The identification of unknown bacterial cultures is one of the major responsibilities of a microbiologist. Samples of blood, tissue, food, water and cosmetics are examined daily in laboratories around the world for the presence of contaminants. Industrial and medical microbiologists are constantly screening materials for new isolates that may produce useful products. Whatever the purpose, an unknown bacterium once isolated must be identified and classified.Bergey’s Manual of Determinative Bacteriology is the official, and internationally accepted reference for bacterial classification since 1923. In Bergey’s Manual, related bacteria are arranged into groups or sections. The interrelationship of the bacteria in each section is based on characteristics such as morphology, staining reactions, nutrition, culture characteristics, and biochemical tests. This manual is an important tool to aid microbiologists in their identification of unknown bacterial cultures.The purpose of this Unknown Project is to allow you to apply the laboratory techniques you have learned and use them to characterize and identify an unknown bacterial isolate. You will be assessed for concise documentation of lab work in a “lab journal”, interpretation of results and logical justification for the ‘path’ that is taken in your project to identify the unknown. Several copies of Bergey’s Manual are available in the micro lab for use in the lab only. Bergey’s Manual will be the information source for creating dichotomous keys for the elimination process to identify your unknown.How to get started:You will be given an Unknown bacterial specimen on a NA slant. Using this original slant as your “working slant”, you will inoculate an “insurance NA slant” and you will prepare a smear for Gram Staining (I recommend making 2 smears). Also include known Gram positive and negative controls on slides too. Store original unknown slant in the 4oC refrigerator. Incubate insurance slant at 25oC for 48 hours.Gram stain your heat fixed smears of the unknown and the controls.Your working slant will be used for day-to-day experiments. Keep track of the age and organism density on the working slant and inoculate a fresh slant as needed.All insurance slants and working slants are kept in the refrigerator when not in use.From your working slant, perform an aerotolerance test using agar shakes.Cell shape and arrangement can be verified with a negative stain. Aerotolerance can be further verified with an Anaerobic Jar test. (FOR THIS PART I ALREADY DID IT AND THE RESULT WAS GRAM POSITIVE ROD AND UNKNOWN WAS OBLIGATE AEROBE OR STRICT AEROBE SO AM ON GROUP 18 ON THE BERGEY’S MANUAL).Once you have the results for Gram stain reaction, cell shape and aerotolerance, you will consult Bergey’s Manual to decide which tests need to be done. When performing specific biochemical testing, a control should always be included to validate your results.Make your own dichotomous flowchart as you systematically compile and analyze data.Careful planning with clear reasoning for the direction of your ‘identity search’ is important to not only identifying your unknown, but also for your final grade! Avoid doing unnecessary or repetitive tests.Part ILab Notebook – virtual notebook (template for you to follow is in Environmental Unknown module. So the days for lab is on Monday and Wednesday and we started on February 2, 2022).All entries into your lab notebook should be recorded EACH TIME you work on your project in the Micro lab. Your lab book is your “scientific blog” and can be documented with a virtual lab notebook and then within Canvas. It should be organized, and complete from the start of the project. You can bring your own laptop or tablet to record your work, or record your work on paper and then transfer data into your virtual notebook once you get home. I will be periodically inspecting lab books to ensure that entries are made properly and that your project is on the right track.Your notebook should start with a Table of Contents that should include the date, descriptive title, and page numbersEvery lab book entry should have the following sections:DATE and descriptive TITLE of experimentPURPOSE of the experiment (e.g. to confirm the motility of my unknown using a Hang Drop).MATERIALS and PROCEDURE – “cite specific pages in Leboffe lab manual” or include procedure from handouts (do not just refer to handouts online). Note specific controls used or any modifications to the written procedure. If using the digital lab manual, there are no page numbers so the name and exercise number of the exercise is sufficient.DATA/OBSERVATIONS – detailed description of test results (and controls) and should include drawings/photo of cultures, biochemical tests or microscopic observations where applicable; e.g. “pink colored rod shaped bacteria observed in Gram Stain”, (drawings or photos should include magnification used)INTERPRETATION/ANALYSIS OF RESULTS – should discuss what the results tell you about the organism and where your identity search is headed. e.g. “Gram Stain results indicate a Gram-negative bacilli, unknown is likely in group 4 or 5. Next test should examine aerotolerance because… All Gram positive organisms were eliminated”NEXT STEPS – e.g. “unknown was inoculated into NB, Motility Stab, and streak plated onto MacConkeys”Each entry should be as detailed and complete as possible and indicate the direction of the project: what do you know so far, and what is the next test to do!You may find it useful to “sum up” your findings within your lab book as you compile and analyze results. This may involve drawing portions of your dichotomous tree, listing bacteria eliminated and those still considered. This will help me to understand why you choose the next step. Careful planning with clear reasoning for the direction of your ‘identity search’ is important.At the end of the project, your lab notebook should explain and then identify your Unknown based on your lab results. This lab ID will then be compared to the confirmatory BLAST ID.Part IIFormal Summary of Biochemical Analysis of Unknown Written summary analyzing results and explaining why particular lab tests were chosen to show the process used to identify your unknown. Explain how you arrived at a Bergey’s Grouping, then a Genus then a species as opposed to explaining results in chronological order. Discuss the test results used to decide why certain genera were being considered in your determination and why particular genera could be eliminated at each stage of the project. Discuss whether test results were reliable or not and troubleshoot any conflicting data. End with the final Unknown identification as determined by your lab tests.Summary Table of all FINAL test results. This should include cell shape and arrangement, staining characteristics, biochemical test results and growth characteristics on special media as well as BLAST analysis. Dichotomous tree/flowchart (can be hand-drawn and scanned or photographed) showing YOUR path for eliminating various organisms. List all groups, genera, or species that are eliminated. Molecular Analysis of UnknownState the name of the organism identified by BLAST search of the nucleic acid sequence given to you. Indicate what nucleic acid sequence is being compared and on what database. Include a screenshot of the BLAST results of the top 10-15 organisms with the highest % identity and highlight YOUR ORGANISM. Compare your Lab ID and the BLAST ID. Do they match? (Assume BLAST ID is correct) If they do not match, take the lab data and analyze which test(s) lead you down the wrong path. Do the other lab results match up with your correct organism?Brief description of your correct Unknown determined by BLAST (cell structure, metabolism, significance, ecology, pathology, etc).
