Isolation and characterization of Neisseria cinerea strain NCT20 from an agriculture soil

Abstract

As a part of academic lab in bacteriology, human skin microbiome was sampled and swabbed onto a half a strength 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 Neisseria cinerea strain NCT20 from an agriculture soil
Background
As a part of academic lab in bacteriology, human skin microbiome was sampled and swabbed
onto a half a strength 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: NCT20 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are white, circular, flat and sticky with smoother edges. Cells are Gram negative, non-sporulative
and non-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 absence of hydrogen sulfide. Catalase and oxidase positive. The strain NCT20
was sensitive to ampicillin, tetracyclin, ciprofloxacin and resistant to azithromycin.
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular characteristics, the strain NCT20 was identified
as Neisseria cinerea. The 16S rRNA gene sequences of NCT20 was 99.65% similar to Neisseria
cinerea (GenBank accession number:
NR_121687) and can be globally
accessed through genbank accession
number OQ255727.
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 1328
positions in the final dataset.
The strain NCT20 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