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National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
1
Isolation and characterization of culturable heterotrophic bacteria from
Synechococcus elongatus NCT65 culture
Objective: To isolate and characterize the heterotrophic bacteria culturing along with
Synechococcus elongates strain NCT65.
Methodology
Synechococcus elongates strain NCT65 (GenBank accession number: OM677385) from NCCCC
was retrieved from glycerol stock in MN broth (fig. 1). After 14 days of incubation under 12hrs
light and dark cycle, the broth was screened for heterotrophic bacteria. Nutrient agar was
prepared in MN broth (MN-NB media) in triplicates and the plates were spread plated in
duplicates with 100µl S. elongates broth (un-inoculated plate was used as control). The plates
were incubated for 24h at 22°C under 12h light conditions. Following incubation, unique bacterial
morpho-types were identified and pure cultured (via quadrant streak) onto MN-NB media.
Theses pure culture isolates were subjected to macro-, micro-morphological, biochemical and
molecular characterizations.
Results
The un-inoculated plates remained sterile following incubation. Eight numbers of unique
morphotypes were recognized in the mother plate and named as NCT319 through NCT326 (fig.
1). The last quadrant metrics of pure cultures were recorded for all 8 strains. Theses strains were
subjected to Gram’s and spore staining procedures, whose results were tabulated in fig. 1. All the
bacterial strains were commercially outsourced for 2-way Sanger’s sequencing reaction for 16S
rRNA gene. The sequences were compiled and subjected to BLAST analysis. All sequences were
more than 1350bp and shared >98% sequence similarity with reference sequences in GenBank
(fig. 1).
National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
2
Fig. 1: Flow chart describing the pathway followed for bacterial isolations and results of
biochemical and molecular characterization.
The present work identified 8 species of
heterotrophic bacteria from S. elongates
broth (fig. 1). Neighbour-joining optimum
tree was generated by K2P method. There
were a total of 1307 positions in the final
multiple sequence alignment. The 16S rRNA
gene sequences produced in the present
study was marked with red triangle.
Bootstrap values were given at the nodes of
the phylogram.
The strains obtained through the present
study was deposited in National College
Culture Collection Centre www.ncccc.in.
These strains can be obtained for research
and academic purposes.