidase enzyme. The only oxidase positive species of the four gram positive species in this lab is Pseudomonas aeruginosa, while the other three species are oxidase negative (The Molecular and Cell Biology Department 64). The oxidase test of isolate B displayed a very faint color change to blue, indicating a negative oxidase result, and eliminating the possibility that isolate B could be Pseudomonas aeruginosa. For the last two tests, an EnteroPluri-Test® was inoculated with isolate B to allow for rapid results of multiple diagnostic

media at one time. To further narrow the possible species contained in isolate B, a lactose fermentation test was used. Fermentation of the sugar lactose by an organism produces lactic acid. Lactic acid can be detected by a change in color from red to yellow of the indicator dye, phenol red, due to a decrease in pH below 7 (The Molecular and Cell Biology Department 69). Within the EnteroPluri tube, a color change of isolate B from red to yellow was observed, indicating a positive lactose fermentation result. Because Salmonella enterica cannot ferment lactose, isolate B could only be one of two remaining species that are able to ferment lactose: Escherichia coli or Enterobacter aerogenes. The final test to determine the identity of isolate B was a citrate test. A citrate test is performed on enteric bacteria to determine if they are able to utilize citrate. When bacteria metabolize citrate, ammonia is produced, increasing the alkalinity. A positive result for citrate utilization is indicated by the color change in bromothymol blue from green to dark blue because of an increase in alkalinity, while a negative result is indicated by no color change from green. Enterobacter aerogenes is citrate positive, while Escherichia coli is citrate negative (The Molecular and Cell Biology Department 80-82). The result of the citrate test in the EnteroPluri tube was negative by the observation of no color change from green. The final result of citrate negative allows the conclusion that isolate B is Escherichia coli. Conclusion The “Differentiation of Microorganisms” lab was essential in identifying the bacteria species in the original mixed culture, as it outlined the correct procedure for a T-streak and each subsequent test conducted, including enzyme and fermentations tests, and use of the EnteroPluriTest®. The information given in the “Differentiation of Microorganisms” lab showed the meaning of results produced in these tests, as well as the differing characteristics of each bacteria species that could be aligned with a particular series of results. It also allowed understanding of

the chemical reactions taking place within the selective and differential media types. Some applications of this lab include food borne pathogens and the efficiency of antibiotic treatments based on what bacteria species is causing an infection. Antibiotics are metabolic products of an organism that can kill, or inhibit the growth of other microorganisms in very small amounts. It is important to know what bacteria species is causing an infection because different antibiotics structural features or biologic pathways specific to certain species (The Molecular and Cell Biology Department 137). Both identified isolate A, Staphylococcus aureus, and isolate B, Escherichia coli, cause foodborne illnesses when ingested by a human host. Staphylococcus aureus has enterotoxins that can cause nausea, vomiting, abdominal cramps and diarrhea, along with more serious conditions such as pneumonia, meningitis, and toxic shock syndrome. Escherichia coli can cause all of the same symptoms, as well as fever and the development of Hemolytic Uremic Syndrome and has led to numerous deaths worldwide due to it’s long survival periods and low infectious doses (Bintsis 2017).

Works Cited

Bintsis T. (2017). Foodborne pathogens. AIMS microbiology, 3(3), 529–563. https://doi.org/10.3934/microbiol.2017.3.529 The Molecular and Cell Biology Department, University Of Connecticut. MCB 2610 Laboratory Manual: Fundamental of Microbiology - University of Connecticut at Storrs - Spring 2021. Macmillan Higher Education, 2020. [Yuzu]....