Fluent Essays

my  Unknown C on   *Lab reports are always written in the past tense **They are always written in the third person; never I; instead, use we. The links to the PDF and the website below will serve as a useful guide for you; however, please remember that they are just that--a guide.  You may not copy directly from them, nor should you follow them to the letter; rather, please use what I have listed here to work on and simply use them for general organization and structure. Here is what I would like you to work on for this week (and I will include this again in the actual assignment document that I post): Find at least two peer-reviewed, primary articles that you will use as reference for both your introduction section and possibly for your discussion section. Google Scholar is a good source (https://scholar.google.com), as is PubMed (https://pubmed.ncbi.nlm.nih.gov) Working on your Introduction.  While I want you to put things in your own words, here is something to get you started and point you in the right direction: The ability to confirm the identity of an unknown bacterium is an important one, especially in the medical field.  In order to be able to appropriately diagnose and treat a patient infection, it is critical that we first know 1) if the infection is bacterial in nature and 2) if it is bacterial, then what specific type of bacterium it is....WITHOUT COPYING EXACTLY, YOU CAN USE MY FIRST FEW SENTENCES AS A TEMPLATE.  THEN YOU WILL CONTINUE TO ADD IN HERE.  Finding an article or two that references what its important to know the identity of a bacterial pathogen or perhaps one that talks about the important tests that are done in order to figure out a bacteriums identity might be useful here.  You can also included something like, In this course, we were each assigned a bacterium of unknown identity (designated Unknown A, Unknown, B, Unknown C, or Unknown D).  Our group (use the plural form here even though it was just you--unless you decide to team up with others who have your same unknown) was assigned Unknown X (list your specific unknown letter here).  Given that this course is completely online, we were not able to perform the actual experiments ourselves.   Instead were given the results of our unknowns for each of the tests listed in the Materials and Methods section.  Based on the combination of results obtained, our group concluded that our assigned unknown was (list here).  (While we do not go into results really in the intro, it is nice to have a concluding statement of sorts, just to wrap it up nicely.  Intro doesnt need to be more than a paragraph or two.) For the Materials and Methods section, usually, you would cite the lab manual or whenever you obtained the various experimental protocols from, and then elaborate on any ways in which you tweaked/amended the protocols or any mishaps that took place that perhaps skewed the results.  However, since you did not actually perform the experiments, we are going to change this section slightly.  Instead, you will structure it as such: The first week we were assigned our unknown bacterium, we given photos of the bacterium growing on nutrient agar plates and in nutrient agar broth.  Using the photos provided, along with a guide from our instructor on colony morphology and growth patterns in broth culture, we identified the colony size, margin, form, and pigment, as well as the growth appearance in broth. During the second week of working with our unknown, we were given the results of a Gram and flagellar stain, as well as the results seen after inoculation of mannitol salt, MacConkey, and Eosin-Methylene Blue agar.  Here, rather than list what you did (since you really didnt do it, you will include a brief explanation of how each test works--youll do the same for Week 7 and, now, Week 8.  You can use the descriptions of the various tests that I have provided from websites, but you always need to cite. We will start working on the Results section next week, as you have basically been provided with all of it and just need to include some of the photos I have been including, along with in a figure legend; however, if you want to start working on that, you may. Week 5 Lab: 6/14-6/20 Study Topic(s): Assignment of Unknown Bacterium and Analysis of Colony Morphology on NA Plate and in Nutrient Broth This week, each of you will be assigned a letter for your Unknown Bacterium—either an A, B, C, or D. Below, you will see a photo of your bacterium 1) on a nutrient agar plate and 2) in a nutrient broth. For this week, please record the following, to the best of your ability from the photos shown (if you have trouble, let me know): · Colony size (plate) · Colony margins (plate) · Colony form (plate) · Colony pigment (plate) · Growth appearance in nutrient broth *Use the Lab 3 PPT and accompanying materials to get you started Unknown A [Fig. 1: Unknown A on nutrient agar plate] [Fig. 