Identification of Two Unknown Bacteria in Mixed CultureA standard part of nearly all lab courses in introductory microbiology is an activity wherein the student must use everything that has been learned in the course to identify an unknown bacterial culture. Your ability to make aseptic transfers, perform the gram stain, identify cell morphology and shape, and conduct metabolic testing is under examination in this process. Your ability to follow the stepwise logic of a dichotomous key will also be tested. Overall, your strategy is to be parsimonious; you should only do needful tests according to your key and should only need to do them once. This Exercise and Report are worth 50 points towards your final grade.These instructions replace those in Exercise 25, but we will be using the worksheets attached to Exercise 25 in the lab manual for writing up the report. An example of the completed lab report using the worksheet pages is attached to this guide to help guide you in recording your data completely and writing up your final report correctly.Making a Dichotomous KeyThe first thing to prepare for an Unknown Identification exercise is to make a dichotomous key. Dichotomous keys are written from known characteristics of the possible organisms you may need to identify. The following six organisms will be used in this activity; you will be given a mixture of two of them together. Characteristics that you can use in making a dichotomous key are listed in the table. Abbreviations: A = Acid, G = gas, P = pink, W = white, +/- = Variable, - = No Growth, - = No Growth. Table I. Morphological, Staining, and Metabolic Characteristics of Six Bacteria Used in This ExerciseOrganism Gram Stain, ShapeCulturecharacteristics onagar slantMannitolfermentationSalt tolerantHemolysisGlucose ferm.Sucrose ferm.Lactose ferm.Indole production Motility UreaCitrate utilz. MR/VPOxidase CatalaseStaphylococcus aureus +CoccusAbundant, opaque,golden growth + ++/A - A - - - +-/+- +Staphylococcus epidermidis +CoccusOff-white, smooth,small, round - +-/A - A - - - - +/- - +Escherichia coli -BacillusWhite, moist,glistening growth + -+/AG AGAG + + - - +/- - +Klebsiella pneumoniae -BacillusTranslucent-creamy, mucoid,round + -+/AG AGAG - - - - +/- - +Pseudomonas aeruginosa-Bacillus Abundant, thin,white growth withmedium turning- -+/- - - - - - --/- ++ Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 1greenEnterobacter aerogenes-BacillusAbundant, thick,white, glisteninggrowth - -+/AG A A + - + - -/+ - + Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 2Dichotomous keys are charts that require decisions at branch points, much like a flow chart in computer logic: If the answer to a question is yes, then do X; if the answer is no, then do Y.Your job is to make the shortest key, with the shortest pathways, to a final identification of an organism. Ideally, any pathway in your key should have no more than four steps (including gram staining and cell morphology as separate steps) leading to an identification.To make a dichotomous key, you first need to sort all the different species in your list into two big groups based on common characteristics between them. Most microbiologists begin with gram stain results, and I encourage you to do the same. Using the Example Dichotomous Key in this Guide, you will see that you should begin your own key by writing out the names of all the possible bacteria in the test near the top of the page. I used 8 bacterial names in my Example Key; you should use the 6 organism names from Table I. I recommend using landscape (sideways) mode in laying out a key since the wider the page, the better.After listing all 6 potential species on your key sheet, you should next draw two arrows downwards that show this group being separated by the gram stain. Write gram positive at the end of one arrow and gram negative after the other. Follow each result with a list of bacteria still remaining in each group. In my Example Key, I was able to divide my list of 8 bacteria into two groups of four.The next dichotomy to further separate the gram positives from each other, and the gram negatives from each other, should be one of salt tolerance, lactose fermentation, or cell morphology (bacilli or cocci) since you will get results of these tests from the selective media (MacConkey and mannitol salt agar) you will use early in the exercise. The important thing is that you are subdividing each group at every dichotomy into two subgroups of nearly equal size. If you separate off only one species at a time at each dichotomy, your key will be far too long and will not be parsimonious. In my example key, I decided to separate the gram negatives using galactose fermentation. I decided on this test after discovering that the four gram negative bacteria differed in their fermentation reactions to galactose. Half fermented the sugar and half did not. That's the strategy you should employ at each step in the key: find a characteristic that differsin the group of organisms you need to separate. Ideally this characteristic or test divides them into roughly equal subgroups. You continue finding tests that subdivide those groups until you only haveone organism left per group. You will need a completed dichotomous key by the first period of the Unknown ID activity. The instructor will not let you progress further unless you can demonstrate that you have a completed and correct dichotomous key to work from. Ultimately you will handwrite or type a copy of your dichotomous key and (if typed) paste it onto the blank page of your lab manual (pg. 210). Try, if you can, to get it all on one page. Use the Example Key on the next pages an example. Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 3Here's the list of 8 bacteria and their characteristics that I used in making my Example Dichotomous Key of Eight Bacteria on the following page. You are not using these bacteria in Ex 25; these are just for demonstration purposes.Table II. Characteristics of 8 Example Bacteria Used to Make a Dichotomous KeyOrganism CharacteristicsAlcaligenes faecalisGram negative bacillus, acid-fast negative, ferments galactose, catalase positive, motile, alkaline with litmus milk, not hemolytic, H2S negativeCitrobacter freundiiGram negative bacillus; acid-fast negative, does not