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N4C
1
Isolation and characterization of Staphylococcus epidermidis
from spectacles
Objective
To isolate and characterize bacteria from the lenses of the spectacles.
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 in Petri plates for
solidification.
1.2. Sample preparation
1. O.8 % saline was prepared in a conical flask and sterilized in
autoclave for 20 minutes at 121⁰C and 15 lbs. pressure.
2. A sterile cotton bud soaked in the saline was used to swab the
spectacles inside the Laminar Air Flow Chamber.
3. The cotton swab was simple streaked across the Petri plate in a zigzag fashion in the nutrient agar
plate.
4. Inoculated plates were incubated for 24 hours at 370C in incubator.
1.3. Result
1. There were140 CFUs observed after incubation along the streak lines.
2. These colonies appeared white, spherical, raised and cohesive, approximately 1-2mm(in
diameter.)
2. SUBCULTURING THE TARGETED BACTERIAL COLONY
1. Half strength nutrient agar medium was prepared in distilled water.
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2. Glass wares, 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
Laminar Air Flow Chamber.
5. Nutrient agar medium was poured in Petri plates and left for
solidification
6. Single colony was picked using inoculation loop and quadrant
streaked in the NA plate.
7. Plates were left for incubation for 24 hours at 37⁰C. The
picture of quadrant plate was shared here.
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 was 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 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 single type of colony confirms the purity of the glycerol stock.
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Two more glycerol stocks
From the revived plates, prepare 2 more glycerol stocks. For a
bacterial species, total of 3 glycerol stocks were prepared. Two
were kept at -80C and one in -20C (which will be used for
further biochemical characterization and for the exploration of
biotechnological potential of the strains).
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. Mark the position of the water droplet in the slide from below with a marker.
3. A quarter loops of cells were taken from an isolated colony and smeared from the centre of the
droplet.
4. The smear was heat fixed by quickly passing over the Bunsen burners 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
8. Then the decolorizing agent was added drop by drop
for 15 seconds
9. The slide was flooded with counter stain; 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 microscope and identified as GRAM POSITIVE COCCI.
Antibiotic sensitivity test
1. The working surface of the LAF was sterilized with disinfectant before performing the
test.
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2. A sterile cotton swab was dipped into the known concentration of bacterial inoculum
and 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 was 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.
8. The plates were incubated upside down for
24 hours at 37ºC.
9. Following incubation, zone of clearance around the disc was measured using ruler to
assess the degree of sensitivity to the particular antibiotic.
R- Indicates resistance
Identification of the 16S rRNA gene sequence
The 16S rRNA gene sequence produced in the current study shared 99.57% identity with
Staphylococcus epidermidis (GenBank acc. no.: NR_036904).
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) is 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
Zone of inhibition in mm
Amoxicillin
Gentamicin
Rifampin
Ampicillin
R
27
10
R
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NR 113351 Staphylococcus intermedius
NR 024666 Staphylococcus delphini
NR 036791 Staphylococcus lutrae
NR 116912 Staphylococcus rostri
NR 116422 Staphylococcus massiliensis
NR 027520 Staphylococcus equorum
NR 117006 Staphylococcus capitis
NR 113957 Staphylococcus epidermidis
NCT108
NR 036904 Staphylococcus epidermidis
NR 024570 Escherichia coli
90
93
97
54
60
99
100
0.02
11 nucleotide sequences. All
ambiguous positions were
removed for each sequence pair
(pairwise deletion option). There
were a total of 1309 positions in
the final dataset. The 16S rRNA
gene sequences produced in the
current study was indicated as
NCT108. The 16S rRNA gene
sequence of E. coli was used as an
out-group.
Inference
The strain was assigned with NCT accession number NCT108 and
identified as Staphylococcus epidermidis using 16S rRNA gene sequencing.
The strain could be now globally accessed through GenBank accession
number ON241804. https://www.ncbi.nlm.nih.gov/nuccore/ON241804