Microbiology unknown project #40
Write a journal about the identification process. I will add an attached file with all details and requirements. I will also add all results and tests I did.
Picture #1 – determine if gram-positive or negative = I said negative
Picture #2 – results from Gelatin Hydrolysis tests (top tube results) = I said negative
Picture #3- Methyl Red test = I said negative
Picture #4 Nitrate Reduction test ( after addition of zinc) = I said negative
From what I understood, my result was Pseudomonas fluorescens.
I also included an example report.
Microbiology unknown project #40 Write a journal about the identification process. I will add an attached file with all details and requirements. I will also add all results and tests I did. Picture #
BIOL& 260 Microbiology Online 1 Identification o f an Unknown Bacteri um – Online version (75 point lab project) I. Introduction. i. The identification of unknown bacteria is a time -honored part of most microbiology courses. It will challenge your knowledge and skills in understanding laboratory techniques, your ability to critically evaluate the information obtained from these techniques, and your ability to effectively communicate this information. II. Rationale. i. Upon completion of this project, the successful student will demonstrat e: a. The ability to apply basic scientific principles in solving real and hypothetical microbiological problems using the scientific method; b. An ability to effectively communicate complex scientific information; c. Effective personal time management, organization, and study skills. ii. The specific learning objectives involve mastery of techniques and concepts presented throughout the course. Although students in online classes will not actually be performing this project in the microbiology la b, students will demonstrate an understanding of the following techniques: a. Perform ing aseptic transfer technique s; b. Observing plate media that has been streaked for isolation and describing the colonies produced; c. Observing Gram stains and correctly interpret ing gram reactions; d. Effective use of general purpose, selective, differential, and other types of culture media ; e. Select ing only the most significant media and reagents for characterization of the biochemical activities of the unknown bacterial species by applying dichotomous logic; f. Collect ion and evaluat ion of data in a logical manner. g. Present ation of work in a detailed daily lab journal , demonstrating skilled observation with information presented logically, orderly, and neatly. III. Project Format. i. Species identification: a. Each student will receiv e a virtual broth stock culture containing a single unknown bacterial organism , and be required to perform a Gram stain as their first test . b. Each student will run a series of tests to determine the identity of the organism in the culture. 1. Descriptions of the available tests/media will be provided on Canvas . 2. The sequence of tests shou ld display DICHOTOMOUS logic. See Figure 1 below. a. Dichotomous logic involves a series of yes/no or true/false questions. b. Only one test may be run at a time, and the outcome of that test will not be revealed until the appropriate incubation period has passed. Incubation periods are specified in the test instructions/ media descriptions provided on Canvas. i. Each student is responsible for keeping track of the incubation period for each tes t they have chosen and for contacting the instructor at the appropriate time to receive information concerning the outcome of that test. c. SHOTGUN logic (running more than one test at a time) should not be used and will result in substantial points loss es. IV. Daily Lab Journal . i. You are required to keep daily notes of your activities, observations, thoughts, etc ., for every virtual lab test you run . These notes will serve as the basis for the Unknowns Report you will turn in when the project is complete . ii. NOTE: it is important to document all of your activities, whether they give good or poor results. This is critical for when you write your Unknowns Report, as you cannot trust your memory to try and recall what you did during the project. BIOL& 260 Microbiology Online 2 V. Unknowns Report – 75 points. i. You must submit a report that includes your thoughts and activities for each day of virtual unknowns work. a. With the exception of the microorganism paragraph (see h. below), the report should be written using bullet points, and NOT in an essay styl e (an exception is your “microorganism paragraph” – see below) . b. Use 12 point font and single line spac ing and 1” mar gins. c. MINIMUM r eport length: 2.5 pages . Your page count CAN include Tables and bibliography, but CANNOT include pictures. d. Each report entry must begin with the date of each day’s virtual unknowns work. e. Your entries should include the tests you run, the information you hope d to gain from th ose tests, the observations you made from tests inoculated on the previous day , etc . f. You do NOT have to describe the step -by -step details of each test you run. For example, if you run a Gram stain it is unnecessary to describe each step of the process ; instead, just say that you made a Gram stain of your culture . g. The thoughts and words in your report should be YOUR OWN. h. It is MANDATORY that your report include a table (see Table 1 below) summarizing the process you used to identify your unknown organism. i. Microorganism paragraph. A description of your microorganism including diseases, modes of transmission, common sources/places it is f ound, treatments, and any other interesting information will be given at the end of your report after you have determined the identity of your microorganism. 