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General laboratory information for researchers and lab staff, not medical advice. Handle cultures under your institution’s biosafety rules.
Short answer
The gelatin hydrolysis test shows whether a bacterium secretes gelatinases, extracellular enzymes that break gelatin down into polypeptides and then amino acids. In the standard nutrient gelatin tube, a tube that stays liquid after chilling is positive and one that sets solid again is negative (American Society for Microbiology, ASM, protocol, checked 2026-10-30).
Definition and scope
Gelatin is a protein derived from collagen, made by boiling connective tissue in water. The test detects the ability of bacteria to produce gelatinases, and ASM lists it as an aid for identifying genera such as Serratia, Pseudomonas, Flavobacterium and Clostridium. It also separates gelatinase-positive Staphylococcus aureus from gelatinase-negative S. epidermidis, and Proteus and Serratia from other Enterobacteriaceae. It is one biochemical test among several, usually read alongside a Gram stain and other differential tests, as part of routine bacteriology.
How the test works in practice
Nutrient gelatin tube method
ASM calls the nutrient gelatin stab the standard and most commonly used method, and notes that it avoids hazardous reagents. The medium contains peptone 5.0 g/L, beef extract 3.0 g/L and gelatin 120.0 g/L, with a final pH of 6.8 ± 0.2 at 25°C, and gelatin serves as both solidifying agent and substrate. A heavy 18- to 24-hour inoculum is stabbed into the tube, and an uninoculated control tube is incubated alongside it at 25°C or the organism’s optimal growth temperature for up to one week, checked daily. Gelatin normally liquefies at 28°C and above, so liquefaction is confirmed by chilling the tubes in an ice bath for 15 to 30 minutes and tilting them: hydrolysed gelatin stays liquid while the control stays solid.
Nutrient gelatin plate method
In the plate variant, ASM describes a medium of 23 g/L nutrient agar and 8 g/L gelatin, incubated at 35°C for 24 hours. A clear zone around the colony indicates gelatin hydrolysis. Plates are sometimes flooded with saturated ammonium sulfate to precipitate unhydrolysed gelatin and make the zones easier to see. A second suggested plate medium (4 g/L peptone, 1 g/L yeast extract, 12 g/L gelatin, 15 g/L agar) usually gives a visible zone without that reagent.
Agar vs gelatin: what each does in the medium
Agar and gelatin are both used to set a medium, but in this test they play different roles. In the tube, gelatin is both the solidifying agent and the substrate. In the plate formulations, agar is listed as a separate component alongside a smaller amount of gelatin. Figures below are the formulations given in the ASM protocol.
| Format | Gelatin | Agar | Incubation | Positive result |
|---|---|---|---|---|
| Nutrient gelatin tube | 120 g/L, solidifier and substrate | None listed | 25°C (or optimum), up to 1 week | Stays liquid after ice bath |
| Nutrient gelatin plate | 8 g/L | 23 g/L (as nutrient agar) | 35°C, 24 hours | Clear zone around colony |
| Alternate plate medium | 12 g/L | 15 g/L | Not specified | Visible zone, often without ammonium sulfate |
Reading results and how long it takes
Positive: the tube is liquid after chilling while the uninoculated control is solid. Negative: the tube sets solid like the control. ASM’s reference table for nutrient gelatin lists Bacillus subtilis, Clostridium perfringens, Proteus vulgaris, Serratia liquefaciens and Staphylococcus aureus as positive, and Escherichia coli as growing without liquefaction.
Timing is the main practical difference between methods. In ASM’s history of the test, Clarke (1953) compared a simplified plate method at 3 days with the stab method at up to 14 days and the Frazier plate method (1926) at up to 4 days. McDade and Weaver (1959) found that methods ranged from 1 hour to 9 weeks, with reagent-based variants carrying hazards from the chemicals used. ASM notes that some species, such as Proteus vulgaris, may need up to 14 days for a positive tube.
Common misunderstandings
- Liquid at room temperature is not a positive. Gelatin liquefies on its own at 28°C and above, so the chilled comparison against the control is what shows enzyme activity.
- No liquefaction at 24 hours is not a negative. Some gelatinase-positive organisms produce the enzyme slowly; ASM suggests heavy inocula, 7 to 14 days of incubation, smaller tubes (13 by 100 mm) and smaller volumes (2 to 3 mL).
- Growth is not hydrolysis. E. coli grows in nutrient gelatin without liquefying it.
Where it matters for labs and supply decisions
A short checklist drawn from the ASM protocol and the FDA classification record. The storage temperature refers to tubed nutrient gelatin medium, not to cultures.
- Run an uninoculated control of the same medium with every batch of tubes; a result only counts once it is compared against that control.
- Store tubed nutrient gelatin at 2 to 8°C and do not use tubes that are contaminated, discoloured or dried out.
- Use screw-capped tubes, as ASM recommends for the stab method.
- Know the regulatory status of the medium. FDA product code JSF, “Culture Media, Single Biochemical Test”, is a Class I device under 21 CFR 866.2320 (differential culture medium), which the regulation exempts from premarket notification, subject to the limitations in 21 CFR 866.9.
Cultures should be handled under your institution’s biosafety rules; see biosafety levels BSL-1 to BSL-4 and the biosafety level (BSL) definition. For incubation equipment, see what a lab incubator is. Gelatin is also the substrate in a different technique, gel zymography.
Frequently asked questions
What is the difference between agar and gelatin in this test?
In the nutrient gelatin tube, gelatin is both the solidifying agent and the substrate that gelatinase digests. In the plate formulations in the ASM protocol, agar is listed as its own component (23 g/L or 15 g/L) alongside gelatin at 8 g/L or 12 g/L, and a clear zone around the colony marks hydrolysis.
How do you tell a positive gelatin hydrolysis result?
In the tube method the medium stays liquid after the ice bath while the uninoculated control stays solid. In the plate method a clear zone appears around the colony.
How long does the test take?
ASM describes up to 1 week of incubation for the tube method, up to 14 days for slow producers such as Proteus vulgaris, and 24 hours at 3500b0C for the plate method.
Is gelatin hydrolysis the same as zymography?
No. Gel zymography uses gelatin as a substrate in a gel to detect protease activity, whereas this test looks for liquefaction or clearing in culture medium.
Sources
- American Society for Microbiology, “Gelatin Hydrolysis Test Protocol” (dela Cruz and Torres), checked 2026-10-30: asm.org protocol PDF.
- eCFR, 21 CFR 866.2320 Differential culture medium, checked 2026-10-30: ecfr.gov.
- FDA product classification database (openFDA), product code JSF, checked 2026-10-30: api.fda.gov.
