Poly-B-hydroxybutyrate (PHB): A Biodegradable Polymer of Microbial Origin - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 47

deteCtioN oF PhB iNClusioNs IN VIVO
Selection of efficient PHB producers among different microorganisms demands sim-
ple staining methods of PHB content estimation in living bacterial cells which can
give preliminary indication for presence of PHB inside the cells. Different staining
techniques are very useful for detection of physical state of PHB inclusion in vivo . The
PHB inclusion is of 0.2-0.5 μm in diameter, in cytoplasm can be viewed microscopi-
cally due to their high refractivity (Dawes and Senior, 1973).
sudan Black B staining
A simple technique of PHB in bacterial cells is of Sudan black b staining in which
bacterial cells were subjected to staining after growth in deficient minimal medium.
Under bright field microscope, granules of PHB which revealed the blue color gran-
ules against pink cytoplasm. The number of granules varies according to microbial
cells. By this staining, a single granule can be stained properly and observed under
microbial field (Schlegel et al., 1970).
Phosphine r staining
This is fluorescent technique of PHB staining in which bacterial colonies are grown
on a solid medium with the presence of phosphine 3R (lipophylic due). According to
this staining, the fluorescence of colonies in UV light shows the presence of the poly-
mer. Colonies of the strains with the high content of the polymer produce bright-green
fluorescence in UV light (Bonartseva, 2007). The method has been used for primary
qualitative selection of the bacterial strains having high PHB content. Colonies of
strains containing PHB in small quantities does not fluoresce.
Nile Blue staining
In this method, oxazine dyes like Nile blue A is used which exhibits a strong fluores-
cence under the fluorescence microscope (Ostle and Holt, 1982). Nile red can also be
used to detect PHB in growing cultures (Spiekermann et al., 1999). However, during
purification, the membrane surrounding PHB granules is lost, therefore, cannot be
stained by this technique (Dawes and Senior, 1973).
PhysioloGiCal role oF PhB
Physiologically, PHB is associated with the growth and cell division of bacteria. As
PHB is stored as restored energy source, it directly relates with the physiological ac-
tive state of cell. Author has observed that PHB accumulation in Alcaligenes sp. and
Pseudomonas sp. increases at the exponential phase of growth. This observation led
to the conclusion that bacteria make and store PHB when nutrients get exhausted and
when cell is at its higher stage of growth. A deficiency of nitrogen can initiate PHB
biosynthesis. Sayyed et al. (2009) Sayyed and Gangurde (2010) have reported that
Pseudomonas sp. RZS1 accumulated higher amount of PHB after 30 hr of growth
(Figure 1) and A. faecalis after 24 hr of growth (Figure 2).