Homework answers / question archive / BIO 220 – UNKNOWN PROJECT GUIDELINES **IMPORTANT** **Be sure to record all methods and observations in your notebook and communicate with your lab partner!! ** Confirm with your instructor all results before recording in your notebook

BIO 220 – UNKNOWN PROJECT GUIDELINES **IMPORTANT** **Be sure to record all methods and observations in your notebook and communicate with your lab partner!! ** Confirm with your instructor all results before recording in your notebook

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BIO 220 – UNKNOWN PROJECT GUIDELINES **IMPORTANT** **Be sure to record all methods and observations in your notebook and communicate with your lab partner!! ** Confirm with your instructor all results before recording in your notebook. ** If you fail to perform a test correctly, it will need to be repeated the next lab period. **Each student pair will lose 1 point of off their lab report grade for each incorrect guess of their unknowns. ** Each student pair will lose 1 point for inoculate media not required for identification of the organisms. (Please ask before taking any media to confirm you are on the right track). Day 1 – Lab partner #1 1. 2. 3. 4. 5. Record unknown #s in your notebook Perform gram stains on each organism Sub-culture each organism onto a TSA slant Perform deep stab of each organism for aerotolerance Gram Negative Bacilli a. Streak for isolation on TSA Plate b. Inoculate all IMViC test media 6. Gram Positive cocci a. Streak for isolation on Blood Agar plate b. Perform catalase test Day 2 – Lab partner #2 1. 2. 3. 4. 5. Record colony morphology on each organism and note any other growth patterns on the plates Identify aerotolerance of organism Perform pertinent tests for IMViC and record all IMViC results Identify the next set of tests that need to be performed to identify the organisms Sub-culture each organism into a broth tube from your slants. Save the slants!! Day 3 – Lab partner #2 1. Perform the next tests needed to identify the organisms and record in your notebook. 2. Begin formulating a flow chart and table of results for each organism 3. Sub-culture organisms onto new slants from old slant Day 4 – Lab partner #1 1. 2. 3. 4. Record test results and/or perform a test needed for identification Continue working on flow charts and tables Outline report with lab partner If needed sub-culture each organism into new broth tubes from slants Day 5 and Day 6 – complete identification LAB REPORT INSTRUCTIONS GENERAL Unknown reports in microbiology are written in scientific format. The results of the exercise or experiment are what are being showcased, not the writing. The purpose of scientific writing is not to entertain, but to inform. The writing should be simple and easy to understand. There is a specific style that must be followed when writing scientific reports. • Scientific writing is typically written in the passive voice. The pronouns "I", "We" and "They" are not typically used. For example, instead of writing "I used a TSA agar plate to isolate my unknown," it is customary to write, "A trypticase soy agar (TSA) plate was used to isolate the unknown." • It is also customary to write in the past tense for most of the report. This includes the introduction, the summary, the description of the materials and methods and the results. The present tense is reserved for the conclusions about the results. See the examples given below. Some other general rules that should be followed are: • Microbial nomenclature: The name of the bacterium should be written out completely and spelled correctly. • The name should be italicized or underlined. Italicized is preferred. For example, Staphylococcus aureus. • The genus is capitalized but the species is not. After the full genus name is given in the paper, it can be written as S. aureus, but still italicized. This is as long as there in no other genera in the paper that starts with the same letter. TITLE PAGE A title page must be created for each experiment. It will serve as the title sheet for your laboratory report. It should include the title of your report, the “unknown” letter or number, your name, the date, and the course name/number/section. It should include required test results – aerotolerance and colony morphology. INTRODUCTION The introduction should describe the objectives of the experiment. It should also summarize the scientific ideas behind the experiment, and be as brief as