2: Unknown A in nutrient broth] Unknown B [Fig. 3: Unknown B on nutrient agar] [Fig. 4: Unknown B in nutrient broth] Unknown C [Fig. 5: Unknown C on nutrient agar] [Fig. 6: Unknown C in nutrient broth] Unknown D [Fig. 7: Unknown D on nutrient agar] [Fig. 8: Unknown D on nutrient agar] [Fig. 9: Unknown D in nutrient broth] Answer Key for Unknowns Week 1: Colony Morphology & Growth Pattern in Broth 1. Unknown 1 a. Colony size: small sized; about 1–2 mm in diameter b. Colony margins: entire c. Colony form: circular/cohesive d. Colony pigment: white e. Growth appearance in nutrient broth: turbid 2. Unknown 2 a. Colony size: medium sized; about 1–50 mm in diameter b. Colony margins: lobate c. Colony form: irregular d. Colony pigment: opaque, fuzzy white or slightly yellow e. Growth appearance in nutrient broth: turbid 3. Unknown 3 a. Colony size: small sized; about 0.5–4 mm in diameter b. Colony margins: entire c. Colony form: circular d. Colony pigment: off-white or beige, shiny e. Growth appearance in nutrient broth: turbid, pellet 4. Unknown 4 a. Colony size: small sized; about 3–4 mm in diameter b. Colony margins: entire c. Colony form: circular d. Colony pigment: off-white or beige, shiny e. Growth appearance in nutrient broth: turbid LAB 7 ASSIGNMENT IMVIC Results of Unknown *Remember…you are using the same unknown each week! So, if you had Unknown A/1 week 5, you will always have that same one. I apologize for switching to numbers last week; Unknown A = Unknown 1; Unknown B = Unknown 2; Unknown C = Unknown 3; and Unknown D = Unknown D. The IMViC series is a group of four individual tests that are commonly used to identify bacterial species, especially coliforms. The capital letters in ‘IMViC’ each stand for one of the four tests: I for Indole test, M for Methyl Red test, V for Voges-Proskauer test, and C for Citrate test. The IMViC tests are used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic rod, which produces gas from lactose within 48 hours. The presence of some coliforms indicates fecal contamination. Indole Test: https://microbiologyinfo.com/indole-test-principle-reagents-procedure-result-interpretation-and-limitations/ Methyl Red Test: https://microbiologyinfo.com/methyl-red-mr-test-principle-procedure-and-result-interpretation/ Voges-Proskauer Test: https://microbiologyinfo.com/voges-proskauer-vp-test-principle-reagents-procedure-and-result/ Citrate Test: https://microbenotes.com/citrate-utilization-test-principle-procedure-and-result-interpretation/ Unknown A/1 IMVIC Results Indole Test = positive Methyl Red Test = negative Voges-Proskauer = positive Citrate = negative Unknown B/2 IMVIC Results Indole Test = negative Methyl Rest Test = negative Voges-Proskauer = positive Citrate = positive Unknown C/3 IMVIC Results Indole Test = positive Methyl Rest Test = positive Voges-Proskauer = negative Citrate = negative Unknown D/4 IMVIC Results Indole Test = negative Methyl Rest Test = negative Voges-Proskauer = positive Citrate = positive Lab 7 Questions For this week, I am switching it up a little. Instead of answering a series of questions, I would like you all to review the results from Week 5-Week 7 and submit a paragraph or two, explaining what you know about your unknown bacterium so far and listing a few possible species that you think might be your unknown, along with justification (e.g. examples from the last three weeks’ results that suggest why this might be). (15 pts total) WEEK 8 LAB Culture of Unknown on SIM Medium and Exposure to Citrate Test and Urease Test · SIM Medium: https://www.austincc.edu/microbugz/sim_medium.php · SIM medium is a combination differential medium that tests three different parameters, which are represented by the three letters in the name: · Sulfur Reduction · Indole Production · Motility · The sulfur reduction test is useful in differentiating enteric organisms.  · As a whole, the SIM test is primarily useful for differentiating Salmonella and Shigella. · SIM medium contains nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which contains amino acids, including tryptophan.  · If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result. · The sulfur and motility test results should be determined before you perform the indole test (you received the indole test results last week) · Citrate Test: https://www.austincc.edu/microbugz/citrate_test.php · Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. · This test is part of the IMViC tests and is helpful in differentiating the Enterobacteriaceae .  · If the medium turns blue, the organism is citrate positive.  If there is no color change, the organism is citrate negative.  Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change. · Urease Test: https://www.austincc.edu/microbugz/urease_test.php · Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide.  · The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red.  · Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment.  · If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. · Many enterics can hydrolyze urea; however, only a few can degrade urea rapidly.  · These are known as “rapid urease-positive” organisms.  · Members of the genus Proteus are included among these organisms.  Unknown A/1: SIM Medium = positive Citrate = negative Urease = positive Unknown B/2: SIM Medium = negative Citrate = positive Urease = negative Unknown C/3: SIM Medium = positive (variable results) Citrate = negative Urease = negative Unknown D/4: SIM Medium = negative Citrate = positive Urease = positive Week 9 Lab Results on Unknown Bacterium: Culture on Blood Agar and Exposure to Coagulase Test and Nitrate Test Blood Agar Blood agar contains the same general nutrients as nutrient and trypticase soy agar, along with 5\% sheep blood. It is useful for cultivating fastidious (has very complicated nutritional requirements, meaning it will not grow without specific factors present or in specific conditions) organisms and for determining the hemolytic (lyse/rupture RBCs) capabilities of an organism. Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins . Bacteria can produce different types of hemolysins. Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind. This is referred to as α-hemolysis (alpha hemolysis). The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin. If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur. This is called γ-hemolysis (gamma hemolysis). The hemolysins produced by streptococci perform better in an anaerobic environment. Because of this, it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of lower oxygen concentration where the streptolysins can more effectively break down the blood cells. https://www.austincc.edu/microbugz/blood_agar_test.php Blood Agar Results of Unknown Bacteria Unknown A/1 (gamma) Unknown B/2 (alpha) Unknown C/3 (gamma) (non-pathogenic strain of Unknown C/3) (beta) (pathogenic strain of Unknown C/3) Unknown D/4 (gamma) Coagulase Test The coagulase test identifies whether an organism produces the exoenzyme coagulase , which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis (being ingested by immune cells) and some other antimicrobial agents. The coagulase slide test is used to identify the presence of bound coagulase or, “clumping factor”, which is attached to the cell walls of the bacteria. Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to agglutinate, or clump together, which creates the “lumpy” look of a positive coagulase slide test. The coagulase tube test is used to identify the presence of either bound coagulase or free coagulase, which is an extracellular enzyme. Free coagulase reacts with a component of plasma called coagulase-reacting factor . The result of this reaction causes the plasma to coagulate. The coagulase test is useful for differentiating potentially pathogenic Staphylococci such as Staphylococcus aureus from other Gram-positive, catalase-positive cocci. Coagulase Results of Unknown Bacteria Unknown A/1: Coagulase-negative Unknown B/2: Coagulase-negative Unknown C/3: Coagulase-negative Unknown D/4: Coagulase-negative Nitrate Test Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase . It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. Nitrate broth contains nutrients and potassium nitrate as a source of nitrate. After incubating the nitrate broth, a dropperful of sulfanilic acid and α-naphthylamine are added. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid. This reacts with the α-naphthylamine to form a red-colored compound. Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction. If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. If the medium does not turn red after the addition of the nitrate reagents, then a small amount of powdered zinc is added. If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present. Therefore, a red color on the second step is a negative result. The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the α-naphthylamine to create the red complex. If the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium either, that means that denitrification took place, and ammonia or molecular nitrogen were formed. (Basically, it means that the bacteria being tested are SUPER good at reducing nitrate and already reduced it to nitrate and beyond before the broth was tested. https://www.cdc.gov/std/gonorrhea/lab/tests/nitrate.htm Nitrate Reduction Results of Unknown Bacteria Unknown A/1: Positive Unknown B/2: Positive Unknown C/3: Positive Unknown D/4: Positive Lab 10 Assessment Catalase and Oxidase Tests on Unknown For this week’s lab assessment, you will answer just a few questions regarding the oxidase and catalase assays and unknown results (listed below) and then you will continue to work on your lab report. For week 10, you will make sure that you are: 1) continuing to work on your introduction section (if you still