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Unknown Lab Report Biol 251...

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Biol 251 Bacterial Unknown Lab Report Unknown #26

2 Section I: Introduction In this microbiology course, we study microscopic organisms such as bacteria, viruses, fungi, protozoa and archaea. While learning about each of these, it is not only important to recognize specific characteristics of each microorganism but it is important to perfect the proper procedures and methods needed to identify a species. In this course, each student was given a pure culture of an unknown bacterium and was told to identify their specific bacterium by running a variety of tests. Students obtained the unknown bacterium's morphological, physiological, and biochemical characteristics by utilizing the results from each test. In order to ensure consistent and accurate results of the pure bacterium, the aseptic technique is utilized for each test. The aseptic technique is a method used to isolate pure cultures, control bacterial growth, and prevent contamination (4). To successfully ensure a sterile culture transfer, the inoculating loop is sterilized between each step as this reduces the risk of contamination and yields accurate results (4). Notably, the unknown culture needs to receive adequate nutrients from a medium in order for it to grow and survive, this is where selective and differential media come into play. Selective media is used to promote the growth of a select few types of bacteria, while differential media is used to differentiate between closely related organisms, further assisting in the identification of an unknown culture (4). In order to observe the organism, microscopy is used for the examination of minute objects that are not visible to the naked eye via a microscope. Microscopic examination is important in distinguishing the unknown bacteria's morphology and typically helps to eliminate possible genera based on the characteristics observed. Microscopic examination requires adequate microscopy in order to classify and understand a species based on its gram stain reaction, cell shape, arrangement, whether it is endospore forming, and so on. In the typical microbiology laboratory setting, the bright field

3 microscope is the most commonly used. Bright field microscopy makes the specimen appear darker than its surroundings by beaming a light from underneath (4). Moreover, positive and negative controls are used for each test and are vital as they serve as a baseline comparison for the unknown test results. A negative control indicates what the result should look like if the bacteria is not capable of performing a reaction and a positive control indicates what the bacteria should appear as if it is able to perform a reaction. These controls are a crucial part of identifying possible errors made while performing the tests on the unknown and ultimately suggest how to interpret the results. This paper will discuss the process of identifying unknown 26 using the methods, techniques, and materials mentioned above. Section II: Materials and Methods The Gram stain is one of the first vital differential tests students needed to perform in order to identify the unknown bacterium. The gram stain reaction was used to observe the cells shape and arrangement and to indicate whether unknown #26 was a gram negative or a gram positive microorganism. The Gram reaction is identified by performing four simple steps which include applying a primary stain using crystal violet to a heat-fixed smear, followed by covering the smears with the mordant iodine, followed by 95% ethanol to decolorize the slide, and lastly, counterstaining with Safranin (4). The organisms were then observed under 1000x magnification (4). Gram positive organisms hold the primary stain of crystal violet due to their thick peptidoglycan cell wall, while Gram negative organisms will be decolorized and take up the Safranin counterstain due to its thin peptidoglycan cell wall (4). Therefore, the positive control, Micrococcus, stained purple while the negative control, Neisseria, stained red. Performing the Gram Stain on unknown #26 yielded the result of a Gram positive, cocci generally found in clusters, tetrads, pairs and even singles. Notably, understanding the gram stain reaction of the

4 unknown allows students to depict which tests need to be performed next to further identify the bacterium. Endospore Stain The Endospore stain is a differential stain that takes into account the differences in cell composition in bacteria. Therefore, the bacteria may be divided into two groups: endospore formers and non-endospore formers. In order to perform this procedure, the Nutrient Sporulation Medium was used and a primary stain of malachite green was driven into the cells using steam, followed by decolorization using water, and a counterstain of safranin (4). Once this was completed, the endospore stained green and the vegetative cells and sporangium stained red, as depicted in the positive control, B. subtilis. The negative control, E. coli, did not show any green endospores. Bloor Agar Test The Blood Agar Test was used to determine whether or not the bacteria produces hemolysins and specifies which hemolysins if so. This procedure required a blood agar plate which contained 5% sheep blood (4). Once the sterile blood agar plate was obtained, the bacteria was inoculated and streaked on the agar utilizing the streak plate method of isolation to create a dilution gradient. The plate was then incubated for 48 hours at 37 degrees celsius (4). The positive controls were teeth cultures and S. aureus, which produced both alpha and beta hemolysins. The negative control was S. epidermidis and did not produce hemolysins. Coagulase Test The Coagulase Tube Test was used to determine whether or not the unknown bacterium produced coagulase. Coagulase is an enzyme produced by some bacteria that interferes with the phagocytosis process of the host, potentially increasing its virulence (4). In order to test for this

