Page 1 of 4BIOL 3702L: Microbiology LaboratorySkills Test: Bacterial UnknownThis skills test will examine your ability to i) isolate two different bacterial from a mixedculture and ii) identify each bacterium using pertinent diagnostic characteristics. Youhave already performed or are presenting performing these diagnostic tests. Therefore,you should be familiar with how they are conducted.To assist you, expected diagnostic test results have been provided in several tables inthe web page document entitled “Diagnostic Features of Selected Bacteria” (available atthe following URL - http://www.as.ysu.edu/~crcooper/DFSB.pdf). Note that not everytest and every test result is listed for each bacterium. The reason is simple. Some ofthese tests are irrelevant and others are not routinely used to identify certain microbes.In addition, the idea of this exercise is to make you think – not to give away theanswers!You will receive a mixed culture unknown containing two different bacteria. They can beof any combination from among those bacteria listed on the attached sheet entitled“Diagnostic Features of Selected Bacteria”. You are to follow the initial instructions asdescribed below to begin this skills test. In addition, you are to keep a log of youractivities and observations in this test. Use the “Skills Test Log Sheet” which availableat the following URL - http://www.as.ysu.edu/~crcooper/STLS.pdf. The third page of thelog sheets may be photocopied as needed. Furthermore, you will need to submit by thestated deadline (as listed in the laboratory syllabus or as provided by your laboratoryinstructor) the “Mixed Culture Unknown Answer Sheet and Flow Chart” (available at thefollowing URL - http://www.as.ysu.edu/~crcooper/UASFC.pdf). Submit both of theseforms stapled together. Finally, students may assist one another so far as providingadvice and encouragement. Any collaboration beyond this level this stands precariouslyat the precipice of academic dishonesty. In short, each individual is to perform his/herown work with regard to this test.To begin, read the following instructions carefully. Begin working immediately. The“Mixed Culture Unknown Answer Sheet and Flow Chart” and the “Skills Test Log Sheet”must be submitted as a stapled unit to your laboratory instructor by the stated deadline.If any of the items is not submitted by the deadline, a 25% penalty will be applied. If anyitem is submitted more than 24 hours late, but less than 48 hours late, a 50% penaltywill be applied. If any item is submitted more than 48 hours late, then the student willreceive a 100% penalty, i.e., a score of “0” will be recorded.Day 11. The course/laboratory instructor will provide a rack of mixed culture unknownsthat have been randomly labeled with a code number. Select one tube andimmediately record the number of that unknown on the report sheet that will alsobe provided by your instructor.BIOL 3702: Bacterial Unknown Skills TestPage 2 of 42. Obtain one (1) of each of the following agar plate media:= MacConkey’s Agar (MAC)= Mannitol Salts Agar (MSA)= Tryptic Soy Agar (TSA) [Nutrient Agar (NA) may be substituted for TSA]3. Using your very best technique, separately streak a loopful of the mixed cultureunknown onto the MAC, MSA, and TSA plates. Incubate these plates at 35°C-37°C overnight.4. [OPTIONAL] Prepare a heat-fixed smear from your mixed culture unknown on aglass slide. Gram stain your smear. Observe and record your results. Theseobservations may give you an idea of what to expect upon isolating yourunknown bacteria. Note, however, it is conceivable that you may isolate twobacteria having the same Gram-stain reaction and morphology, in which case itmay be difficult to distinguish between the different isolates.Day 24. Observe your streak plates for isolated colonies and record your results.NOTE 1: MAC medium is a differential and selective growth medium. Only Gram-negative bacteria grow on this medium. In addition, bacteria that ferment lactoseappear as pink colonies (occasionally purplish) or as white colonies with a pinkcenter. Non-lactose fermenters appear clear, colorless, or white only.NOTE 2: MSA medium is also a differential and selective growth medium. OnlyGram-positive bacteria grow on this medium. In addition, those Gram-positivebacteria that ferment mannitol grow as white colonies and cause the surroundingmedium to appear yellow. Gram-positive, non-mannitol fermenters appear clear,colorless, or white only with the exception of Micrococcus luteus. The lattermicrobe appears naturally as a yellow colony.NOTE 3: TSA and NA media are not differential or selective growth medium.Both Gram-positive and Gram-negative bacteria grow on this medium. Hence,the purpose of this medium is to select bacteria based upon differences in colonymorphology.6. From your different media, identify two different bacteria. On the underside ofyour medium, mark the location of two distinct, and well-isolated colonies. Usinga loop, pick a small portion of these two colonies and inoculate each onto TSA orNA slants. Incubate the slants at 35°C-37°C. These slants will serve as yourstock cultures to inoculate your diagnostic media.NOTE: Depending upon the nature of your mixed culture unknown, two differentcolony types may not appear on MAC or MSA plates. Remember, these mediaare selective. If you have two Gram-negative bacteria, they will grow on the MACplate, but not the MSA plate. Conversely, if you have two Gram-positive bacteria,BIOL 3702: Bacterial Unknown Skills TestPage 3 of 4they will grow on the MSA plate, but not the MAC plate. If you have one Gram-positive and one Gram-negative bacterium in your mixed culture unknown, thenyou should see the growth of only one colony type on the MAC and only onecolony type on the MSA plate. In most cases, two colony types should appear onthe TSA (or NA) plate.If you cannot find two different types of bacteria based upon their growth patterns onthe selective media or by their colony types on the TSA (or NA) plate, see yourinstructor immediately. Due to technical problems, it is possible that yourparticular mixed culture unknown was not capable of supporting the isolation oftwo different bacteria. In this case, you will be given another mixed cultureunknown or you will be provided the isolated unknown bacteria (this is at theoption of the instructor). No penalty will be assessed.However, if the two bacteria were not isolated due to your lack of