possible. One paragraph describing the purpose of the experiment – Why is it important to identify microorganisms? How did you go about identifying your organism? MATERIALS AND METHODS This is where the details of the study are listed. Where did the specimen come from, and what methods were used to identify it? Briefly describe the experimental method used and the names of any chemicals or equipment used. It is important to indicate all temperatures in °C and measurements in metric units. Be specific, but do not re-write the lab manual. You can assume that the reader is familiar with the various techniques, especially if they are standard, so do not give details. For instance, do not include “cook book” directions. One way is to mention the names of the materials used and reference the lab manual for the procedure or method and then continue to elaborate when necessary. See example 1. Example 1: "An unknown labeled as letter G was given out by the lab instructor. The methods that have been learned thus far for identifying bacteria have been applied to this unknown. Procedures were followed as stated in the course laboratory manual by Laboffe and Pierce (1), unless otherwise noted. The first procedure that needed to be done was to streak the unknown out on a Tryptic Soy Agar plate, using the quadrant streak method described in the lab manual. This needed to be done in order to test the purity of the unknown. After the plates were incubated and grown, the morphology was observed and recorded and a Gram stain was performed.” If there is a procedure that the instructor added or made changes to, or the student used another procedure not in the course lab manual, then it should be written out and referenced. RESULTS In the results, you present your observations and data with no interpretations or conclusions about what they mean. Tables and graphs should be used to supplement the text and to present the data in a more understandable form. The written text of the results section may be as short as one sentence summarizing the highlights and directing the reader to specific Tables and Figures. Use past tense to describe your results shown in table format (see examples below). This is also where the flow chart showing how you arrived at the answer is discussed. Example: Unknown G had the following morphology on a TSA plate: medium sized opaque cream colored colony. After determining that it was a Gram negative rod, an indole test was performed resulting in a red ring at the top of the broth (Table 1; Figure 1)….etc. A citrate test was also performed with the agar slant turning blue after incubation (Table 2). Table 1: Biochemical Test Results TEST PURPOSE Gram stain To determine the Gram reaction of the bacterium To determine the presence of cytochrome c To determine the ability of an organism to split indole from tryptophane Oxidase test Indole Test OBSERVATIONS RESULTS Crystal violet, Iodine, Alcohol, Safranin Oxidase paper REAGENTS Pink rods Kovac's added to 1 ml of tryptone broth Red Ring at top of broth Gram negative rods Positive oxidase test Positive indole test Purple / black color change ETC. Table 2: Physiological and Biochemical Results TEST REAGENTS OR MEDIA TEMP OBSERVATIONS RESULTS INTERPRETATIONS Citrate Citrate slant (green) 35 C 0 Color changed from green to blue Positive Methyl Red MRVP 350C After adding methyl red to one ml, color changed from light yellow to a darker yellow Negative Eschericia coli Proteus spp. Organism is able to utilize citrate as a carbon source Organism is not able to produce large amounts of acid from glucose fermentation Klebsiella pneumoniae Enterobacter aerogenes Figure 1 – Flow chart of tests performed to identify unknown organism G. DISCUSSION/CONCLUSION This is the most important part of your lab report. This is where you show how well you understand what went on in your experiment and draw conclusions based on the observations you made. This section interprets the meaning of the results. The following questions should be answered here: How did the test result lead to identification? Was it the correct identification? If not, why not. What problems were encountered? This is also where the background information on the organism (environment/pathogenicity) that was identified is mentioned. Example of a discussion: After several differential tests, it was concluded that unknown G was Escherichia coli. After performing the Gram stain to determine that the unknown was