The Carbohydrate Fermentation or Phenol Red Broth Test, Atlas 57-59

The Carbohydrate Fermentation test uses Phenol Red Broth to test for the fermentation of different sugars.  It is one of the fermentation tests, others include the Methyl Red test and the Voges-Proskauer test.  Phenol Red Broth is a general purpose fermentation media that includes the pH indicator Phenol Red and a series of tubes each with a different sugar.  Different people use different sugars, but glucose, sucrose, lactose, and mannitol are often studied.  One inoculates bacteria into each tube, if the strain of bacteria ferments that sugar, an acid will build up, changing the color of Phenol Red.  Sugars such as glucose undergo "fermentation" when it acts as an electron donor, such as in glycolysis, and one of its metabolic products (such as pyruvate) act as an electron acceptor in a fermentation reaction.  In reality, most sugars other than glucose are said to undergo  "fermentation" when it is either hydrolyzed into glucose or converted into glucose or both and then the glucose is fermented.  Fermentation reactions often begin with glycolysis, but this is not always the case, especially with prokaryotes.  The second step in the process is the fermentation reaction that generates end products such as different acids, ethanol, hydrogen gas, carbon dioxide gas, and other organic compounds, depending on the specific reactions.  Which reactions will occur depends on the species of bacteria.

In this test, one looks for the production of acids by observing a change in color of the medium to yellow.  Each tube should also have an inverted and filled Durham tube which will allow one to look for the production of gas.  Phenol Red Broth also conatains protein that may allow for the detection of the degradation of peptones and their resulting alkaline end products.  These alkaline end products change the broth color to pink, but this is often only observed at the very top of the tube (where oxygen is most abundant) when the tubes are totally undisturbed.  Because the degradation of proteins is often difficult to observe and the tubes are likely to be disturbed in a normal classroom, this part of the test is often ignored unless it is blatantly obvious.

This test is used in identifying Gram negative bacilli, especially Enterobacteria.

Procedure:

  1. Obtain one tube for each sugar, usually one for glucose, lactose, sucrose, mannitol.  Do not get them confused, they look the same, it is suggested that they be labelled immediately if they are not already labelled.
  2. Observe the tubes before you begin.  They should have an inverted Durham tube (see Atlas p 59) that is filled fully with broth (they are to test for gas production). 
  3. Do not use a tube if it does not have a full and inverted Durham tube or it looks wrong or contaminated.
  4. Label each tube appropriately including the sugar initial if needed.
  5. Use your wire loop to aseptically inoculate each tube with your unknown bacteria.  Do not cross contaminate your tubes with the wrong sugar, flame your loop between inoculations. 
  6. Remind your instructor to set up uninoculated control tubes.  You will need these for a comparison.  Place your tubes in the rack provided for this test.  Your instructor will incubate them.  
  7. They should be incubated for 18-24 hrs at 37 degrees.  If the results are not clear (the color is orange) or you have a slow growing bacteria, increase the incubation period by a day or two.  Do not over incubate because once the sugars become depleted, many species will start degrading the proteins which will erase an acidic pH and turn it alkaline.

Results:

Results can be positive (or negative) for acid production, gas production, and alkaline product production (the last is often ignored unless obvious).  Results are determined from the tube color and presence of bubbles in the Durham tube.  It is best to observe the color of the broth outside of the Durham tube if the two differ.  Score this test as: