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Other features that form part of the description of many bacterial taxa include the accumulation of poly-hydroxybutyrate (PHB) granules or enzyme activities.

Necessary material

Active culture of the strain (Petri dish or slant).

We recommend inclusion of at least four strains in the study.


Poly-hydroxybutyrate (PHB) is detected by optical microscopic observation of fresh cultures grown on basal medium supplemented with 0.2% (w/v) of a carbon and energy source to promote growth of the strain. The presence of PHB granules is analysed by staining with the Nile Blue A dye (Smibert and Krieg, 2007). The test is considered positive when refractile granules are observed in the cells under fluorescence microscopy.

The activity of extracellular enzymes, such as agarase, chitinase, alginase, lipase, DNase, esterase, etc., is detected in the laboratory to evaluate the ability of the bacteria to break down compounds on the culture plate. To do so, the microorganism is grown in a general environment, provided with the basic nutrients required for proper growth, adding the macromolecule whose hydrolysis is under study (proteins, polysaccharides, lipids, nucleic acids). After incubation, an appropriate reagent is used to detect the presence of the macromolecule. Sometimes the macromolecule gives the medium a characteristic appearance (opacity, solidity), thus disappearance of this characteristic when observed with the naked eye indicates that hydrolysis has taken place.

In all cases, results are provided in jpg image format.


Smibert RM, Krieg NR. Phenotypic characterization. 2007. In: Manual of Methods for General Bacteriology, pp. 607-654. Edited by: Reddy CA, Beveridge TJ, Breznak JA, Marzluf G, Schmidt TM, Snyder LR. Washington DC: American Society for Microbiology.