a Gram negative rod, the organism was grown on a TSA slant for use in inoculating the rest of the biochemical tests. All of the biochemical tests worked well except for the indole test. It gave a false negative result at first. This was determined since it was inconsistent with the rest of the result. The instructor suggested that the test be repeated and it was repeated. The repeated test gave a positive result, consistent with the other data. Therefore it was concluded that the unknown was Escherichia coli. THIS CAN BE ELABORATED MORE FOR EACH TEST. E. coli is in the Enterobacteriaceae family. It is typically found in the human intestines, as well as other animals. It can cause disease in the right host. THE REST OF THIS INFORMATION SHOULD BE RESEARCHED FROM THE TEXTBOOK, INTERNET OR OTHER MICROBIOLOGY RESOURCES. References Correct reference format must be used. References should be numbered and the number added to the report when necessary. See example 1 under materials and methods. Spelling of the authors of the references must be correct. References should be numbered in order as they appear in the text. 1. Microbiology: Laboratory Theory and Application 4th edition, Leboffe, M.J. and Pierce, B.E., Morton Publishing, Englewood, CO, 2010. To reference a journal article, whether found in a printed bound journal or a pdf online, the reference should be the same. Do not provide online access dates and websites for journal articles online. This is a legitimate publication and should be treated as such. An example would be: Hemingway, J.D., Hilton, R.G., Hovius, N., Eglinton, T.I., Haghipour, N., Wacker, L., Chen, MC.,Galy, V.V. 2018. Microbial oxidation of lithospheric organic carbon in rapidly eroding tropical mountain soils. Science 360: 209-212 Figures and Tables All Figures are numbered consecutively throughout the report and are referred to by Figure #. Remember, the terms Figure and Table are always capitalized and are referenced in the body of the text of the report. To do all Tables try to use the table editor in Microsoft Word. Unknown Lab Report Grading Rubric Electronic copy to be uploaded to blackboard on or before May 13th. 10 point bonus added to those that submit their report on or before May 7th. Title Page - Contained creative title - Contained “unknown” number/letter - Contained results of all required tests –aerotolerance and colony morphology - Contained your name, date, course information 5 points /1 /1 /2 /1 Introduction (1 paragraph) - Specific purpose of study was stated - Background information on importance of identifying microorganisms that cause disease – in general…not specific to your organism - Brief explanation of how microorganisms are identified in general…not specific to your organism 15 points /3 Materials & Methods - Methods were described in chronological order & paragraph form - Reference to lab manual included - Thoroughness and repeatability. Someone could read this section and perfectly replicate what you actually did, not just want your lab handout asked you to do. 10 points /2 /2 Results - Table(s) of tests and selective media - Figure of completed flow chart - Results were merely stated, not discussed, in paragraph form - Tables and figures were properly labeled, captioned, and referenced in the text 20 points /6 /6 /4 /4 Discussion (2 to 3 paragraphs) - Explanation of results and how this led to identification of organism 30 points - - Explained any problems/mistakes/errors made and how they were corrected. – if no mistakes, provide suggested improvements if project were repeated. Background information of organism identified – references are required /6 /6 /6 /10 /10 /10 Literature Cited - At least 3 sources - lab manual and 2 peer-reviewed journal articles - Sources were cited in a consistent manner according to instructions In-text citations were present and consistently formatted Format & Grammar - Proper lab report format was used throughout the report – 12 pt font, double spaced, and 1” margins Correct spelling & punctuation were used throughout the report Each section was written in paragraph form Points deducted for mis-identification or use of incorrect media Total 10 points /3 /4 /3 10 points /10 /100 points BIO 220 – UNKNOWN PROJECT GUIDELINES **IMPORTANT** **Be sure to record all methods and observations in your notebook and communicate with your lab partner!! ** Confirm with your instructor all results before recording in your notebook. ** If you fail to perform a test correctly, it will need to be repeated the next lab period. **Each student pair will lose 1 point of off their lab report grade for each incorrect guess of their unknowns. ** Each student pair will lose 1 point for inoculate media not required for identification of the organisms. (Please ask before taking any media to confirm you are on the right track). Day 1 – Lab partner #1 1. 