Purpose

Differentiate microorganisms based on the ability to use acetamide as the sole source of carbon.

Principle

 Bacteria capable of growth on this medium produce the enzyme acylamidase, which deaminates acetamide to release ammonia. The production of ammonia results in an alkaline pH, causing the medium to change color from green to royal blue.

 Media: NaCl (5 g), NH4H2PO4 (1 g), K2HPO4 (1 g), agar (15 g), bromthymol blue indicator (0.8 g), per 1000 mL, acetamide (10 g), pH 6.8.

 Method

1. Inoculate acetamide slant with a needle using growth from an 18- to 24-hour culture. Do not inoculate from a broth culture, because the growth will be too heavy.

2. Incubate aerobically at 35°-37°C for up to 4 days. If equivocal, the slant may be reincubated for 2 additional days.

Expected Results

Positive: Deamination of the acetamide, resulting in a blue color (Figure 1, A).

Negative: No color change (Figure 1, B).

Limitations

Growth without a color change may indicate a positive test result. If further incubation results in no color change, repeat test with less inoculum.

Quality Control

 Positive: Pseudomonas aeruginosa (ATCC 27853)—growth; blue color

Negative: Escherichia coli (ATCC 25922)—no growth; green color

Fig1. Acetamide utilization. A, Positive. B, Negative.