5 enzyme, a rabbit or sheep blood plasma medium was used. Upon obtaining the plasma tube, an inoculating loop was first sterilized using the microincinerator, then used to remove a loopful of bacteria from the unknown slant and the bacteria was inoculated into the plasma tube and capped tightly (4). In order for this test to work, a heavy inoculum was necessary. Following inoculation, the tube was then incubated for 24 hours at 35-37 degrees celsius (4). The positive control, S. aureus, formed a gel-like or firm clot indicating the bacterium produced coagulase and the negative control, S. epidermidis, resulted in a runny tube indicating the bacterium did not produce coagulase. Aerotolerance Test The Aerotolerance test was used to determine the bacterium's aerotolerance capacity. Thus, the organism's ability to live in the presence of molecular oxygen (4). This test was performed using a Thioglycollate agar deep which allows for the growth of both aerobes and anaerobes (4). Using the aseptic technique, the thioglycollate agar deep was inoculated with the unknown bacterium. The agar deep was then stabbed in the center of the agar deep using an inoculation needle. The needle was removed without disturbing the surrounding agar to ensure the medium did not crack. The tubes were recapped and incubated for 48 hours at 35 ± 2 degrees celsius (4). Once the incubation was completed, the growth patterns were observed. Growth at the surface or top of the agar indicated an obligate aerobe (Micrococcus luteus), growth toward the bottom indicated an obligate anaerobe (Clostridium sporogenes) and growth throughout the entire stab indicated a facultative anaerobe (Escherichia coli) (4). Bacitracin Susceptibility Test The Bacitracin Susceptibility Test was used to differentiate between Micrococcus and Staphylococcus which are morphologically similar (4). In order to get the desired results, a

6 bacterial lawn was made using the aseptic technique, and a single disk of BAC 0.04 was placed in the middle of the lawn. A bacteria is considered susceptible to bacitracin if it creates a zone clearance of 10 mm or greater and resistant bacteria create a zone clearance that is less than 10 mm (4). The unknown bacterium’s results were compared to the positive or resistant control, Staphylococcus aureus, and the negative or susceptible control, Micrococcus luteus. 16S rRNA Sequence: Lastly, students were given the full length (1,500nt) 16S rRNA sequence to their unknown and ran it through the U.S. National Library of Medicine National Center for Biotechnology Information database to identify the specific species. Using the Basic Alignment Search Tool (BLAST), students were able to compare the query sequence to sequence databases and identify their unknown. More specifically, the BLASTn was used to search the nucleotide database using the given nucleotide query (1). The program calculates the statistical significance of matches and provides an Expected value (E) which indicates the number of hits one can anticipate to see by chance (1). The E-value significantly decreases as the match score increases. Therefore, an E-value that is significantly less than one can be classified as biologically significant (1). Section III: Results and Discussion Performing the Gram Stain on unknown #26 yielded the result of a Gram positive, cocci generally found in clusters, tetrads, pairs and even singles. Following this test, all Gram negative and rod-shaped bacteria were eliminated from the possibilities in the identification process. Following the gram stain, the catalase test was performed in order to indicate whether or not unknown #26 produced catalase. The results showed that the bacterium was catalase positive indicating that the unknown could be from the Micrococcus or Staphylococcus genera,

7 eliminating Enterococcus. The positive control was Micrococcus and Staphylococcus, and the negative control was Lactococcus. Next, the oxidase test was performed and used to identify bacteria that produce cytochrome c oxidase generally found in the bacterial electron transport chain. The oxiswab from unknown #26 resulted in no color change after undergoing the oxidase test, indicating that the bacterium was oxidase negative. Based on these results, Micrococcus was eliminated from the possible genera because it is oxidase positive. The positive control was Pseudomonas fluorescens and the negative control was Escherichia coli. The Mannitol Salt Agar (MSA) is a selective and differential medium used to detect whether or not unknown bacterium #26 could grow and/or undergo mannitol fermentation. Unknown #26 turned the MSA bright yellow surrounding the colonies, indicating that the bacteria can grow on the medium and ferment mannitol (4). Staphylococcus epidermidis was eliminated due to its inability to ferment the mannitol. Anthrobacter was also eliminated due to its inability to grow on MSA. The positive controls used for this test were Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Enterobacter aerogenes. The negative control was Pseudomonas fluorescens. Unknown #26 showed no clotting and resulted in a watery tube, indicating that it was coagulase negative. Receiving a negative result on the coagulase test following the MSA results indicated that unknown #26 was a Coagulase Negative Staphylococcus (CoNS). To concur, only certain Staphylococcus can ferment the mannitol, however, only CoNS do not produce a positive coagulase test. Given these results, the positive control, S. aureus, could be eliminated. The negative control for this test was S. epidermidis. Additionally, the results from the endospore stain indicated that unknown #26 was a non-endospore former. The positive control for this test was Bacillus subtilis and the negative control was Escherichia coli.