2. 3. 4. 5. Record unknown #s in your notebook Perform gram stains on each organism Sub-culture each organism onto a TSA slant Perform deep stab of each organism for aerotolerance Gram Negative Bacilli a. Streak for isolation on TSA Plate b. Inoculate all IMViC test media 6. Gram Positive cocci a. Streak for isolation on Blood Agar plate b. Perform catalase test Day 2 – Lab partner #2 1. 2. 3. 4. 5. Record colony morphology on each organism and note any other growth patterns on the plates Identify aerotolerance of organism Perform pertinent tests for IMViC and record all IMViC results Identify the next set of tests that need to be performed to identify the organisms Sub-culture each organism into a broth tube from your slants. Save the slants!! Day 3 – Lab partner #2 1. Perform the next tests needed to identify the organisms and record in your notebook. 2. Begin formulating a flow chart and table of results for each organism 3. Sub-culture organisms onto new slants from old slant Day 4 – Lab partner #1 1. 2. 3. 4. Record test results and/or perform a test needed for identification Continue working on flow charts and tables Outline report with lab partner If needed sub-culture each organism into new broth tubes from slants Day 5 and Day 6 – complete identification The professor gave me a list of organisms one of them is my organism which is Alcaligenes faecalis Enterobacter aerogenes Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Lactococcus lactis **Micrococcus luteus this one is my organism after I did the tests Morganella morganii Proteus mirabilis Proteus vulgaris Providencia stuartii Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Streptococcus agalactiae Streptococcus bovis Streptococcus gallolyticus Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes And I must identify my organism from these organisms by doing many tests First test was gram stain and after that I put my organism under the microscope and cell arrangement is ROUND IN CLUSTERS AND TETRADS. Second test was TSA test, and the result is the growth was good Third test was CATALASE test the result is positive and the color changed from green to blue Fourth test is MSA test the result is no growth Fifth test is coagulase, and the result is negative This schedule shows the results I got from the tests under Micrococcaceae>Micrococcus + (Micrococcaceae) Catalase test Staphylococcus aureus Streptococcus spp. Staphyloccus epidermidis Enterococcus spp. Cocci in Pairs Lactococcus spp. Cocci in Chains Enterococcus faecalis Streptococcus sp Micrococcus luteus TSA Deep Stab Facultative Anaerobes Staphylococcus aureus Staphylococcus epidermidis Mannitol Salt Agar + Staphylococcus Staphylococcus epidermidis aureus - (Streptococcaceae) LactococcusBile lactus Aerobes Micrococcus luteus Esculin Agar + LactococcuEnterococcus faecalis s lactus This chart gives you a good example about the process for just two tests which is catalase and TSA test those are the last two steps to identify the organism I want to tell you this beside the report I have to have a flow chart about all organisms and put the tests in it, and eliminate the organisms from the beginning based on those tests and show the process how did I find my organism Identification of Unknown Lab Report Klebsiella pneumoniae Staphylococcus epidermidis Introduction Identification and complete understanding of a microorganism had played a vital role in microbiology. The identification of microorganism is a systematic and watchful process that uses several tests to center on the types of microorganisms specially bacteria in an unknown sample. It yields assistance for several aspects of the microorganism’s research and provide assistance to physicians to treat a patient appropriately. In this study several different tests were conducted on the basis of all the methodologies that have been implemented during the laboratory class so that identification and complete identification of an unknown microorganism can be made. Materials and Methods The sample was enriched on different types of media and to isolate discrete colonies of bacteria. The media includes: 1. Nutrient agar 2. MacConkey agar 3. Mannitol salt agar 4. EMB agar Following different biochemical tests were perform to identify the bacteria. 