Purpose

 Fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. Andrade’s formula is used to differentiate enteric bacteria from coryneforms, and bromo cresol purple is used to distinguish enterococci from streptococci.

Principle

Carbohydrate fermentation is the process microorganisms use to produce energy. Most microorganisms convert glucose to pyruvate during glycolysis; however, some organisms use alternate pathways. A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose, or sucrose) for fermentation. However, the medium may contain various color indicators, such as Andrade’s indicator, bromocresol, or others. In addition to a color indicator to detect the pro duction of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism.

Basal media: Pancreatic digest of casein (10 g), beef extract (3 g), NaCl (5 g), carbohy drate (10 g), specific indicator (Andrade’s indi cator [10 mL, pH 7.4] or bromocresol purple [0.02 g, pH 6.8]).

Method

A. Peptone Medium with Andrade’s Indicator  (for Enterics and Coryneforms)

1. Inoculate each tube with 1 drop of an 18- to 24-hour brain-heart infusion broth culture.

2. Incubate at 35°-37°C for up to 7 days in ambient air. Note: Tubes are held only 4 days for organisms belonging to the Enterobacteriaceae family.

 3. Examine the tubes for acid (indicated by a pink color) and gas production.

4. Tubes must show growth for the test to be valid. If no growth in the fermentation tubes or control is seen after 24 hours of incubation, add 1 to 2 drops of sterile rabbit serum per 5 mL of fermentation broth to each tube.

Expected Results

 Positive: Indicator change to pink with or without gas formation in Durham tube (Figure 1, A, left and middle).

 Negative: Growth, but no change in color. Medium remains clear to straw colored (Figure 1, A, right).

B. Broth (Brain Heart Infusion Broth May Be  Substituted) with Bromcresol Purple Indicator  (for Streptococci and Enterococci)

1. Inoculate each tube with 2 drops of an 18- to 24-hour brain-heart infusion broth culture.

 2. Incubate 4 days at 35°-37°C in ambient air.

3. Observe daily for a change of the bromcresol purple indicator from purple to yellow (acid).

Expected Results

 Positive: Indicator change to yellow (Figure 1, B, left).

 Negative: Growth, but no change in color. Medium remains purple (Figure 1, B, right).

Limitations

 Readings after 24 hours may not be reliable if no acid is produced. No color change or a result indicating alkalinity may occur if the organism deaminates the peptone, masking the evidence of carbohydrate fermentation.

Quality Control

Note: Appropriate organisms depend on which carbohydrate has been added to the basal medium. An example is given for each type of medium.

A. Peptone  Indicator Medium with Andrade’s  Dextrose:

Positive, with gas: Escherichia coli (ATCC25922) Positive, no gas: Shigella flexneri (ATCC12022)

B. Brain-Heart Infusion Broth with Bromocresol Purple Indicator Dextrose:

Positive, with gas: Escherichia coli (ATCC25922) Negative, no gas: Moraxella osloensis (ATCC10973)

Fig1. Fermentation media. A, Peptone medium with Andrade’s indicator. The tube on the left ferments glucose with the production of gas (visible as a bubble [arrow] in the inverted [Durham] tube); the tube in the middle ferments glucose with no gas production; and the tube on the right does not ferment glucose. B, Heart infusion broth with bromocresol purple indicator. The tube on the left is positive; the tube on the right is negative.