need to) · if you are still struggling to find articles, here are a few suggestions to get you started….you will need to find at least one other on your own, preferably 3-4 · https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560418/ · https://pubs.rsc.org/en/content/articlehtml/2017/cs/c6cs00693k · https://onlinelibrary.wiley.com/doi/full/10.1111/1541-4337.12618 2) making sure your materials and methods look good · see any feedback I may have provided · make sure to only include the name of the technique/assay and how it works (no listing of results or interpretations in the materials and methods section) 3) continue working on your results section · again, as I mentioned last week, your results section is just going to be a listing of your results and any photos with figure legends that you include · you are also free to include a table of your results Questions 1) What are the bubbles that you see in a positive (+) catalase reaction? 2) Bacteria capable of synthesizing the enzyme catalase hydrolyze __________ into _________ and gaseous oxygen. 3) The oxidase test is used to identify microorganisms containing the enzyme _________________. 4) Cytochrome oxidase transfers electrons from the electron transport chain to ________ (the final electron acceptor) and reduces it to water. Catalase Test The metabolic activity of aerobic and facultatively anaerobic microorganisms produce toxic by-products like hydrogen peroxide and superoxide radical (O2—). These products are toxic to the organisms and might even result in cell lysis if not broken down. In the case of pathogenic organisms, different mechanisms are found that break down these products to non-toxic substances. Bacteria capable of synthesizing the enzyme catalase hydrolyze hydrogen peroxide into water and gaseous oxygen, which results in the liberation of gas bubbles. H2O2         ———–>        H2O + O2 catalase The production of catalase thus protects the organism against the lethal effect of hydrogen peroxide accumulated at the end of the aerobic metabolism. The presence of the catalase enzyme can be demonstrated by adding hydrogen peroxide to the bacterial inoculum, which results in the rapid liberation of oxygen bubbles. The lack of enzyme is demonstrated by the absence of such bubbles. The catalase test has been extensively used over the years as it allows the differentiation of catalase-positive organisms like Staphylococci from catalase-negative species like Streptococci. Unknown A/1 = catalase-positive Unknown B/2 = catalase-positive Unknown C/3 = catalase-positive Unknown D/4 = catalase-positive https://www.youtube.com/watch?v=VKaa24sOqwo https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/26\%3A_Catalase_Test Oxidase Test This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae. Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. https://www.youtube.com/watch?v=YSPbMdR2K70 Unknown A/1 = oxidase-negative Unknown B/2 = oxidase-variable Unknown C/3 = oxidase-negative Unknown D/4 = oxidase-negative Lab 11 Assessment Antimicrobial Susceptibility Test on Unknown Bacterium 1. Click on this link, and read up on the theory behind and the protocol for performing a Kirby Bauer Antibiotic Sensitivity Assay https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/09\%3A_Kirby-Bauer_(Antibiotic_Sensitivity) 2. Review the given antibiotic sensitivity results for each of your unknowns. Please understand that I could not find all identical assays, performed with the same antibiotics. 3. Your assignment this week is to: a. Finish your results section, with the antibiotic sensitivity results from this week included i. I understand that some of these results are in diameter of zone of inhibition (in mm), some report S = susceptible or NS = not susceptible, some report \% resist, and others a photo of the zones of inhibition ii. The goal here is not to have perfect results but, rather, to help support what you know about if your unknown is gram positive or gram-negative, and how its biochemical characteristics from the past couple week’s tests. For example, if you have Unknown C/3, you know it is Gram-negative; therefore, would you expect it to be susceptible to penicillin? Why or why not? b. Write at least the first two paragraphs of your Discussion and Conclusion Section i. *Remember, in your Results section, you are simply stating WHAT you observed. 