The Case of the Mystery Bacteria: Part III

 WE HAVE NO GROWTH!!!

In the last episode of 'The Case of the Mystery Bacteria' I had inoculated a pair of MacConkey plates with the unknown bacteria.  The lack of growth is a success in that is supports the hypothesis of the Gram-positive status of the bacteria.

With the narrowing down of possibilities I turned to a dichotomous key to set up a sereis of tests to determine the identity of the bacteria.  A diachotomous key is a tool used to identify a substance through a series of branching choices that lead to the correct name of the given substance.  Dichotomous means "two branching", at each branch there are only two possible results from teh proposed test (usually positive or negative) and as one continues along the key the possible results are increasingly narrowed until no further tests are needed to identify the substance.

From earlier testing I knew the mystery bacteria was a Gram-positive cocci.  Below is the dichotomous key I used:

With the materials and reagents available I collected what was necessary to prepare a catalase, glucose fermentation, and MSA test.

I performed the catalase test first.  This test is used to identify whether an organism possesses possess the catalase enzyme. Catalase converts toxic forms of oxygen (hydrogen peroxide) into oxygen and water.  Oxygen is seen as bubbles, so a positive test results in bubbles and a negative test results in no bubbles being present.

To perform the test a small amount of the bacteria is transferred from a plate to a slide and drops of hydrogen peroxide are added.

If the result was negative I would have been done, and the bacteria would be identified as Endercoccus faecalis.  As you can see above the test was positive.  As such, I moved on to the next test.

The rest of the tests require a period of incubation, the bacteria must be stored in an incubator for a period of time before viewing the results.  Rather that perform one  test per day.  I prepared three tests that would move me further along the key: 

• Glucose Fermentation 
•  Fermentation is the metabolism of a simple sugar into acids and/or gases.  
• The test involves inoculation a tube containing a sugar solution with phenol red indicator dye. 
•  If the bacteria metabolizes the sugar acid is released and the pH of the solution drops turning the dye from red to yellow.  
• To determine if any gas was released during fermentation a Durham tube is placed in the test tube.  A bubble will appear in the Durham tube if gas is released during fermentation.
• No change indicates a negative test.
• MSA Plate
• MSA - Mannitol Salt Agar Plate  
• MSA is a selective and differential growth medium, encouraging the growth of certain bacteria while inhibiting the growth of others.
• This plate encourages the growth of bacteria that can metabolize mannitol (sugar) and grow in an environment with a high salt concentration.
• The test involves inoculation of a plate containing MSA and observing the changes.
• Changes of color from read to yellow indicate sugar metabolism and growth indicates salt tolerance.
• No change indicates a negative test.
• Motility
• The media in the test tube is a semi-solid which contains the indicator tetrazolium chloride (TTC).  
• TTC reacts with hydrogen ions changing its color to red.
• Since flagella, a microscopic appendage that allows bacteria and other organisms to swim, pump hydrogen ions flagella action shows up as red, indicating a mobile bateria.
• A negative resulte indicates a non-mobile bacteria.

Here are the prepared tests ready to enter the incubator:

See you next time for the results, and possible the solution to this mystery!