Isolation and characterization of Bacillus badius strain NCT3 from human oral cavity Background

Abstract

As a part of academic lab in bacteriology, oral cavity samples were crowd plated and morphological distinct colonies were purified using quadrant streaking. The pure culture was preserved using glycerol stocking in triplicates. This glycerol stocked cultures were revived and used for further morphological and molecular characterization.

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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 badius strain NCT3 from human oral cavity
Background
As a part of academic lab in bacteriology, oral cavity samples were crowd plated and
morphological distinct colonies were purified using quadrant streaking. The pure culture was
preserved using glycerol stocking in triplicates. This glycerol stocked cultures were 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
Strain description: NCT3 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are white, sticky and swarming in nature. Colonies were on average 0.6mm in size (last quadrant
metrics). Cells are Gram positive, sporulative motile rods. Biochemical reaction implies Indole
negative, methyl red negative, voges proskauer negative and citrate negative. Triple Sugar Iron
implies acid butt and alkaline slant with no gas production. Catalase positive and oxidase positive.
The strain was resistant to Ampicillin and sensitive to Ciprofloxacin (diameter of the zone =
13mm), and Azithromycin (diameter of the zone =8mm), Rifampicin (6mm). The genomic DNA of
NCT3 was more than 16KB in size.

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
3
16S rRNA gene sequencing:
Based on the colony, biochemical and
molecular characteristics, the strain NCT3
was identified as Bacillus badius. NCT3
was 98.84% similar to Bacillus badius
(GenBank accession number: NR_036893).
16S rRNA gene sequence of NCT27 can be
globally accessed through genbank
accession number OQ271409.
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 1382
positions in the final dataset.
The strain NCT3 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