1. For example, you may have a photo of your bacterium growing on MSA and the figure legend will read, “Figure X. Growth of Unknown A on mannitol salt agar (MSA). And your description of your results would describe what you observed (e.g. Unknown A exhibited ample growth on MSA with colonies pink in colony.) ii. For the Discussion and Conclusions, this is where you say when the results MEAN or imply. For example, “Based on the fact that Unknown A exhibited ample growth on MSA, it appears that this bacterium is halotolerant, and can survive in high salt conditions. In combination with the cocci morphology and the fact that this bacterium is gram-positive, this strongly suggests that Unknown A is a Staphylococcus species. Then, based on the lack of color change of the MSA from pink to yellow implies that Unknown A is….” c. If you need help with APA formatting, please visit: https://owl.purdue.edu/owl/research_and_citation/apa_style/apa_formatting_and_style_guide/general_format.html Unknown A/1 Unknown B/2 Unknown C/3 Unknown D/4 Lab Report Guide and Final Checklist For the Week 12 Lab, we will focus on completing all the sections of your lab reports, including your references section. In this document, I will attempt to clarify areas that are still giving folks a little bit of trouble or confusion. Please also remember to go back and review the weekly lab documents from Weeks 5-11. Introduction As mentioned previously, this section should provide the reader with the following information: 1. Reasons why it is important for one to be able to figure out the identity of an unknown bacterium 2. What you did in this particular “study” and why you did it 3. One statement about what you believe your unknown bacterium to be, based on your collective results 4. One or two statements about the bacterium that you think it is. 5. MAKE SURE THAT YOU USE REFERENCES FOR ANY STATEMENTS MADE THAT ARE NOT YOUR ORIGINAL THOUGHT! For example, (and do not copy this verbatim, as that is plagiarism—this is just to give you an idea): “There are an estimated 30,000 identified bacterial species in the world (Dykhuizen, 2005). Of all the microbes in the world (viruses, bacteria, archae, fungi, protozoa and helminths), only about 1,400 species are pathogenic to humans (“Microbiology by numbers,” 2011). In this study, each lab group was assigned an unknown bacterium by our instructor. Each week for seven weeks, we were provided with lab results of our unknown, including its colony morphology on nutrient agar and growth pattern in nutrient broth; a Gram and flagellar stain; growth appearance on a variety of selective, differential, and enriched media; and results of a multitude of biochemical tests. Based on all of the results provided, we concluded that our unknown was Pseudomonas aeruginosa. P. aeruginosa is a serious concern in the medical field, as it is an aggressive opportunistic pathogen that targets immunocompromised individuals, especially patients suffering from cystic fibrosis (CF) (Moradali, M.F. et al, 2017).” Materials and Methods Remember, in this section, you will be listing each test that you did, along with an explanation of how it works/what it tells you. Example (and note that I did simply choose a few tests; you must include all tests for which you were given results): “During week one of working with our unknown bacterium, we were provided with results of what our bacterium looks like when cultured on nutrient agar and in nutrient broth. Culturing bacteria on solid agar allows one to study its colony characteristics, which can help narrow down the possibilities for our unknown’s identity. Culturing in nutrient broth also provides insight into the growth requirements of our unknown. “During week two of working with our unknown, we were provided with results of culture on mannitol salt agar. Mannitol salt agar is both a selective and differential medium. It is selective based on….(you will need to provide a citation for each description that you include. You can certainly use the same source for multiple test descriptions (like a lab manual or text book), as long as the source describes the test in question) Results Again, this section is meant to just describe the results that you observed/were provided with—no interpretation here. For example, if you are describing the results of your unknown bacterium when cultured on mannitol salt agar, you will simply describe what you saw. “Unknown X exhibited growth on mannitol salt agar and also turned the medium a yellow color.” If you are including photos, you will need to include a figure legend under each. For example, if you have a photo of your unknown on MSA, you could write, “Figure X. Unknown B growing on mannitol salt agar.” Discussion and Conclusions THIS is where you start to talk about what the results you were given TELL YOU about your unknown bacterium’s identity. For example, we know that bacteria who exhibit growth on mannitol salt agar must be able to tolerate high salt conditions; otherwise, they would die and no colonies would be observed. Also, if your bacterium grew and turned the MSA yellow, that means your bacterium ferments the carbohydrate mannitol. Make sure to “round out” your discussion and conclusion with a statement something to the effect of: “Based on the collective results observed, we inferred that our unknown bacterium, Unknown P, was Pseudomonas aeruginosa. And then include a couple sentences about your unknown, where