Purpose

 The production of gelatinases capable of hydrolyzing gelatin is used as a presumptive test for the identification of various organisms, including Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli.

Principle

 This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue.

Nutrient gelatin medium differs from traditional microbiology media in that the solidifying agent (agar) is replaced with gelatin. When an organism produces gelatinase, the enzyme liquefies the growth medium.

Media: Enzymatic digest of gelatin (5 g), beef extract (3 g), gelatin (120 g), per 1000 mL, pH 6.8.

Method

1. Inoculate the gelatin deep with 4 to 5 drops of a 24-hour broth culture.

2. Incubate at 35°-37°C in ambient air for up to 14 days. Note: Incubate the medium at 25°C if the organism grows better at 25°C than at 35°C.

 3. Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabbing four or five times, 0.5 inch into the medium.

4. Remove the gelatin tube daily from the incubator and place at 4°C to check for liquefaction. Do not invert or tip the tube, because sometimes the only discernible liquefaction occurs at the top of the deep where inoculation occurred.

 5. Refrigerate an uninoculated control along with the inoculated tube. Liquefaction is determined only after the control has hardened (gelled).

Expected Results

 Positive: Partial or total liquefaction of the inoculated tube (the control tube must be completely solidified) at 4°C within 14 days (Figure 1, A).

 Negative: Complete solidification of the tube at 4°C (Figure 1, B).

Limitations

Some organisms may grow poorly or not at all in this medium

Gelatin is liquid above 20°C; therefore determination of results must be completed following refrigeration.

Quality Control

Positive: Bacillus subtilis (ATCC9372)

 Negative: Escherichia coli (ATCC25922)

Uninoculated control tube: medium becomes solid after refrigeration.

Fig1. Gelatin hydrolysis. A, Positive; note liquefaction at top of tube. B, Uninoculated tube.