1. Catalase 2. Motility 3. Mannitol 4. Lactose 5. Esculin Hydrolysis 6. Oxidase 7. Citrate 8. Sulfur 9. Indole 10. MR (Methyl Red) 11. VP (Voges Proskauer) 12. ONPG (ß-galactosidase) 13. Lysine decarboxylases 14. Ornithine Decarboxylase 15. Urease Results Colony Morphology on Different Medias: Different colony morphology were observed on different culture medium such that circular pink colonies on MacConkey agar medium and Pinkish red mucoid colonies on EMB agar as shown in Table 01. Table 01: Colony Morphology and Gram Staining Characteristics Culture medium Colony morphology on different media Results Nutrient agar Small transparent colonies Bacteria is present Mannitol Salt agar No growth No gram positive bacteria MacConkey agar Circular pink colonies EMB agar Pinkish red mucoid colony Gram negative bacteria is observed Escherichia coli is not present Biochemical Identification of Bacteria Different biochemical tests like gram staining, citrate, MR & VP, lactose, Oxidase, mannitol and catalase were performed for the identification of bacteria as shown in Table 02 and Flowchart. Test Gram stain Catalase Motility Mannitol Lactose Table 02: Biochemical Test Results PURPOSE REAGENTS OBSERVATIONS To determine the Crystal violet, Pink rods Gram reaction of the Iodine, Alcohol, bacterium Safranin Differentiating A drop of H2O2 on Evolution of O2 Aerobic and obligate colony are observed anaerobic bacteria To determine None No movement flagellated and aflagellated bacteria To differentiate Phenol red Color changes to mannitol fermenting mannitol broth yellow bacteria medium To differentiate Phenol red lactose Color changes to lactose fermenting broth medium yellow RESULTS Gram negative bacilli Positive Negative Positive Acid/Acid Esculin Hydrolysis Oxidase Citrate Sulfur Indole bacteria To differentiate enterococci and nonenterococcus group D streptococci To differentiate enterobacteriaceae to other genera To differentiate citrate fermenting bacteria To differentiate members of enterobacteriaceae on the basis of sulfide production To determine the ability of an organism to split indole from tryptophan Esculin and bile salt in nutrient agar medium To determine mixed acid fermenting bacteria Positive Tetramethyl-pNo color phenylenediamine formation Negative Slants of simmon citrate agar medium were streaked Color change Positive SIM (Sulphur Indole Motility) Media Absence of blackening of media Negative Kovac's added to 5ml of tryptone broth no Ring at top of agar Negative indole test Drop of methyl red solution was added in 5ml of trypton broth To determine if an Barrit solution A organism produce and B with few ml VP (Voges acetyl methyl of NaOH was Proskauer) carbinol from added in 5ml of glucose tryptone broth ONPG disk added ONPG (ßTo detect late lactose to bacterial galactosidase) fermenting bacteria suspension To determine if Lysine Lysine organism use amino decarboxylase decarboxylases acid lysine or not broth media To determine if ornithine Ornithine organism use amino decarboxylase Decarboxylase acid ornithine or not broth media To differentiate urea Urease Urease agar media hydrolyzed bacteria MR (Methyl Red) Color changes to dark brown Yellow coloration Negative was observed Pink coloration was observed Positive Color changes to yellow Positive Color changes to yellow Positive No color change Negative Color changes to pink Positive Figure 2: Colony Morphology of Bacteria Figure 1: Gram Staining of Bacteria Figure 4: MR Test Figure 3: Indol Test Identification through Biochemical Test Gram Staining Pink Rods G+ve Lactose test Negative Salmonella Shigella Pseudomonas Positive E. coli Klebsiella Enterobacter Motility Catalase Positive Klebsiella Enterobacte r Negative MR Test Positive E. coli Proteus Positive E. coli Serratia Negative Klebsiella Enterobacte r VP Test Negative Klebsiella Enterobacter Positive Klebsiella Enterobacter Indole Test Negative E. coli Proteus Positive E. coli Proteus Negative Klebsiella Enterobacter Oxidase Positive E. coli Proteus Negative Klebsiella Enterobacter Mannitol Positive Klebsiella Enterobacter Esculin Negative Positive Klebsiella Enterobacter Negative Lysine Positive Klebsiella Pneumoniae Positive Klebsiella Enterobacter Negative Positive Klebsiella