1. This part of the report only needs to be a paragraph or two in length; do not exceed one typed page for this section. 2. All background literature/sources you use for the microorganism paragraph should be cited. You must give credit to the source of any piec e of information that is not your own original thought. a. Use APA citation format for IN -TEXT CITATIONS and for LITERATURE CITED . This Clark library Class Guide has the required information: http://libraryguides.library.clark.edu/biol260 -anitori b. Plagiarism will result in a score of zero for the project. j. Your finished report must be submitted on Canvas by the due date. Late submissions will be penalized at 20% per day. The file must be in either Word (.doc, .docx) or PDF (.pdf) format. Use this as a file name: Your last name_ Unknown . For example: Anitori _Unknown . VI. Plagiarism I wish to remind you that I take plagiarism very seriously, as does the college. The following definition of plagiarism is from the section entitled “Academic Dishonesty” , found at: http:// www.clark.edu/clark -and -community/about/policies -procedures/student_code.php “Plagiarism includes taking and using as one’s own, without proper attribution, the ideas, writings, or work of another person in completing an academic assignment.” ** If plagi arism is identified, I will subtract 20 % at a minimum . The more egregious the plagiarism, the greater will be the points subtracted, up to a maximum of 75 points.** Be aware that repeated instances of plagiarism by a student can be punished more severely, including loss of all points in an assignment up to (the most severe) expulsion from the college. VII. Grading Criteria. Listed below are the grading criteria, starting with the most important and ending with the least important. 1. Logic of tests performed — only the most relevant tests, using a dichotomous approach, should be BIOL& 260 Microbiology Online 3 performed. A shotgun approach will score poorly. a. Running tests to confirm the results of earlier tests should only be performed under certain circumstances, for example, if there was a procedural error. If you DO repeat a test, you must be able to adequately describe your rationale for repeating the test in your Final Report. If not, points will be deducted. 2. Completeness, organization, clarity, and accuracy of the daily lab entries are very important. a. All information must be typed in your own words. b. Each entry must be dated. c. What tests were run? Why? What is the incubation period of the selected test? What were the possible outcomes? What were the actual results of the tests? What conclusions did you draw from them? What organisms were eliminated by performing each test? d. Effe ctiveness of written communication. i. Your report should be presented chronologically and be free of grammatical and spelling errors. e. Your report should be concise, easy to read, and easy to understand. f. Successful identification of your unknown organism is worth a small percentage of the grade. i. It is much more important to show logical thinking than it is to correctly identify your organism . ii. Make sure to include a microorganism paragraph (discussed above). EXAMPLE TABLE 1. * This table is mandatory and worth 4% of your report * This table is NOT the s ame thing as the table(s) shown in the “Sample Report ” by Dr Kibot a Table 1: An Example Table for Presenting Test Outcomes Obtained During the Unknowns Project. TEST: Gram stain Catalase PR -mannitol PR -glucose Citrate YOUR RESULT: G+ Coccus Positive Negative Acid Positive SPECIES: E. faecalis M. luteus M. luteus M. luteus M. varians M. varians M. varians M. varians M. varians S. aureus S. aureus S. epidermidis S. epidermidis S. epidermidis S. epidermidis S. agalactiae S. bovis S. mitis S. pneumoniae S. pyogenes ** NOTE: for each species, keep it in the same row throughout the table. ** BIOL& 260 Microbiology Online 4 Figure 1. Example of a Dichotomous Process. ** NOTE – you DO NOT need to submit answers to these questions. They are for your own benefit. ** Refer to Figure 1 (above) to answer the following hypothetical questions by using dichotomous logic. 1) If your organism is a bacillus shaped bacterium , would the PR Glucose test be a logical test to run? Why or why not? 2) If your organism is a coccus shaped bacterium, would the PR Glucose test be a logical test to run? Why or why not? 3) If your organism is a coccus shaped bacterium that is negative for lactose fermentation, what test should you run to determine the identity of your organism? If you do not understand this process (dichotomous logic) discuss it with your lab instructor as soon as possible! A B C D E F G Species Cell Morphology PR Sucrose + – + – – – – PR Lactose + – – – + + – PR Glucose + + + – + + +