it comes from, what infections it is usually associated with, and what the treatments for infections with it are. **note that you should include reference in this section! References (for in-text citations provided in the Introduction) Dykhuizen D. (2005). Species Numbers in Bacteria. Proceedings. California Academy of Sciences, 56(6 Suppl 1), 62–71. Microbiology by numbers, Issue 9, pg. 628 (2011). Nature Reviews Microbiology. Moradali, M.F., Ghods, S., and Rehm, B.H. (2017). Paradigm for Adaptation, Survival, and Persistence. Frontiers in Cellular and Infection Microbiology. For citing sources without an author listed https://owl.purdue.edu/owl/research_and_citation/apa_style/apa_formatting_and_style_guide/in_text_citations_author_authors.html (in-text citations) https://owl.purdue.edu/owl/research_and_citation/apa_style/apa_formatting_and_style_guide/reference_list_author_authors.html (references) Final Checklist · Introduction · Materials and Methods · Results · Discussion and Conclusions · References Unknown Lab Report #1 Unknown number 1 xxxxxxxxxxx Date Katie Ruggieri Microbiology Spring 2016 BIO 239 - B02 Introduction Microorganisms exist everywhere. They are living on the surface of a steering wheel in a car, on the food that is eaten, and even inside of the human body. It is important to know that not only do bad microorganisms exist, but good microorganisms also. It is crucial to be aware of bad microorganisms and their effects on humans when considering working in the medical field. It is essential to determine which microorganism may be infecting a patient in order to properly diagnose and effectively treat the patient. Determining the microorganism is important due to the fact that some treatments may fight against a particular bacteria, where others may not affect the bacteria. The purpose of this study was to identify an unknown bacteria from a culture using the methods previously conducted and learned in the microbiology laboratory this semester. Ultimately the unknown was identified as Staphylococcus epidermidis. Materials and Methods An unknown labeled number 1 was given out by the microbiology professor. The methods that have been learned in lab this semester for identifying a bacteria have been used on this unknown. The following procedures were applied as stated in the laboratory manual The Pearson Custom Library for the Biological Sciences, unless changed by the instructor and noted. The first procedure that was applied to the unknown was to streak the unknown on a Trypticase Soy Agar plate, using the method stated in the lab manual. This was done to test the purity of the unknown. After the plates were incubated and grew, the morphology of the unknown was easily observed and recorded in the lab manual. The results were round, entire, and flat macroscopic morphology. A Gram stain was then performed on the unknown. Quality controlled bacteria were Gram stained as well as the unknown to ensure that the Gram stain reaction was correctly performed. The Gram reaction was then recorded into the lab manual. The unknown was streaked using proper aseptic technique on a Blood Agar plate, Mannitol Salt Agar plate, MacConkey Agar plate, and Eosin-methylene Blue Agar which were all incubated for two days at 37 degrees Celsius. The unknown was observed in the isolation streak on a Blood Agar Plate to look for the hemolytic reaction and recorded in the lab manual. A Mannitol Salt Agar test was used on the unknown to assure it was a Staphylococcus bacteria because they are salt tolerant and differentiates between species based on agar color.  A MacConkey agar test was used in order to isolate a gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. Eosin methylene blue agar was streaked to test for fecal coliforms and observe if it would inhibit the growth of the bacteria indicating if it was gram positive. All results were observed and documented in the lab manual. Biochemical tests were used from the unknown identification tabled provided in the lab manual. All tests were performed by the methods listed in the lab manual ( ). Table 1.2 lists the test, purpose, reagents, and results. All of the following tests were performed on this unknown: 1. Mannitol Salt Agar 2. Blood Agar 3. Eosin Methylene Blue Agar 4. MacConkey Agar 5. Gram Stain 6. Glucose 7. Lactose 8. Mannatol 9. Sucrose 10. Indole 11. Citrate Utilization 12. Hydrogen Sulfide 13. Methyl Red 14. Voges-Proskauer Results Table 1.1 Structural Characteristics Gram Stain: Gram Positive/Purple Cell Shape: Coccus Cell Arrangement: Grape-like Clusters Pigmentation: Yellow Colony Appearance: Yellow colonies, Small dots Table 1.2 Test Media/or Reagents Temp. Observation Results Interpretations Gram Stain Crystal Violet, Iodine, Alcohol, Safranin Purple cocci clusters Gram Positive Organism’s cell wall retained dye TSA 37C Yellow colonies Mannitol Salt Agar (MSA) Mannitol 37 C Growth with