Isolation and characterization of Brevibacillus laterosporus strain NCT75 from a agri soil

Abstract

As a part of academic lab in microbiology, agri soil samples were collected, serially diluted and spread plated onto a nutrient agar media. 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.

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National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
1
N4C
Isolation and characterization of Brevibacillus laterosporus strain NCT75 from a agri soil
Background
As a part of academic lab in microbiology, agri soil samples were collected, serially diluted and
spread plated onto a nutrient agar media. 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 www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
N4C
Strain description: NCT75 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are small, round, yellowish white, shiny with rough edges and are 9mm in diameter (last quadrant
metrics). Cells are Gram positive, non-motile rods. Biochemical reaction implies Indole positive,
methyl red positive, voges proskauer positive and citrate positive. The strain is positive for
amylase and lipases enzymes. Triple Sugar Iron implies alkaline butt and slant with no gas
production. Catalase negative and oxidase negative. The strain NCT75 was resistant to penicillin,
streptomycin, erythromycin and resistant to rifampicin.
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular characteristics, the strain NCT75 was identified
as Brevibacillus laterosporus. The 16S rRNA gene sequences of NCT75 was 99.58% similar
Brevibacillus laterosporus (GenBank accession number: NR_037005) and can be globally
accessed through genbank accession number OQ255769.
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 1325
positions in the final dataset.
The strain NCT75 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