Microbiology unknown project #40 Write a journal about the identification process. I will add an attached file with all details and requirements. I will also add all results and tests I did. Picture #
Please list your Unknowns culture clearly on your cover page. Identification of Unknown #6 by Travis Kibota Biology 240 Winter 2004 This is a journal of activities that I performed on one of the Unknowns cultures during Winter 2004. I identified BOTH organisms in mixed culture #6. You report will contain your work on identifying ONE organism. Note that this example report is not complete. Biol 240 Winter 2004 Unknowns ID Kibota 2 Journal: Unknown #6 Notice that entries are labeled with the Day 1 (February 3, 2004) Day of observation AND the date. · Streaked 2 TSA and 2 EMB plates (there were no Azide or MacConkey plates). · Incubated one TSA/one MacConkey at 30°C, other set at 37°C. Day 2 (February 4, 2004) Observations · TSA (37°C) –see Figure 1. On Day 2, I described my observations of colony characteristics, in detail, for each of the cultures. o Colony A was ~ 1 mm diameter, entire margin, slightly raised and convex, unpigmented, somewhat translucent. o Colony B was ~1 mm diameter, entire margin, slightly raised and convex, colored slightly off-white or yellow, more opaque than colony A. Colony Colony B Photos are helpful but not required. Figure 1. Unknown 6 on TSA incubated at 37°C. · TSA (37°C) o Colony A was very similar to 37°C plate. o Colony B was similar to 30°C plate except colonies were only 1/2 to 2/3 the size. · EMB (30°C and 37°C) o The colonies were pinpoint. Because of the small size, I couldn’t reliably determine other characteristics. Activities · I picked cells from the colony labeled “A” and streaked onto a TSA plate. I picked cells from the colony labeled “B” and streaked a second TSA plate. I did this to make sure that my cultures are pure. I incubated both plates at 37°C. · Because my Day 1 plates have only been incubated for 24 hours, I am re-incubating them to encourage the colonies to grow to larger size. Continuing on Day 2, I described activities I performed AND why I performed them. Biol 240 Winter 2004 Unknowns ID Kibota 3 Day 3 (February 5, 2004) Observations · The TSA Plates from Day 1 (both 30°C and 37°C) look very similar to their appearances on Day 2. The only difference is that Colony B is now ~2-3 mm in diameter. Colony A and B are definitely different colors. Colony A is white (unpigmented). Colony B is more yellowish and may be pigmented (although I’m not sure about this). · The EMB Plates from Day 1 have grown a little more but the colonies are still small (> 1mm). They appear light colored indicating no lactose fermentation. However, their small size makes this determination difficult. · The Day 2 TSA Isolation Plates have good growth. Both the Colony A and Colony B plates look homogenous. The colonies look like they do on the Day 1 TSA plates. I am confident I have good isolation (Figure 2A, B). A B Figure 2. Day 2 TSA plate with isolated cells from (A) colony A and (B) colony B. Activities · I will select cells from one colony on my Colony A plate. I will use these cells to inoculate two TSA slant cultures: one slant will become my working stock, the other will be my reserve. · I will select cells from one colony on my Colony B plate. I will use these cells to inoculate two TSA slant cultures: one slant will become my working stock, the other will be my reserve. · I will incubate all four TSA slant cultures at 37°C. Day 4 (February 6, 2004) Observations · Colony A Stock and Reserve slants had good homogenous translucent white growth. · Colony B Stock and Reserve slants had good homogenous opaque yellow growth (Figure 3). Figure 3. Stock cultures of Colony A (left) and Colony B (right). Biol 240 Winter 2004 Unknowns ID Kibota 4 Activities · Performed Gram-stain for both Colony A and Colony B (using cells from stocks). Both appear to be Gram-positive cocci (Figure 4A, B). · Refrigerated stock and reserve colonies. A B Figure 4. Gram-stains of (A) Colony A and (B) Colony B. Both appear to be Gram-positive cocci. Day 5 (February 9, 2004) Observations · None. Activities · Given that both colonies appear to be Gram-positive cocci, I ran catalase tests on both cultures. I did this by putting a drop of hydrogen peroxide on a slide (one drop per culture) and mixing cells into the drop. I ran these tests twice for both cultures so I am fairly confident of the results. Conclusions · Colony A is catalase