Isolation and characterization of Paenibacillus alvei strain NCT76 from an agri soil

Abstract

As a part of academic lab in microbiology, agriculture soil samples were collected, serially diluted in sterile saline and spread plated on to nutrient agar. Following incubation, 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 Paenibacillus alvei strain NCT76 from an agri soil
Background
As a part of academic lab in microbiology, agriculture soil samples were collected, serially diluted
in sterile saline and spread plated on to nutrient agar. Following incubation, 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: NCT76 grows luxuriantly in nutrient agar plate at 37°C in 24 hours. Colonies
are round, raised with undulate margins, yellowish white, sticky and mucoid in nature. Cells are
Gram positive, non-motile, spore forming bacilli. Biochemical reaction implies Indole negative,
methyl red negative, voges proskauer negative and citrate positive. Triple Sugar Iron implies acid
butt and alkaline slant with no gas production. Catalase negative and oxidase positive. Flocculent
like growth with pellicle formation was observed in nutrient broth. The strain NCT76 was
sensitive to fluconazole, ketoconazole and resistant to amoxicillin, ampicillin.
16S rRNA gene sequencing:
Based on the colony, biochemical and
molecular characteristics, the strain
NCT42 was identified as Paenibacillus
alvei. The 16S rRNA gene sequences of
NCT76 was 99.42% similar to
Paenibacillus alvei (GenBank accession
number: NR_113577) and can be globally
accessed through genbank accession
number OQ255772.
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 1401 positions in the final dataset.
The strain NCT76 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