Isolation and characterization of Sporosarcina pasteurii strain NCT85 from an agri soil

Abstract

As a part of academic lab in microbiology, agri soil sample was collected, serially diluted and spread plated onto a nutrient agar plates. 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 Sporosarcina pasteurii strain NCT85 from an agri soil
Background
As a part of academic lab in microbiology, agri soil sample was collected, serially diluted and
spread plated onto a nutrient agar plates. 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:
NCT85 grows stringently
in nutrient agar plate at
37°C in 24 hours.
Colonies are small, round,
milky white, shiny with
rough edges. Cells are
Gram positive, sporulative, non-motile rods. Biochemical reaction implies Indole negative, methyl
red positive, voges proskauer negative and citrate positive. Triple Sugar Iron implies acid butt
and alkaline slant with no gas production. Catalase negative and oxidase positive. Micro-
aerophilic type of growth was observed in nutrient broth. The strain NCT85 was sensitive to
ketoconazole, amoxicillin and streptomycin and resistant to ampicillin.
16S rRNA gene sequencing:
Based on the colony, biochemical and
molecular characteristics, the strain NCT85
was identified as Sporosarcina pasteurii.
The 16S rRNA gene sequences of NCT85
was 99.51% similar to Sporosarcina
pasteurii (GenBank accession number:
NR_104923) and can be globally accessed
through genbank accession number
OQ255786.
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 1384 positions in the final dataset. The strain
NCT85 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