Screening for PHA Producing Bacteria
Sudan black B positive isolates were checked for PHA production by Nile blue A staining, a more specific stain for Polyhydroxyalkanoic acids (PHAs) by a more rapid and sensitive, viable colony method [14]. This dye at concentrations of only 0.5 μg/mL was directly included in carbon rich nutrient agar medium (glucose 1%, beef extract 0.3%, peptone 0.5%, sodium chloride 0.8%, and agar 1.5%) and growth of the cells occurred in the presence of the dye. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The PHA accumulating colonies, after Nile blue A staining, showed bright orange fluorescence on irradiation with UV light and their fluorescence intensity increased with the increase in PHA content of the bacterial cells. The isolates which showed bright orange fluorescence on irradiation with UV light after Nile blue A staining were selected as PHA accumulators