Enterobacter Positive Enterobacter Serratia Negative Enterobacter ONPG Test Ornithine Negative Sulphur Urease Citrate Negative Klebsiella Pneumoniae Positive Klebsiella Pneumoniae Figure 01: Flowchart of tests performed to identify unknown organism. Negative Serratia Positive Negative Klebsiella Enterobacter Conclusion After performing Gram stains, a gram negative rod was observed and according to the unknown chart, the bacterium was either any Gram negative bacteria may be lactose fermenter (E.coli, Klebsiella, Serratia, Enterobacter) or non-fermenter (Salmonella, Shigella, Pseudomonas). For this reason Lactose fermentation test was performed and observed that the unknown bacteria is lactose fermenter, produced acid in the medium and changes its color to yellow [1]. From lactose test it is concluded that the unknown bacteria was a lactose fermenter bacteria either, E.coli, Serratia, Klebsiella or Enterobacter. For further isolation the next step was to know either bacteria has catalase enzyme or not and bacteria is motile or not. For this reason catalase test and motility test was performed and found that the unknown bacteria was catalase positive but non-motile. From this finding it was concluded that the unknown bacteria is not related to E.coli, Enterococcus and Serratia because they are motile. Furthermore it was found that the given unknown bacteria is MR negative, indole negative and oxidase negative which supports the above conclusion and favours the presence of Klebsiella or Enterobacter specie [1]. It is also found that the unknown sample of bacteria is Indol negative which supports the presence of Klebsiella or Enterobacter specie of bacteria. For further identification different fermentation tests were performed such that Mannitol test, Esculin Hydrolysis, Citrate utilization test, and Sulphur test and all of these tests show positive result except sulphur test. It is concluded from the above findings that the unknown bacteria may be Klebsiella or Enterobacter. After performing Urease test it is concluded that the given unknown bacteria may be Klebsiella because Klebsiella is urease positive whereas Enterobacter is urease negative and the unknown sample shows positive result when performing urease test. Furthermore Lysine test, Ornithine and ONPG tests were performed for the confirmation of Klebsiella pneumonia and found that the unknown sample showed positive result for Lysine test and ONPG test whereas negative result for Ornithine test. It is confirmed from the biochemical testing that the given unknown sample may contain Klebsiella pneumonia bacteria. This was the correct identification of bacterium. Klebsiella pneumoniae Klebsiella pneumoniae belong to the genus Klebsiella and family Enterobacteriaceae. These are rod shaped Gram negative bacteria that can form capsule around them (2). Klebsiella pneumoniae usually live in the normal flora of intestines (3), and remain inoffensive there, But if they blowout to another part of the body, they can cause different infections. Likewise, these bacteria can also become virulent in patients with compromised immune systems (4). Klebsiella pneumoniae generally cause pneumonia and urinary tract infections (5), moreover death is conceivable in patients with compromised immune systems. For the first time in 1982, Carl Friedlander defined Klebsiella pneumoniae as an encapsulated bacillus when he isolated it from the lungs of a patient who had deceased from pneumonia (6). Klebsiella pneumoniae is not an air-borne bacteria, it always transfer by direct contact with patients. Healthcare staff can assist to lessen the blowout of Klebsiella pneumoniae by taking hygiene measures during the taking care of patients. Identification of Unknown Lab Report Unknown Number #88 Staphylococcus epidermidis Introduction There are several reasons for understanding and identifying any microorganisms. In clinical situation, it is important to understand the reason behind a patient’s ailment, and how to treat it by understating the symptoms. The identification of spot-on microorganism can help in precise treatment of a patient and making of certain antibiotics or foods. This study was conducted by applying all the approaches that have been performed so for during laboratory class in order to understand the spot-on identification of any unknown microorganism from an unknown lab sample. Materials and Methods The sample was enriched on different types of Media and to isolate discrete colonies of bacteria. The media includes: 1. Nutrient agar 2. MacConkey agar 3. Mannitol salt agar Following different biochemical tests were perform to identify the bacteria. 