Isolation and characterization of Paenibacillus larvae strain NCT73 from an agri soil

Abstract

As a part of academic lab in microbiology, agriculture soil samples were serially diluted in sterile saline and spread plated in half a strength nutrient agar media. 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
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Isolation and characterization of Paenibacillus larvae strain NCT73 from an agri soil
Background
As a part of academic lab in microbiology, agriculture soil samples were serially diluted in sterile
saline and spread plated in half a strength nutrient agar media. 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.
Strain description: NCT73 grows slowly in nutrient agar plate at 37°C in 24 hours. More
prominent growth was observed after 48hours of incubation. Colonies are round, yellowish
white, flat, smooth and swarming. Cells are Gram positive, sporulative and motile rods.
Biochemical reaction implies Indole negative, methyl red positive, voges proskauer negative and
citrate positive. The strain was positive for amylase and lipase production. Triple Sugar Iron
implies acid butt and alkaline slant with no gas production. Catalase negative and oxidase
positive. The strain NCT73 was resistant to rifampicin, erythromycin, penicillin and
streptomycin.

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
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular characteristics, the strain NCT73 was identified
as Paenibacillus larvae. The 16S rRNA gene sequences of NCT73 was 99.52% similar
Paenibacillus larvae (GenBank accession number: NR_042947) and can be globally accessed
through genbank accession number OQ255767.

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
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 1346 positions in the final dataset.
The strain NCT73 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