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Isolation and characterization of
Bacillus subtilis
from the garden soil
OBJECTIVE:
To isolate and characterize bacteria from garden soil.
METHODOLOGY:
1. Isolation of bacterial community
1. Half strength NA media and 0.7% NaCl solution were prepared and autoclaved along with a petri plate and
the test tubes.
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 cleaned and wiped with 70% ethanol and UV light was turned on for about
15 minutes.
4. After sterilization, glass wares and Petri plates were brought to LAF.
5. Media was poured on the plate allowed to settle in clean room conditions.
6. One gram of soil sample was taken in a sterile beaker and mixed with 100ml of saline water shaken for 1
minute to get the slurry.
7. The serial dilution tubes were prepared (autoclaved) with 9ml of saline water each and serially diluted
serially with the 1ml of slurry (10-2, 10-3, 10-4, 10-5, 10-6).
8. Finally, 100µL of the diluted samples were spread over the NA
plates and properly labeled (with dilution factor, date and time).
9. The plates were incubated at 37℃ for 24 hours.
Result:
1. Following incubation about 5 to 6 punctiform colonies which are
opaque and nearly white in colour were observed in all plates after
10-3.
2. Colonies had a smooth margin with raised elevation, and slimy,
moist texture.
3. After 48 hours of incubation, the colony size increased significantly.
2. Sub-culturing the targeted bacterial colony:
1. Half strength nutrient agar medium was prepared in distilled water.
2. Nutrient agar medium was poured in petri plates and left them to solidify under clean room conditions.
3. Single colony was selected was quadrant streaked into the NA plates from the mother plate.
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4. Plates was left for incubation for 24 hours at 37⁰C and observed.
Result:
The pure culture was isolated by quadrant streaking method.
3. Preparation of glycerol stock:
1. A total of 1.5 ml of 50% glycerol stock was prepared and 0.5 ml was
transferred to 3 sterile Eppendorf tubes.
2. 1.5 ml of nutrient broth was prepared and 0.5 ml was transferred to
each Eppendorf tube containing glycerol stock (1:1 ratio).
3. Three or few loop full of pure culture was taken from isolated colonies and transferred to the glycerol
stock prepared (add more colonies until the stock appear turbid).
4. The prepared glycerol stock was then labeled with appropriate NCT number.
5. 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).
4. Re-viability test:
1. Half strength nutrient agar medium was prepared in distilled
water.
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 cleaned and wiped with 70%
ethanol and UV light was turned on for 15 minutes.
4. After sterilization, glass wares and Petri plates were brought to
LAF.
5. Nutrient agar medium was poured in petri plates and left for
solidification.
6. A sterile loop was dipped in the glycerol stock in order to pick up colonies and then quadrant streak was
performed on a fresh plate.
7. The streaked plate was incubated at 37℃ for 24 hours.
8. After incubation the colonies were analyzed in order to check that the prepared stock contains pure
culture.
Result:
The colonies were observed following incubation confirmed the viable nature of the glycerol stock.
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5. Gram staining:
1. A clean glass slide was taken and a drop of distilled water was placed at the centre of the slide. A sterile
loop was dipped on the plate containing pure culture to pick
bacterial cells and a smear was prepared and heat-fixed.
2. The smear was flooded with crystal violet stain for one minute.
3. Then the slide was washed gently with tap water for 2 seconds.
4. The slide was flooded with Gram’s iodine (mordant) for one minute
and washed gently with tap water.
5. Then the decolorizing agent (alcohol) was added drop by drop for
15 seconds.
6. The slide was flooded with a counterstain, safranin for 30 seconds.
7. The slide was washed gently and blot dried with absorbent paper.
8. The slide was observed under oil immersion (100x) using a bright
field microscope.
Result:
By gram staining, it is observed that the bacteria are gram positive
rods. Presences of endospores are speculated and the culture was
again Gram stained after 48 hours. The second Gram staining had increased number of exospores.
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 an isolated colony and smeared from the center of the droplet.
4. The smear was heat-fixed by quickly passing over the Bunsen
burners flame.
5. The smear was flooded with malachite green stain 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:
The bacterium had central endospores. Vegetative cells were pink and spores were green in color.
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NR 116017 Bacillus subtilis
NR 027552 Bacillus subtilis
NR 113265 Bacillus subtilis
NR 112629 Bacillus subtilis
NCT99
NR 112116 Bacillus subtilis
NR 024690 Bacillus carboniphilus
NR 132725 Bacillus borbori
NR 133973 Bacillus fengqiuensis
NR 149175 Bacillus mesophilus
NR 024570 Escherichia coli
93
45
40
63
100
0.02
7. Antibiotic profiling:
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 excess medium was
removed by pressing the swab onto the wall of the tube.
3. The surface areas of the Muller-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. Following incubation, zone of
clearance around the disc was measured using ruler
to access the degree of sensitivity to the particular
antibiotic.
Identification of the 16S rRNA gene sequence
The 16S rRNA gene sequence produced in the
current study shared 99.71% similarity with
Bacillus subtilis
(GenBank acc. no.: NR_112116).
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
Zone of inhibition in mm
Rifampicin (5 μg)
Gentamicin (50 μg)
Ampicillin (25 μg)
Amoxicillin (10 μg)
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26
R
2. R
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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 1351 positions in the
final dataset. The 16S rRNA gene sequence produced in this study was indicated by NCT99. The 16S rRNA gene
sequence of
Escherichia coli
was used as an out group.
Inference
:
The strain was assigned with NCT accession number NCT99 and
identified as
Bacillus subtilis
using rRNA gene sequencing. The strain
could be now globally accessed through GenBank accession number
ON243966. https://www.ncbi.nlm.nih.gov/nuccore/ON243966