Isolation and characterization of Paenibacillus lentimorbus strain NCT5 from garden soil

Abstract

As a part of academic lab in bacteriology, fertile garden soil collected, serially diluted 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.

Full text

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
1
Isolation and characterization of Paenibacillus lentimorbus strain NCT5 from garden soil
Background
As a part of academic lab in bacteriology, fertile garden soil collected, serially diluted 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.
Results

National College Culture Collection Centre
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
Strain description: NCT5 readily grows in nutrient agar plate at 37°C in 24 hours. Colonies
appeared white, round and shiner with smooth edges. Colonies are 0.3mm in size (last quadrant
metrics). Cells appears Gram positive, sporulative, motile rods in chains. Biochemical reaction
implies Indole negative, methyl red negative, voges proskauer negative and citrate negative
reactions. Triple Sugar Iron implies acid butt and slant with no gas production. Catalase and
oxidase were positive. Primary enzyme screening implies positive for amylase and lipase
synthesis. NCT5 was sensitive to amoxicillin,
streptomycin, clindamycin and resistant to
neomycin (11mm), ampicillin (15mm).
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular
characteristics, the strain NCT35 was identified as
Paenibacillus lentimorbus. NCT5 was 99.65%
similar to Paenibacillus lentimorbus (GenBank
accession number: NR_115981). 16S rRNA gene
sequence of NCT27 can be globally accessed
through genbank accession number OQ271411.
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 1449 positions in the final dataset.
The strain NCT5 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