8.4: Fermentation of Carbohydrates w. Procedures and Results

Carbohydrates are complex chemical substrates which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen, and oxygen (with hydrogen and oxygen being in the same ratio as water; [CH₂O] and are usually classed as either sugars or starches.

Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism.

We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:

1. acid end products, or
2. acid and gas end products.

In order to test for these fermentation products, you inoculate and incubate tubes of media containing a single carbohydrate (such as lactose or maltose), a pH indicator (such as phenol red) and a Durham tube (a small inverted tube to detect gas production).

• If the bacterium ferments that particular carbohydrate producing acid end products alone, the acid will lower the pH, causing the pH indicator phenol red to change form its original red color at a neutral pH to a yellow or clear color see Fig. \(\PageIndex{2A}\)).
• If the bacterium ferments that particular carbohydrate producing both acid and gas, the pH indicator phenol red to change form its original red color at a neutral pH to a yellow or clear color and the gas will collect in the Durham tube as a substantial gas bubble appearing at the top of the Durham tube (see Fig. \(\PageIndex{2B}\)).
• If the carbohydrate is not fermented by the bacterium, no acid or gas will be produced and the phenol red will remain red (see Fig. \(\PageIndex{2C}\)).

Fig. \(\PageIndex{1}\) : An Uninoculated Fermentation Tube.	Fig. \(\PageIndex{1A}\) : A Fermentation Tube Showing Bacterial Fermentation of the Carbohydrate Producing Acid but No Gas	Fig. \(\PageIndex{2B}\) : A Fermentation Tube Showing Bacterial Fermentation of the Carbohydrate Producing both Acid and Gas.	Fig. \(\PageIndex{2C}\) : A Fermentation Tube Showing No Bacterial Fermentation of the Carbohydrate Producing No Acid or Gas.
Fermentation tube before inoculation. The phenol red remains red at a neutral pH and there is no gas bubble at the top of the Durham tube.	Acid end products from carbohydrate fermentation lower the pH causing the pH indicator phenol red to turn from red (neutral) to yellow (acid), but there is no gas bubble seen at the top of the Durham tube.	Acid end products from carbohydrate fermentation lower the pH causing the pH indicator phenol red to turn from red (neutral) to yellow (acid). A large bubble seen at the top of the Durham tube indicates gas end products.	Fermentation tube showing no carbohydrate fermentation. The phenol red remains red indicating no acid, and there is no bubble seen at the top of the Durham tube indicating no gas.
(Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

Organisms

Trypticase Soy agar cultures of

• Bacillus subtilis,
• Escherichia coli, and
• Staphylococcus aureus.

Materials

• 3 tubes of Phenol Red Lactose broth and
• 3 tubes of Phenol Red Maltose broth

PROCEDURE (to be done in pairs)

1. Label each tube with the name of the sugar in the tube and the name of the bacterium you are growing.

2. Inoculate one Phenol Red Lactose broth tube and one Phenol Red Maltose broth tube with Bacillus subtilis.

3. Inoculate a second Phenol Red Lactose broth tube and a second Phenol Red Maltose broth tube with Escherichia coli.

4. Inoculate a third Phenol Red Lactose broth tube and a third Phenol Red Maltose broth tube with Staphylococcus aureus.

5. Incubate the tubes in your test tube rack on your shelf of the 37°C incubator corresponding to your lab section until the next lab period

Results

As mentioned above, we can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:

1. acid end products, or
2. acid and gas end products.

The results of fermentation may be acid alone or acid plus gas, but never gas alone.

Phenol red pH indicator appears red or orange at neutral pH and appears yellow or clear at an acidic pH.

• A change in color in the tube from red or orange to yellow or clear indicates that the organism has fermented that particular carbohydrate, producing acid end products.
• A substantial gas bubble at the top of the Durham tube, the inverted test tube within the broth, indicates gas was also produced from the fermentation of the carbohydrate.
• If the phenol red remains red, no acid was produced and the carbohydrate was not fermented.

Possible results are as follows:

• Carbohydrate fermentation producing acid but no gas: acidic (yellow or clear); no substantial gas bubble in the Durham tube (see Fig. \(\PageIndex{8A}\)).
• Carbohydrate fermentation producing acid and gas: acidic (yellow or clear) ; a substantial gas bubble in the Durham tube (see Fig. \(\PageIndex{8B}\)).
• No carbohydrate fermentation. No acid or gas (neutral pH (red or orange); no substantial gas bubble in the Durham tube (see Fig. \(\PageIndex{8C}\)).

Carbohydrate Fermentation
Record your results below (+ = positive; - = negative).

Organism	Phenol Red Maltose	Phenol Red Lactose
Bacillus subtilis
Acid
Gas

 

Fermentation
Escherichia coli
Acid
Gas

 

Fermentation
Staphylococcus aureus
Acid
Gas
Fermentation

 

Contributors and Attributions

• Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)