Isolation and characterization of Bacillus cereus strain NCT47 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 cereus strain NCT47 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
Strain description: NCT47 grows well in nutrient agar plate at 37°C in 24 hours. Colonies are
white, round with smooth edges and swarm heavily in nutrient agar. Cells are Gram positive,
spore forming, and non-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. The strain NCT47
was sensitive to ciprofloxacin (22mm), azithromycin (12mm), rifampicin (15mm) and resistance
to ampicillin (7mm). Primary screening for amylases and lipases reveals positive reactions. Beta-
hemolytic patterns were observed in blood agar. Carbohydrate fermentation test revealed
organic acid production with gas formations for the sugars, arabinose, glucose, maltose and
sucrose.

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
16S rRNA gene sequencing:
Based on the colony and biochemical characteristics, the strain NCT6 was identified as Bacillus
stercoris. NCT47 was 99.6% similar to Bacillus cereus (GenBank accession number:
NR_115526). 16S rRNA gene sequence of NCT47 can be globally accessed through genbank
accession number OQ271435.
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.