1. Catalase 2. Motility 3. Coagulase 4. Bacitracin Susceptibility 5. Novobiocin Susceptibility 6. Optochin Susceptibility 7. Hemolysis 8. CAMP 9. Mannitol 10. Lactose 11. Esculin Hydrolysis 12. Oxidase Results Colony Morphology on Different Medias: Different colony morphology were observed on different culture medium such that small circular pink colonies on Mannitol Salt Agar medium as shown in Table 01. Table 01: Colony Morphology and Gram Staining Characteristics Culture medium Colony morphology on different media Results Nutrient agar Small transparent colonies Bacteria is present Mannitol Salt agar Small Pink Colonies Gram positive bacteria MacConkey agar No Growth No Gram Negative Bacteria is present Biochemical Identification of Bacteria Different biochemical tests like gram staining, catalase, citrate, MR & VP, lactose, Oxidase, mannitol were performed for the identification of bacteria as shown in Table 02 and Flowchart. Test Gram stain Catalase Motility Mannitol Lactose Table 02: Biochemical Test Results PURPOSE REAGENTS OBSERVATIONS To determine the Crystal violet, Violet cocci in Gram Iodine, Alcohol, grape shape reaction of the Safranin clusters bacterium Differentiating A drop of H2O2 on Evolution of O2 Aerobic and obligate colony are observed anaerobic bacteria To determine None No movement flagellated and aflagellated bacteria To differentiate Phenol red mannitol fermenting mannitol broth No Color changes bacteria medium To differentiate Phenol red lactose Color changes to lactose fermenting broth medium yellow bacteria RESULTS Gram Positive cocci Positive Negative Negative Acid/Acid Esculin Hydrolysis Oxidase Coagulase Hemolysis CAMP Bacitracin Susceptibility Novobiocin Susceptibility Optochin Susceptibility To differentiate enterococci and nonenterococcus group D streptococci To differentiate enterobacteriaceae to other genera To differentiate staphylococcus aureus from other staphylococcus To determine what hemolysin enzyme bacteria possess Esculin and bile salt in nutrient agar medium Color changes to dark brown Negative Tetramethyl-pNo color phenylenediamine formation Negative Coagulase reagent No clot was (Rabbit Plasma) formed Negative 5% sheep blood Beta-lysin To identify Group B producing S. Streptococci aureus To differentiate Beta hemolytic group A Bacitracin disk Streptococcus from other Stretococcus To differentiate Novobiocin disks coagulase negative Staphylococcus To differentiate S. pneumonae from Optochin disks other alpha hemolytic viridians No notable zones Gamma around the colony No enhanced hemolysis Negative No zone of inhibition Resistant Zone of inhibition Susceptible No zone of inhibition Resistance Figure 1: Gram staining of bacteria Figure 2: Hemolysis of bacteria Figure 4: Coagulase Test Figure 3: Novobiocin susceptibility test Identification through Biochemical Test Gram Staining Violet cocci Gram+ Bacteria Lactose test Negative Streptococcus specie Positive Staphylococcus specie Motility Catalase Positive Staphylococcus Negative Enterococcus Streptococcus Positive Negative Staphylococcus Streptococcus Enterococcus Coagulase Positive Staphylococcus aureus Negative Staphylococcus epidermidis Staphylococcus saprrophyticus Hemolysis Test Gamma Hemolysis S. epidermidis Resistance Staphylococcus saprophyticus Novobiotin Susceptibility Test Sensitive Staphylococcus Epidermidis Figure 01: Flowchart of tests performed to identify unknown organism. Conclusion After performing Gram stains, a gram positive cocci was observed and according to the unknown chart, the bacterium was either any Gram postive bacteria may be lactose fermenter (Staphylococcus) or non-fermenter (Streptococcus, Enterococcus) [1]. For thisreason Lactose fermentation test was performed and observed that the unknown bacteria is lactose fermenter, produced acid in the medium and changes its color to yellow[2]. From lactose test it is concluded that the unknown bacteria was a lactose fermenter bacteria either any specie of Staphylococcus. For further isolation the next step was to know either bacteria has catalase enzyme or not and bacteria is motile or not. For this reason catalase test and motility test was performed and found that the unknown bacteria was catalase positive but non-motile. From this finding it