Isolation and characterization of Klebsiella oxytoca strain NCT83 from agriculture soil

Abstract

As a part of academic lab in bacteriology, agriculture soil sample was collected, serially diluted and plated on to nutrient agar plates. 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
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Isolation and characterization of Klebsiella oxytoca strain NCT83 from agriculture soil
Background
As a part of academic lab in bacteriology, agriculture soil sample was collected, serially diluted
and plated on to nutrient agar plates. 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: NCT83 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are small, greyish white, round with smooth edges. Colonies were on average 0.4mm in size (last

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
quadrant metrics). Cells are Gram negative, sporulative motile rods. Biochemical reaction implies
Indole positive, methyl red positive, voges proskauer negative and citrate positive. Triple Sugar
Iron implies acid butt and alkaline slant with no gas production. Catalase positive and oxidase
positive. The strain NCT49 was sensitive to ampicillin (13mm) and resistant to clindamycin,
neomycin and ampicillin.
16S rRNA gene sequencing:
Based on the colony and
biochemical characteristics, the
strain NCT83 was identified as
Klebsiella oxytoca. NCT83 was
99.57% similar to Klebsiella
oxytoca (GenBank accession
number: NR_119277). 16S rRNA
gene sequence of NCT27 can be
globally accessed through genbank
accession number OQ271434.
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 1380 positions in the final dataset.
The strain NCT83 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