no change in media negative Did not ferment mannitol, Negative for Staphylococcus aureus MacConkey Agar Neutral Red 37 C Growth with no change in media Gram Positive Eosin-methylene Blue Agar 37 C Growth with no change in media Blood Agar Neutral Red 37 C White Growth no change in media negative Microorganism is not hemolytic Lactose Lactose fermentation tube 37 C Orange, no bubble Positive for Acid, Negative for Gas Fermented with the production of acidic waste, no evolution of Gas Mannatol Mannatol fermentation tube 37 C Pink, no bubble Negative for Acid and Gas Did not ferment and no evolution of Gas Sucrose Sucrose fermentation tube 37 C Peach, no bubble Variable, Negative for Gas Unable to determine if Acid was in tube, no evolution of Gas Glucose Glucose fermentation tube 37 C Yellow, no bubble Positive for Acid, Negative for Gas Fermented with the production of acidic waste, no evolution of Gas, Positive for Staph Indole SIM Agar, Kovac’s reagent 37 C Reagent color yellow Negative Tryptophan was not hydrolyzed Methyl Red Methyl Red indicator 37 C Color of medium was red Positive Voges-Proskauer Barrett’s Reagent A and B 37 C Medium was pink-red Positive Citrate Simmons citrate agar slant 37 C No presence of growth, medium was green Negative No growth on slant surface, no Sodium Carbonate present Hydrogen Sulfide Production (H2S) 37 C Medium was yellow Negative for H2S production, Negative fir motility No anaerobic respiration or motility Catalase Hydrogen Peroxide, TSA plate 37 C Bubbled Positive Aerobe bacteria Urease Urea broth 37 C Peach Negative Hydrolysis *Should have been a positive result Oxidase Trypticase soy agar plate, p-Aminodimethylaniline oxalate 37 C No color change Negative for oxidase production Enterobacteriaceae Figure 1 Figure 2 Figure 1: Inoculated Unknown colony in agar plate Figure 2: Mannitol Salt Agar or MSA plate featuring Unknown, Staphylococcus aureus, Staphylococcus epidermis Figure 3 Figure 4 Figure 3: Blood Agar plate featuring Unknown and Enterococcus faecalis Figure 4: Eosin methylene blue agar or EMB plate featuring Unknown, Staphylococcus aureus, and Salmonella typhimurium Figure 5 Figure 6 Figure 5: MacConkey Agar Plate featuring Unknown, Enterobacter aerogenes, and Escherichia coli Figure 6: Mannitol Salt Agar or MSA plate featuring Unknown, Escherichia coli, and Enterobacter aerogenes Figure 7 Figure 8 Figure 7: Unknown Gram Stain Figure 8: Unknown Glucose observation Figure 9 Figure 10 Figure 9: Unknown Lactose observation Figure 10: Unknown Mannatol Observation Figure 11 Figure 12 Figure 11: Unknown Sucrose Observation Figure 12: IMVIC-SIM (Indole production test) Figure 13 Figure 14 Figure 13: Citrate Utilization Test Figure 14: Hydrogen Sulfide Production Test Figure 15 Figure 16 Figure 15: Voges-Proskauer Test Figure 16: Methyl Red Test The Gram stain result was positive for the unknown. The unknown in the Blood Agar plate were found to be round, entire, and flat colony morphology as well as an alpha hemolytic reaction pattern which indicated red blood cell ion leakage which is characteristic of S. epidermidis. The Mannitol Salt Agar remained red after incubation with the unknown culture which indicated that the Mannitol-D sugar was not fermented. Discussion/Conclusion Following several differential tests, it is determined that unknown 1 is Staphylococcus epidermidis. Unknown 1 was identified as Staphylococcus epidermidis, due to the gram-positive cell wall, coccus shape, yellow colonies, positive catalase test, positive glucose, alpha hemolytic reaction pattern, and negative mannitol salt agar plate, which are all characteristics of Staphylococcus epidermidis. Staphylococcus epidermidis is important to identify because it’s overwhelming presence on human skin and mucous membranes as well as most surfaces that have been in contact with human skin. “In particular, S. epidermidis represents the most common source of infections on indwelling medical devices.” (Michael Otto, Ph. D.) This pathogen is also usually associated with intravascular devices, urinary tract infections, septicemia, endocarditis, and most importantly biofilms. “Frequent antibiotic resistance, in particular to methicillin (methicillin-resistant S. epidermidis, MRSE), makes S. epidermidis an important threat in nosocomial infections.” (Gordon Y C Cheunga, Michael Ottoa) Although S. epidermidis is not as well-known as many other infectious pathogens, it is extremely common in the medical field and it is important to distinguish this pathogen from others in order to treat the pathogen effectively. References Gordon Y C Cheunga, Michael Ottoa. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874874/. 23 June 2010. 19 April 2016. Michael Otto, Ph. D. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/. August 2009. 19 April 2016. Paul D Fey, Michael E Olson. http://www.futuremedicine.com/doi/abs/10.2217/fmb.10.56. n.d. 19 April 2016.