was concluded that the unknown bacteria is not related to Streptococcus and Enterococcus species because they do not have catalase enzyme [2]. Furthermore it was found that the given unknown bacteria is CAMP negative, Esculin negative, Mannitol and oxidase negative which supports the above conclusion and favors the presence of Staphylococcus specie. It is also found that the unknown sample of bacteria is Coagulase negative which supports the presence of Staphylococcus specie of bacteria and from this test it is concluded that the unknown bacteria is not Staphylococcus aureus because staphylococcus aureus is coagulase positive, the unknown bacteria is either Staphylococcus epidermidis or Staphylococcus saprohyticus [1]. After performing Hemolysis test it is concluded that the given unknown bacteria showed Gamma (?) Hemolysis and affirms the presence of S. epidermidis. Furthermore the unknown organism showed sensitivity against Novobiocin susceptibility test which confirms the presence of Staphylococcus epidermidis, and rejects the presence of Staphylococcus saprophyticus because S. saprophyticus is resistance against Novobiocin. It is confirmed from the biochemical testing that the given unknown sample contain Staphylococcus epidermidis bacteria. This was the correct identification of bacterium. Staphylococcus epidermidis Staphylococcus epidermidis belongs to the genus Staphylococcus. It can be present within mucous membranes as well as a part of skin microflora (3). Due to contamination, it has become a common microorganism in laboratory tests. Habitually, Staphylococcus epidermidis does not have antagonistic virulence factors (4), but it may cause infection to a person with compromised immune system. Infections caused by Staphylococcus epidermidis can be both community acquired and nosocomial, but nosocomial infections are more threatened. In 1884, Friedrich J. Rosenbach distinguished Staphylococcus epidermidis from Staphylococcus Aureus (5). Staphylococcus epidermidis is a Gram+ bacteria having a cocci shape, organized in grape-like clusters. Staphylococcus epidermidis can form biofilms on plastic devices (6), triggering an important virulent factor. The biofilm that propagates on plastic devices commonly occurs on medical prostheses and catheters (7). These biofilms produced by Staphylococcus epidermidis may support the antibiotics resistance including methicillin, penicillin, and amoxicillin. References 1. Hmeraj V, Diksha S and Avneet G (2013). A review on commonly used biochemical test for bacteria. Innovare Journal of life sciences, 1(1): 01-07 2. McDonald, Thoele, Salsgiver, Gero (Lab Manual for Gen. Microbiology STLCC Meramec). 3. Nguyen, T. H., Park, M. D., & Otto, M. (2017). Host response to Staphylococcus epidermidis colonization and infections. Frontiers in cellular and infection microbiology, 7, 90. 4. Otto, M. (2009). Staphylococcus epidermidis—the 'accidental' pathogen. Nature reviews microbiology, 7(8), 555-567. 5. Licitra, G. (2013). Etymologia: Staphylococcus. Emerging infectious diseases, 19(9), 1553. 6. Knobloch, J. K. M., Bartscht, K., Sabottke, A., Rohde, H., Feucht, H. H., & Mack, D. (2001). Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of thesigB operon: differential activation mechanisms due to ethanol and salt stress. Journal of bacteriology, 183(8), 2624-2633. 7. Pascual, A. (2002). Pathogenesis of catheter-related infections: lessons for new designs. Clinical microbiology and infection, 8(5), 256-264. 8. Ashurst, J. V., & Dawson, A. (2019). Klebsiella Pneumonia. In StatPearls [Internet]. StatPearls Publishing. 9. KLIPSTEIN, F. A., HOLDEMAN, L. V., CORCINO, J. J., & Moore, W. E. C. (1973). Enterotoxigenic intestinal bacteria in tropical sprue. Annals of Internal Medicine, 79(5), 632-641. 10. Fang, C. T., Lai, S. Y., Yi, W. C., Hsueh, P. R., Liu, K. L., & Chang, S. C. (2007). Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clinical infectious diseases, 45(3), 284-293. 11. Caneiras, C., Lito, L., Melo-Cristino, J., & Duarte, A. (2019). Community-and hospital-acquired Klebsiella pneumoniae urinary tract infections in portugal: virulence and antibiotic resistance. Microorganisms, 7(5), 138. 12. Vaidya, M. Y. (2019). Antimicrobial efficacies for metal ions and graphenebased compounds against Klebsiella pneumoniae, Acinetobacter baumannii and Enterococcus faecium in the absence and presence of conditioning films (Doctoral dissertation, Manchester Metropolitan University).