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National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
1
Isolation and characterization of Bacillus amyloliquefaciens strain NCT35 from a marine
fish gut
Background
As a part of academic lab in bacteriology, fertile garden soil collected, serially diluted and spread
plated on to nutrient agar. Morphologically distinct colonies were purified using quadrant
streaking. The pure culture was preserved using glycerol stocking in triplicates. One of these
glycerol stocks was revived and used for further morphological and molecular characterization.
Work plan
Bergey’s manual was used to determine the lowest possible taxonomic hierarchy and the strain
was commercially outsourced for bidirectional 16S rRNA gene sequencing for species level
resolution.
Results
National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
Strain description: NCT35 readily grows in nutrient agar plate at 37°C in 24 hours. Colonies
appeared greyish white, round and shiner with smooth edges. Colonies are 0.6mm in size (last
quadrant metrics). Cells appears Gram positive, sporulative, motile rods. Biochemical reaction
implies Indole positive, methyl red positive, voges proskauer negative and citrate negative
reactions. Triple Sugar Iron implies acid butt and alkaline slant with no gas production. Test for
catalase and oxidase were positive. Primary enzyme screening implies negative for amylase and
lipase synthesis. NCT35 was sensitive to ciprofloxin and streptomycin.
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular characteristics, the strain NCT35 was identified
as Bacillus amyloliquefaciens. NCT35 was 99.44% similar to Bacillus amyloliquefaciens
(GenBank accession number: NR_041455).
16S rRNA gene sequence of NCT27 can be
globally accessed through genbank
accession number OQ271429.
The Neighbor-Joining method tree with
percentage of replicate trees in which the
associated taxa clustered together in the
bootstrap test (500 replicates) are shown.
The tree is drawn to scale, with branch
lengths in the same units as those of the
evolutionary distances used to infer the
phylogenetic tree. The evolutionary
distances were computed using the
Maximum Composite Likelihood method
and are in the units of the number of base
substitutions per site. This analysis involved
11 nucleotide sequences. All ambiguous
positions were removed for each sequence
pair (pairwise deletion option). There were
a total of 1239 positions in the final dataset.
The strain NCT35 was deposited in National
College Culture Collection Centre (ncccc)
and are freely available for academic and
research purposes. The strain can be obtained here www.ncccc.in