8 Upon undergoing the aerotolerance test, unknown #26 showed abundant growth towards the top and surface of the tube and growth toward the bottom of the deep. Therefore, the bacterium could be identified as a facultative anaerobe. However, these results may have shown inconsistencies due to cracks in the agar. This inconsistency may have resulted from not using a straight needle or not removing the needle without disturbing the surrounding agar. Stabbing the agar must be performed accurately or oxygen could be allowed to diffuse into the medium resulting in false results. The positive control was Escherichia coli (facultative anaerobe) and the negative control was Clostridium sporogenes (obligate anaerobe). All the results from the preliminary tests indicated that unknown bacterium 26 was Staphylococcus saprophyticus. However, the blood agar hemolysis test result reported complete lysis of red blood cells with transparent areas on the agar, insinuating that the organism produced beta hemolysis. S. saprophyticus is a non-hemolytic organism. This inconsistency may have occurred due to over incubating the blood agar plate or a contaminated inoculating loop. Prolonged incubation time could lead to clearing in the agar. A contaminated inoculating loop could transfer other specimens onto the blood agar plate and result in false positive results. The positive control for this test was Staphylococcus aureus and teeth cultures, the negative control was Staphylococcus epidermidis. Additionally, the bacitracin test was performed on unknown 26 and resulted in a zone of inhibition of 13 mm, insinuating that the bacterium was susceptible. However, Staphylococcus are resistant to bacitracin and Micrococcus are susceptible to bacitracin. Unknown 26 falls under the Staphylococcus genera and should be resistant to bacitracin. This inconsistency may have occurred due to working with an old culture, inaccurate measurements or prolonged incubation periods.

9 The top three matches for unknown bacteria #26 following the 16S rRNA BLAST were Staphylococcus saprophyticus strain UTI-045 (Max Ident: 100%, E-value: 2e^-124), Staphylococcus saprophyticus strain UTI-050 (Max Ident: 100%, E-value: 2e^-124), and Staphylococcus saprophyticus strain UTI-058y (Max Ident: 100%, E-value: 2e^-124). These results indicated that each match was biologically significant because the E-value was significantly lower than one and each identity match was 100%. The low E-value suggests that there is a low probability of the sequence match occurring in the database solely by chance.

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11 Conclusion: Unknown bacterium #26 was identified as Staphylococcus saprophyticus. S. saprophyticus is an opportunistic pathogen that is a common cause of human urinary tract infections (UTIs) (2). An infection that typically occurs in young sexually active females (2). S. saprophyticus is less commonly associated with complications including urethritis, prostatitis, epididymitis and acute pyelonephritis (3). As part of the normal human flora, S. saprophyticus colonizes the cervix, rectum, perineum, gastrointestinal tract, and urethra (3). S. saprophyticus’ host range varies from humans to lower mammals, birds and primates and are typically found in small, transient populations on the skin of these organisms (2). In fact, S. saprophyticus may be transferred to humans by consuming pig and cow meat because it is a common gastrointestinal flora in these animals (3). According to a study conducted by the Centers for Disease Control and Prevention (CDC), 35% of slaughterhouse samples were contaminated with S. saprophyticus (3). Thus, indicating that the meat industry poses a threat to the spread of this infection. Beyond causing human infections, this pathogen is important due to its ability to persevere in the environment, obtain and transmit plasmids that can bestow antibiotic resistance (5). S. saprophyticus have phenotypic characteristics and protein machinery that can cause the pathogen to invade and persist in the human host (5). Studying S. saprophyticus’ virulence factors can reveal the significant protein targets for future drug development (5).

12 References 1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2 2. Brenner DJ, Krieg NR, Staley JT, Garrity GM. 2005. Manual of Systematic Bacteriology, 2nd ed., Volume 3 The Firmicutes, Springer, Berlin, Germany. 3. Lawal OU, Fraqueza MJ, Bouchami O, Worning P, Bartels MD, Gonçalves ML, Paixão P, Gonçalves E, Toscano C, Empel J, Urbaś M, Domínguez MA, Westh H, de Lencastre H, Miragaia M. 2021. Foodborne origin and local and global spread of staphylococcus saprophyticus causing human urinary tract infections. Emerging Infectious Diseases 27. https://wwwnc.cdc.gov/eid/article/27/3/20-0852_article 4. Shyama Malwane & Sharon D. Malwane. 2018. A Laboratory Manual, Microbiology, Morton Publishing Company, Englewood, Colorado. 5. Silva KCS, Silva LOS, Silva GAA, Borges CL, Novaes E, Paccez JD, Fontes W, Giambiagi-deMarval M, Soares CMdA, Parente-Rocha JA. 2020. Staphylococcus saprophyticus Proteomic Analyses Elucidate Differences in the Protein Repertories among Clinical Strains Related to Virulence and Persistence. Pathogens. 9(1):69. https://doi.org/10.3390/pathogens9010069...