NUTRIENT GELATIN (CM135a)

Code: CM0635

For determination of gelatin liquefaction, and for the 20°C plate count.

Directions
Suspend 128g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Mix well before pouring and cool below 20°C or leave to set in a refrigerator.

Description
Nutrient Gelatin, a solid medium at 22°C and below, is employed for the determination of gelatin liquefaction and for the 20°C plate count¹.
Gelatin is liquefied in a characteristic manner by certain proteolytic organisms; whereas gelatin media have been largely superseded by agar media for most purposes, nutrient gelatin is still employed for differentiation of micro-organisms by their proteolytic effects.

Technique
Test for gelatin liquefaction by incubating a Nutrient Gelatin stab or plate culture at 20-22°C. Alternatively, incubate at a higher temperature (usually optimum for the organism under investigation) and then transfer the tube to a refrigerator or into cold water before observation. The latter method not only allows determinations to be carried out on organisms which grow slowly or not at all at 20-22°C but also usually avoids false positive results produced by the release of enzymes after the death of the organisms².

If the medium is incubated at a higher temperature it is necessary to employ uninoculated controls to allow for the hydrolytic effect of heat and other factors. Rates of liquefaction vary considerably, so that some organisms produce liquefaction within a few days whereas others require several weeks. For practical purposes, a maximum incubation period of 14 days is suggested^3,4.

Considerably longer incubation may be necessary, some strains of Enterobacter cloacae liquefied gelatin only after 3 months at 20-22°C⁵. Especially where prolonged incubation is necessary, it is important to ensure adequate closure of the containers in order to prevent dehydration of the medium. Besides its presence or absence, the shape and nature of the liquefied portion of the stab culture are often useful identifying characteristics.

Particularly with plate cultures, gelatin liquefaction may be detected sooner by the `Stone reaction’ ( Stone<sup>6</sup> ): add a drop of saturated aqueous ammonium sulphate solution, or of fresh 20% aqueous sulpho-salicylic acid solution, to an individual colony growing on Nutrient Gelatin. A positive reaction (i.e. gelatin liquefaction) is indicated by the presence of a clear zone round the colony after 10 minutes contact with either reagent. This method is slightly less sensitive but several strains may be tested on one plate. The `Stone reaction’ is also employed with Staphylococcus Medium No.110 CM0145 for the differentiation of staphylococci.

For the standard gelatin plate count on water ( American Public Health Association¹ ) dilute the original sample with sterile tap water and place 0.5 or 1ml of the dilutions in each dish of at least two duplicate sets of sterile Petri dishes. Cool the sterile prepared Nutrient Gelatin to approximately 42°C and aseptically add 10ml to each Petri dish. Mix the contents by tilting and rotation, allow to solidify as soon as possible after pouring and immediately place in an incubator at 19-21°C. Incubate for 48 ± 3 hours and count at least two plates made from the dilution giving between 30 and 300 colonies per plate.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder/granules
Prepared medium: Straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

Precautions
Do not shake the gelatin tubes whilst they are warm because growth and liquefaction of gelatin frequently occurs on the surface layer only⁷.
In routine diagnostic work report gelatin liquefaction or not. The type or shape of liquefaction is of less importance².

References
1. American Public Health Association (1946) Standard Methods for the Examination of Water and Sewage. 9th Edn. APHA Inc. Washington DC.
2. American Society for Microbiology (1981) Manual of Methods for General Bacteriology. ASM. Washington DC.
3. Cowan S. T. and Steel K. J. (1966) Manual for the Identification of Medical Bacteria. Cambridge University Press. Cambridge. pp. 19. 27-28, 116 and 156.
4. Wilson G. S. and Miles A. A (1964) Topley and Wilson’s Principles of Bacteriology and Immunity. 5th Edn. Vol.1. Edward Arnold. London. pp. 493- 494.
5. Windle Taylor. E. (1958) `The Examination of Waters and Water Supplies’ 7th ed., Churchill Ltd., London, pp. 414 and 422.
6. Stone R. V. (1935) Proc. Soc. Exper. Biol. Med. 185-187.
7. Frobisher M. (1957) Fundamentals of Microbiology. 6th Edn. W. B. Saunders. Philadelphia. p. 39.