Isolation and characterization of Aeromonas salmonicida strain NCT48 from a marine fish gut

Abstract

As a part of academic lab in bacteriology, marine fish gut samples were serially diluted in 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
1
Isolation and characterization of Aeromonas salmonicida strain NCT48 from a marine fish
gut
Background
As a part of academic lab in bacteriology, marine fish gut samples were serially diluted in 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.
Results
Strain description: NCT48 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are greyish white and sticky in nature. Cells are Gram negative, non-sporulative and motile cocco-
bacilli. Biochemical reaction implies Indole positive, methyl red positive, voges proskauer
negative and citrate positive. Triple Sugar Iron implies acid butt and slant with gas production
but no hydrogen sulfide. Catalase and oxidase positive. NCT48 was positive for lipase production
and negative for amylase. The genomic DNA of NCT48 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
16S rRNA gene
sequencing:
Based on the colony,
biochemical and
molecular
characteristics, the
strain NCT48 was
identified as Aeromonas
salmonicida. The 16S
rRNA gene sequences of
NCT48 was 99.57%
similar to Aeromonas
salmonicida (GenBank
accession number:
NR_113635) and can be
globally accessed through genbank accession number
OQ271433.
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 1326
positions in the final dataset.
The strain NCT48 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