Bacillus megaterium PHB inclusions.
Sudan black staining.
PHB inclusions
DESCRIPTION
Procedure for the examination of bacterial capacity to accumulate poly-beta-hydroxybutyrate inclusions. Polyhydroxyalkanoates (PHA)
serve primarily as a carbon and energy storage material when exogenous carbon sources are depleted; also enhances the
resistance of bacterial cells to various stress conditions including low temperatures and freezing. Among the wide variety of PHAs, the
polyester of 3-hydroxybutyrate, poly(3-hydroxybutyrate) (PHB), is the most common and of interest as bio-derived and biodegradable
plastics. PHB is produced by various microorganisms, like  
Bacillus megataerium, Methylobacterium rhodesianum, Ralstonia
eutropha
, Pseudomonas (Pseudomonas extremaustralis) and Pseudomonas-like organisms.
PROCEDURE
Cultures are grown in a medium with high carbon-nitrogen ratio.
Sudan black solution is prepared as 0.3% solution in 70% ethanol, shaken and
allowed  to stand overnight.

Make a smear from the culture, dry and heat-fix it then flood it with Sudan black
solution and wait 15 minutes.

Drain off excess stain, dry the slide, then rinse in water and dry again.
Counterstain with safranin, rinse and dry. Examine with oil immersion microscope.
RESULTS
Poly-beta-hydroxybutyrate inclusions are stained blue-grey to black. Result may vary
depending on strain tested: Acidovorax or Burkholderia strains give weaker reaction
than Ralstonia.
REFERENCES:
1. Pseudomonas Pathogens of Stone Fruits and Nuts: Classical and Molecular Phytobacteriology. COST873 ESR Training Course
Handbook and Presentations. 6th-9th April 2010, Belgrade,Serbia.
2. Stanislav Obruca, Petr Sedlacek, Vladislav Krzyzanek, Filip Mravec, Kamila Hrubanova, Ota Samek, Dan Kucera, Pavla Benesova,
and Ivana Marova: Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing. PLoS One. 2016; 11(6):
e0157778.
3. Jari M, Khatami SR, Galehdari H, Shafiei M: Cloning and Expression of Poly 3-Hydroxybutyrate Operon Into Escherichia coli.
Jundishapur J Microbiol (2015).
(c) Costin Stoica
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