Jorge Vargas
Ugoo Anieto
April 4, 2013
Identifying Unknown Bacteria
In my microbiology lab I was given a detectives’ task to choose a broth which contained two
micro-organisms and I had to find out their identities, so I choose Japan. Our goal was to basically find
the type of bacteria present in this broth. We knew that there were two types, gram positive and gram
negative. We were also given a list of possible organisms to choose from and a paper for the negative
chemical test with results for each strain of bacteria. This sheet was going to come in handy really soon,
it was basically a cheat sheet. The possibilities for the gram negative bacteria were greater than that of
the gram positive, the gram negatives were also challenging to figure out. The list of gram positives are
as followed: Staphylococcus epidermis, Staphylococcus saprophyticus, Enterococcus faecalis, or Bacillus
megaterium and the list for gram positives are as followed: Escherichia coli, Citrobacter freundii,
Morganella morganii, Providencia stuartii, Serratia marcescens, Edwardsiella tarda, or Psuedomonas
fluorescens. I will now explain the procedure.
First I grabbed my Japan labeled broth and started with the gram stain in order to see the
morphology and type of peptidoglycan layer. I found a gram positive cocci and gram negative bacillus.
The next step would be to do a four quadrant streak in order to achieve single colonies; after the streak
was complete I moved the plate into the autoclave and left it there until the next class day. After I took
my plate out of the autoclave I looked closely to see any size or color differences in the colonies and then
picked out two that looked different from one another and then did a gram stain again to make sure I
have pure colonies that were different from each other. I then grabbed two agar plates and took one of
the different colonies and did a four quadrant streak in order to gain single colonies and then be able to
start with the testing. I did the same for the other colony; I then did a four quadrant streak and put both
of them in the autoclave and allowed them to grow. After they were allowed to grow for a day or two; I
took them out, looked at them, and saw that they looked different and separated so I skipped the gram
staining in order to save time.
I started with the positive test. I had cocci first off so my options became Staphylococcus
epidermis, Staphylococcus saprophyticus or Enterococcus faecalis I preceded with the catalase test and it
came out negative, so I was sure I now have Enterococcus faecalis. Now I have to do the negative tests.
At first I decided not to create a broth because it would be quicker to use the organisms I have on the
agar plate and after doing the chemical tests I kept getting weird results, so I decided to make a broth. I
grabbed some nutrient agar and inoculated my gram negative bacteria into the nutrient agar, of course
using the correct aseptic techniques. After the broth was complete I started on the chemical test. I used
the inoculating loop to grab some of the bacteria and transferred a drop into two MR-VP test tubes,
urea, lactose, glucose. For the SIM we used the needle coated with the bacteria and did a quick stab, for
the citrate we simply streaked the bacteria onto the medium. I then waited to see the results.
My results were very confusing and didn’t match up with anything. I got gas production for the
lactose medium, glucose was positive because it turned yellow; lactose was negative because it remained
red. For the results from the SIM we can figure out three things: motility, indol production and H2S
production. My SIM wasn’t black so it was negative for H2S, I couldn’t tell at the time if it was motile
or not, this could be because there wasn’t enough time for the organism to move or grow. For the indol
test we had to add 10 drops of Kovac’s solution to the SIM tube and see if it forms a red ring on the
Jorge Vargas
Ugoo Anieto
April 4, 2013
surface, mine did not form a red ring so it was negative for indol. For the citrate tube we could tell if it
was positive by the color change from green to blue, mine turned blue so it’s positive for citrate. Urea
remained yellow and not red so it was negative. To determine the MR and VP tests we have to add 4
drops of methyl red into the MR and wait, and then add 9 drops of alpha-naphthol and 9 drops of KOH
into the VP tube and wait ten minutes for both. MR turned a light red and VP turned a light pink, so I
concluded that it was MR positive and VP negative. I then did the oxidase test by using two cotton
swaps to get some of the bacteria and then rubbed them together. I then added the reagent to see if it
would turn purple and it didn’t so it was negative for oxidase.
Based on these wacky results the only one that matched was Serratia Marcescens, Glucose was
positive , I did have a small gas bubble, I’m positive. It was negative for indol, I wasn’t sure about
motility, it was positive for motility, urea was also negative, H2S was also negative, and the MR-VP’s
were confusing. This was my best and closest one. I loved doing this lab and would like to know why
my results didn’t correlate as exact as it should have. There it is Serratia Marcescens and Enterococcus
faecalis.