Microbiology Unknown

ID of Unknown Bacteria

The last and most stressful project of the year was to identify two unknown organisms. I was very apprehensive about this project and fearful about making a mistake, but when it was all said and done this project made the whole semester make since.

On day one I received unknown #5 broth. I began my identification process by inoculating my unknown broth onto three different petri plate mediums. The first plate I inoculated was a tryptic soy agar plate. After the bacteria grow on this type of medium I should be able to distinguish between the two colonies based on shape, color and size. The next plate I inoculated was a phenyl ethanol agar this is medium is selective for gram positive organisms, therefore if I were to get growth on this plate I could almost guarantee that I have a gram positive organism. This medium does have a disadvantage because some gram negative organism is able to grow on this medium. The last plate I inoculated on day one was a MacConkey Agar Plate; this medium is selective exclusively for gram negative organisms. This agar is extremely selective for gram negative organism so if I get growth on this plate it will absolutely tell me that I for sure have a gram negative organism. The final thing I did on day one was to do a gram stain on my unknown broth , the gram stain would allow me to see if my organisms were gram negative or gram positive or both. Doing a gram stain would also allow me to see the morphology of the cell, it's shape, knowing whether it is cocci or bacillus shaped would further help me in determining my organims' identity. After performing my gram stain I was able to identify a gram positive cocci, I was able to tell that I had a positive cocci due to the dark purple circles that appeared under the microscope. I was also able to identify a gram negative bacillus because I also saw light pink rod shaped bacteria. After I finished my gram stain I placed my three plates in the incubator to incubate for 48 hours. I planned to do another gram stain from my plates after I had isolated colonies.

After 48 hours of incubation I was excited to see good growth on all of my plates. My TSA plate had two separate isolated colonies on it. But it was very hard to tell the difference between the two. Both growths appeared to be white although one was a little creamer white. One was also a little bit smaller. I moved on to my next plate PEA. I had good isolated colonies on this plate; the colonies were small round, cream colored circles. I had good growth on my MAC plate as well, my growth was colorless and not pink which let me know that my bacteria was not a lactose fermenter. I then performed a gram stain from my PEA which reveled gram positive cocci, no gram negative bacteria had grown on my PEA plate. I then gram stained a sample from my MAC plate which revealed a gram negative bacillus. The last thing that I did on this day was Inoculate two TSA plates one with a colony from the MAC plate and the other with a colony from the PEA plate. I placed the two TSA plates in the incubator and allowed them to incubate for 48 hrs.

On my third day of trying to identify my organisms I did several biochemical tests on my two bacteria. These are tests that can be very helpful in trying to identify unknown bacteria. There are certain test for gram negative and certain tests for gram positive. I stared with my gram positive bacteria I did a catalase test which tests for the catalase enzyme. My unknown tested positive for catalase. I then did the coagulase test on my catalase positive gram positive unknown. The coagulase test came out negative as my unknown bacteria did not clot with rabbit plasma. I knew at this point that my gram positive bacteria were either Staphylococcus epidemis or Staphylococcus saprophytic us. I knew the best way to differentiate between the two would be to do a novobiocin sensitivity test. I streaked another TSA plate with my gram positive unknown and then placed a novobiocin disk in