NEGATIVE. Thus, I will tentatively conclude that Colony A is either Streptococcus or Enterococcus. · Colony B is catalase POSITIVE. Thus, I will tentatively conclude that Colony B is either Staphylococcus or Micrococcus. At this point, I have begun to narrow down the possibilities. Notice that I listed the possible organisms. Here I listed only the genus names. Day 6 (February 10, 2004) This indicates that I can have any of the species Observations within those genera. · None. Activities · Because the catalase result is so critical to my next steps, I want better confirmation. On a TSA plate, I heavily inoculated Colony A on the left side and Colony B on the right Biol 240 Winter 2004 Unknowns ID Kibota 5 side. After incubation, I will put drops of hydrogen peroxide on each area of growth. This will provide a much more sensitive test for catalase ability. · I streaked Colony A and Colony B on blood agar plates to test for hemolytic ability. · I would like to test Colony B on 7% NaCl, but no plates are available. Day 7 (February 11, 2004) Observations · TSA had heavy growth of both Colony A and Colony B (Figure 5). This plate shows the difference in pigmentation. I am now confident that Colony A is unpigmented and Colony B produces a yellow, water-insoluble pigment. Hydrogen peroxide with no bubbles Large, vigorous bubbles Figure 5. This shows a TSA plate that has heavy growth of Colony A and Colony B. Hydrogen peroxide was dropped on both regions of growth. No bubbles appeared in the Colony A region. Vigorous bubbling appeared in the Colony B region. · Colony A showed no hemolysis on blood agar. · Colony B showed complete (!) hemolysis on blood agar (Figure 6). A B Figure 6. Blood agar plates. (A) Colony A formed small white colonies that show no hemolysis. (B) Colony B formed small white colonies that show complete (!) hemolysis. Biol 240 Winter 2004 Unknowns ID Kibota 6 Summary of observations Characteristic Gram-Reaction Cell Shape Pigment Catalase Hemolysis Possible Species Here I present a table of characteristics and possible organisms based on these characteristics. Notice that I’ve now begun to list individual species (instead of just the genus names) because I’m narrowing down the possibilities. Colony A Gram-positive Coccus Negative Negative Negative Streptococcus pneumoniae Streptococcus mitis Streptococcus bovis Enterococcus faecalis !-hemolyzers (tentatively ruled out) Streptococcus pyogenes Streptococcus agalactiae Colony B Gram-positive Coccus Yellow Positive Beta Staphylococcus aureus Micrococcus luteus is pigmented yellow but not beta-hemolytic. Staphylococcus epidermidis in unpigmented and not beta-hemolytic. Activities · For Colony A, I inoculated a Bile Esculin slant. This will separate E. faecalis/S. bovis from S. mitis/S. pneumoniae. · For Colony B, I am fairly confident that it is Staphylococcus aureus. I inoculated a mannitol broth. If positive for mannitol fermentation, this will add to my confidence. When 7% NaCl becomes available, I will test this (to defintively rule out Micrococcus). Day 8 (February 12, 2004) Observations · Colony A Bile Esculin: The culture turned black from top to bottom. Bile esculin is considered positive if more than half the culture is black, so Colony A is definitely positive (Figure 7). · Colony B Mannitol Broth: The broth turned bright yellow with no gas bubble. Thus, Colony B is an acid-former in Mannitol (Figure 8). Summary of Observations Characteristic Colony A Colony B Gram-Reaction Gram-positive Gram-positive Cell Shape Coccus Coccus Pigment Negative Yellow Catalase Negative Positive Hemolysis Negative Beta Bile Esculin Positive — Mannitol — Positive Possible Species Streptococcus bovis Enterococcus faecalis Staphylococcus aureus Biol 240 Winter 2004 Unknowns ID Kibota 7 Figure 7. Bile Esculin Test. Control (left) and Colony A (right). Note the dark coloration of the Colony A tube relative to the control. Figure 8. Mannitol Broth. Control (left) and Colony B (right). Colony B shows acid production (yellow) but no gas bubble. Conclusions/Activities · Colony A is narrowed down to Streptococcus bovis or Enterococcus faecalis. To differentiate between these two species, I inoculated a 6.5% NaCl Broth—E. faecalis should be positive for growth, S. bovis should be negative. · I am fairly confident that Colony B is Staphylococcus aureus. However, Micrococcus luteus is currently only ruled out by hemolytic ability. To increase confidence, I inoculated a 7% NaCl plate—S. aureus should be positive for growth, M. luteus should not grow. I didn’t finish these identifications. If I did, I would draw final conclusions about my organisms. I would present these conclusions in a separate section that would clearly list: the Unknowns Number and the organisms that I think I have. Following this conclusion section, I would provide a brief (one paragraph) write up about my organisms. This write-up would be based on literature research about my organisms. I would include literature citations for this research.