Isolation and characterization of Psychrobacillus insolitus strain NCT74 from an agri soil

Abstract

As a part of academic lab in microbiology, agriculture soil sample was collected, serially dilued in sterile saline and spread plated on to nutrient agar. 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.

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National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
1
N4C
Isolation and characterization of Psychrobacillus insolitus strain NCT74 from an agri soil
Background
As a part of academic lab in microbiology, agriculture soil sample was collected, serially dilued in
sterile saline and spread plated on to nutrient agar. 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.

National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
N4C
Strain description: NCT74 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are large, with irregular margins, raised, milky white, opaque and swarming in nature. Cells are
Gram positive, 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 and oxidase positive.
Sedimentary growth was more
prominent in nutrient broth. The
strain NCT74 was sensitive to
rifampicin, erythromycin,
streptomycin and resistant to
penicillin.
16S rRNA gene sequencing:
Based on the colony, biochemical
and molecular characteristics, the
strain NCT74 was identified as
Psychrobacillus insolitus. The
16S rRNA gene sequences of

National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
3
N4C
NCT74 was 99.58% similar to Psychrobacillus insolitus (GenBank accession number:
NR_042709) and can be globally accessed through genbank accession number OQ255768.
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 1416 positions in the final dataset.
The strain NCT74 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