Isolation and characterization of Brevibacillus borstelensis strain NCT37 from a marine fish gut

Abstract

As a part of academic lab in bacteriology, marine fish gut samples were serially diluted in sterile saline and spread plated. 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.

Full text

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
N4C
Isolation and characterization of Brevibacillus borstelensis strain NCT37 from a marine
fish gut
Background
As a part of academic lab in bacteriology, marine fish gut samples were serially diluted in sterile
saline and spread plated. 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.

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
N4C
Results
Strain description: NCT37 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are greyish color, smooth and swarming in nature. Colonies are 0.5mm in size (last quadrant
metrics). Cells are Gram positive, sporulative, capsulated motile bacilli. Biochemical reaction
implies Indole negative, methyl red positive, voges proskauer negative and citrate negative
reactions. Triple Sugar Iron implies acid butt and alkaline slant with no gas production. Catalase
and oxidase were positive. NCT37 resistant to streptomycin and sensitive to ampicillin (9mm),
rifampicin (11mm), and azithromycin (12mm).
16S rRNA gene sequencing:
Based on the colony, biochemical and
molecular characteristics, the strain NCT37
was identified as Brevibacillus borstelensis.
The 16S rRNA gene sequences of NCT37 was
100% similar to Brevibacillus borstelensis
(GenBank accession number: NR_029131) and
can be globally accessed through genbank
accession number OQ271427.
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 1292 positions in the final dataset.
The strain NCT37 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