Isolation and characterization of Bacillus velezensis strain NCT44 from marine fish gut Background

Abstract

As a part of academic lab in bacteriology, marine fish gut samples were serially diluted and plated onto nutrient agar plates. Following incubation, morphologically unique colonies were segregated via quadrant streaking. These pure cultures were preserved using glycerol stocking in triplicates. One of these glycerol stocks were 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
1
Isolation and characterization of Bacillus velezensis strain NCT44 from marine fish gut
Background
As a part of academic lab in bacteriology, marine fish gut samples were serially diluted and plated
onto nutrient agar plates. Following incubation, morphologically unique colonies were
segregated via quadrant streaking. These pure cultures were preserved using glycerol stocking
in triplicates. One of these glycerol stocks 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

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
Strain description: NCT44 grows well in nutrient agar plate at 37°C in 24 hours. Colonies are
grey, round with smooth edges and swarms heavily in nutrient agar. Colonies are 0.5mm in size
(last quadrant metrics). Cells are Gram positive, spore forming, capsulated and motile rods.
Biochemical reaction implies Indole negative, methyl red positive, voges proskauer negative and
citrate negative. Triple Sugar Iron implies acid butt and alkaline slant with no gas production.
Catalase positive and oxidase positive. Beta-hemolytic patterns were observed in blood agar. The
strain NCT47 was sensitive to ampicillin (22mm) and resistant to azithromycin, rifampicin and
ampicillin. Primary screening for amylases and lipases reveals positive reactions.
16S rRNA gene sequencing: NC44 was 99.6% similar to Bacillus velezensis (GenBank accession
number: NR_075005).
Based on the colony and biochemical characteristics, the strain NCT6 was identified as Bacillus
stercoris. NCT44 was 99.6% similar to Bacillus velezensis (GenBank accession number:
NR_075005). 16S rRNA gene sequence of NCT44 can be globally accessed through genbank
accession number OQ271432.
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 1400 positions in the final dataset.
The strain NCT44 was deposited in National
College Culture Collection Centre (ncccc) and
are freely available for academic and
research purposes. The strain can be order
here www.ncccc.in.