Day 3 - Utilization of Carbohydrates

Sugar, sugar, sugar. 

We tested for our bacteria's utilization of carbohydrates: fermentation of carbohydrates, MR-VP tests, and Simmon's citrate test.

Fermentation of Carbohydrates:  Bacteria frequently ferment carbohydrates to use as energy, but what particular kinds of carbohydrate do our bacteria use for energy?  This is what we are aiming to discover.

Materials:
Tubes of phenol sugar broth with Durham tube: lactose, sucrose, glucose, and mannitol
Pure culture of our bacteria from broth culture

Steps:





 

Inoculating Carbohydrate for Fermentation

1.  Label the tubes with the sugars with our group number and type of test.

2.  Use aseptic technique to inoculate the sugar tubes with our bacteria.

3.  Incubate the sugar tubes at 35 degrees for 24 hours.

4.  Examine the tubes for acid production or acid and gas production.  A yellow color would be a positive test for acid production, and a gas bubble inside the Durham tube would be a positive result for gas production.

5.  Discard the tubes properly




Results:

Lactose and sucrose were fermented with gas production.  Mannitol and glucose were fermented but there was no gas production.

Discussion:  Our bacteria can ferment lactose, sucrose, glucose, and mannitol for energy.



MR-VP Tests:
This determines whether our bacteria can ferment glucose.  The methyl red test indicates whether the bacteria can ferment glucose via mixed-acid fermentation.  The Voges-Proskauer test indicates whether the bacteria can ferment glucose via butanediol fermentation.

Materials:
Methyl red/Voges-Proskauer (MR-VP) broth tube
Pure culture of our bacteria on an agar slant
One extra test tube
Methyl red (pH indicator) in a dropper bottle
Barritt's (VP) solution A in a dropper bottle
Barritt's (VP) solution B in a dropper bottle

Steps:
1.  Label the MR-VP tube with our group number and type of test.
2.  Use aseptic technique to inoculate the MR-VP tube from our agar slant.
3.  Incubate at 35 degrees for 24 hours.
4.  Pour part of the MR-VP broth into another clean tube.
5.  Add 15 drops of methyl red to the second tube.  Gently mix.


Methyl Red

6.  Read the reaction immediately.  A red color would be a positive result.
7.  Into the original tube of MR-VP broth, add 15 drops of solution A and 5 drops of solution B.


Voges-Proskauer

8.  Tap the bottom of the tube vigorously to mix.  A red color at the top of the broth is a positive result.

Result:

 

Negative Methyl Red Result





Our VP Test while mixing in both solutions A and B.  (waiting for a reaction)

Our positive VP (left) after a reaction had taken place (due to solutions A and B). On the right is another group's negative VP (right). Solutions A and B had no effect on theirs..



Discussion:  Our test is negative for methyl red and positive for Voges-Proskauer.  So our bacteria can ferment glucose via butanediol fermentation but not via mixed fermentation.




Simmon's Citrate Test:  This test determines whether our bacteria can use citrate as its only source of carbon and energy.

Materials:
Simmons citrate agar slant tube
Pure culture of our bacteria in a broth tube

Steps:
1.  Label the Simmons tube with our group number and type of test.

2.  Use aseptic technique to inoculate the Simmons citrate agar slant with our bacteria.




Simmon's Citrate test

3.  Incubate the Simmons tube at 35 degrees for up to 4 days, checking every 24 hours for color change and bacterial growth.  A blue color is a positive result.

4.  Discard the tube properly.



Results:

Positive Result

Our positive result (right) with another group's negative result (left)

Discussion:  Our bacteria gave a positive result, so it can use citrate as its only source of carbon and energy.