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Isolation and characterization of Serratia fonticola from human scalp
Objective
To isolate and characterize a bacterial pure culture from scalp region
1. ISOLATION OF BACTERIA:
1.1. Media preparation
1. Half strength nutrient agar (HiMedia) was prepared
by autoclaving at 121⁰C, 15 lbs pressure for 15
minutes.
2. Laminar air flow chamber floor was surface
sterilized with 70% ethanol and UV sterilized for 15
minutes.
3. After sterilizations, autoclaved glass wares and Petri
plates were brought to Laminar Air Flow Chamber.
4. Nutrient Agar media was allowed to cool down to
hand bearable temperature (~55C) and poured
onto Petri plates for solidification.
1.2. Sample preparation
1. 0.8% saline was prepared in a conical flask and
sterilized in an autoclave for 20 minutes at 121˚C and 15 lbs. pressure.
2. Cotton swab was dipped in saline solution and then carefully rubbed on the scalp.
3. The swab containing the sample was rubbed gently on the plate to transfer the bacterial
colonies.
4. Inoculated plates were incubated for 24 hours at 37˚C
in the incubator.
2. SUBCULTURING THE TARGETED BACTERIAL
COLONY:
1. Half strength nutrient agar medium was prepared in
distilled water.
2. Glass wares and Petri plates were kept for
sterilization in autoclave for 20 minutes at 121⁰C and
15 lbs. pressure.
3. Laminar air flow chamber was prepared as
mentioned above.
4. After sterilization, glassware and Petri plates were
brought to LaminarAir Flow Chamber.
5. Nutrient agar medium was poured into Petri plates
and left for Solidification
6. Single colony was picked using an inoculation loop and quadrant streaked in the NA plate.
7. Plates were left for incubation for 24 hours at 37⁰C. The picture of the quadrant plate was
shared here.
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3. PREPARATION OF GLYCEROL STOCK
1. 5 ml of distilled water is mixed with 5 ml of 100%
glycerol to make 10ml of 50% glycerol solution.
2. 0.5 ml of 50% glycerol was transferred to a sterile 1.5ml
Eppendorf tubes.
3. 0.5 ml of nutrient broth was prepared and transferred
to the Eppendorf tubes containing glycerol stock (1:1)
4. Two to three loops full of pure culture were taken from
a colony and transferred to the glycerol stock.
5. Glycerol stock was then stored in deep freeze at -800C.
4. RE-VIABILITY TEST
1. Reviving ability of the glycerol stock is checked in a half-strength nutrient agar medium.
2. Media was sterilized and laminar was prepared as
mentioned above (section 1.1.).
3. Inoculation loop was dipped inside the glycerol stock
and quadrant streaked onto NA plates.
4. Plates were observed after 24 hours of incubation at
37C.
Result:
Observation of a single type of colony confirms the purity of
the glycerol stock.
Two more glycerol stocks
From the revived plates, 2 more glycerol stocks were prepared
as backups. For a bacterial species, a total of 3 glycerol stocks
were prepared. Two were kept at -80C and one at -20C
(which will be used for further biochemical characterization
and the exploration of the biotechnological potential of the strain).
5. GRAM STAINING
1. A clean glass slide (grease-free) was taken and a drop of
sterilized distilled water was placed on the center of the
slide.
2. The position of the water droplet was marked in the
slide from below with a marker.
3. A quarter loops of cells were transferred from an
isolated colony in the plate to the center of the droplet
in the slide and smeared.
4. The smear was heat-fixed by quickly passing over the
Bunsen burner’s flame.
5. The smear was flooded with primary stain crystal
violet for one minute.
6. Then the slide was washed gently with tap water for 2 seconds.
7. The slide was flooded with Gram’s iodine for one minute and washed gently with tap
water.
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8. Then the decolorizing agent was added drop by drop for 15 seconds.
9. The slide was flooded with counterstain; safranin for 30 seconds.
10. The slide was washed gently and blot dried with absorbent paper.
11. The slide was observed under oil immersion (100x) using a bright field microscope.
Result
The bacterial cells were observed in the microscope and identified as GRAM-NEGATIVE RODS.
6. SPORE STAINING
1. Clean glass slide (grease-free) was taken and a drop of sterilized distilled water was placed on the
center of the slide
2. Mark the position of the water droplet on the slide below the
marker.
3. A quarter loops of cells were taken from anisolated colony and
smeared from the center of the droplet.
4. The smear was heat-fixed by quickly passing over the Bunsen
burner’s flame.
5. The smear was flooded with malachite greenstain solution
and the slide was steamed over hot water for 5 minutes.
6. Excess stain was removed with tap water.
7. The slide was flooded with counterstain; safranin for 30
seconds
8. The slide was washed gently and blot dried with absorbent paper.
9. The slide was observed under oil immersion (100x) using a bright field microscope.
Result
Spores were observed in green color.
Antibiotic sensitivity test
1. The working surface of the LAF was sterilized with
disinfectant before performing the test.
2. A sterile cotton swab was dipped into the known
concentration of bacterial inoculum and the excess
medium was removed by pressing the swab onto the wall
of the tube.
3. The surface areas of the Mueller-Hinton agar plates were
swabbed completely by rotating the plate to produce
lawn culture.
4. The plates were allowed to dry for 5 minutes for the proper absorption of the inoculums.
5. The forceps were
sterilized with
alcohol before
picking up antibiotic
discs.
6. The antibiotic discs were placed at a distance of about 24mm from each other.
7. Each disc was slightly pressed with forceps to ensure that it is in good contact to avoid
misplacement.
Zone of inhibition in mm
Penicillin
Amoxicillin
Gentamicin
Cefotaxime
Streptomycin
R
19
24
22
25
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NCT94
NR 025339 Serratia fonticola
NR 114156 Serratia fonticola
NR 114577 Serratia fonticola
NR 116808 Serratia fonticola
NR 117512 Serratia symbiotica
NR 114578 Serratia odorifera
NR 036886 Serratia marcescens
NR 169468 Serratia surfactantfaciens
NR 157762 Serratia oryzae
NR 024570 Escherichia coli
100
63
51
47
99
81
33
100
0.01
8. The plates were incubated upside down for 24 hours at 37ºC.
9. Following incubation, the zone of clearance around the disc was measured using a ruler to
assess the degree of sensitivity to the particular antibiotic.
R- Indicates resistance
Species identification
The 16S rRNA gene sequence shared
99.71% sequence similarity with
Serratia fonticola (GenBank acc. no.:
NR_025339). The evolutionary history
was inferred using the Neighbor-
Joining method. The optimal tree is
shown. The percentage of replicate
trees in which the associated taxa
clustered together in the bootstrap test
(500 replicates) are shown next to the
branches. 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 Kimura 2-parameter 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 1397 positions in the final
dataset. The 16S rRNA gene sequences
produced in the current study was indicated by NCT94 and the 16S rRNA gene sequence of
Escherichia coli was used as an out group.
Inference
The strain was assigned with NCT accession number NCT94 and
identified as Serratia fonticola using 16S rRNA gene sequencing. The
strain could now be globally accessed through GenBank accession
number ON242376
https://www.ncbi.nlm.nih.gov/nuccore/ON242376