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N4C
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Isolation and characterization of
Bacillus cereus
from the
fermenter cooling system waters
Objective
To isolate and characterize a bacterium from the fermenter cooling system water.
I. Isolation of bacteria:
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 (~55°C) and
poured in Petri plates for solidification.
Sample preparation:
1. One millilitre of fermenter cooling system water was made
up to 10ml using sterile distilled water.
2. Inside the Laminar Air Flow Chamber, incubated sample
was serially diluted up to 10-6 dilutions.
3. 100 microlitre of diluted sample from 10-5 tube was spread
plated in Nutrient Agar medium using L-Rod.
4. NA plates inoculated with sterile distilled water acted as
control.
5. Inoculated plates were incubated for 24 hours at 370C in
incubator.
Results:
1. Following 24hrs incubation, 540 opaque, white, medium,
round, convex shaped colonies were observed.
2. Control plates were free of any bacterial growth.
3. Fermenter cooling system water contained 5.4 x 108 CFUs per ml.
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II. Sub-culturing the targeted bacterial colony
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 prepared as mentioned
above.
4. After sterilization, glasswares 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
discontinuously quadrant streaked in the NA plate.
7. Plates were left for incubation for 24 hours at 37⁰C.
III. 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% sterile glycerol was transferred to a sterile 1.5mlEppendorf tubes.
3. 0.5 ml of nutrient broth was prepared and transferred to theEppendorf tubes containing
glycerol stock (1:1).
4. Two to three loops full of pure culture was taken and transferred to the glycerol stock (or
more number of colonies could be transferred until the stock looks cloudy).
5. This first glycerol stock was stored overnight at -80C.
IV. 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
mentionedabove
3. Inoculation loop was dipped inside the glycerol
stock and quadrant streaked onto Nutrient agar
plates.
4. Plates were observed after 24 hours of incubation
at 37°C.
Result:
Observation of single type of colony with swarming
type of growth was observed.
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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 -80°C and one in -20°C (which will be used
for further biochemical characterization and for the exploration of biotechnological potential
of the strains).
V. Gram staining
1. A clean glass slide (grease-free) was taken and a drop of sterilized distilled water was
placed on the centre of the slide.
2. Mark the position of the sterile 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 recognised as Gram Positive rods.
VI. 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 inoculums and excess
medium was removed by pressing the swab onto
the wall of the tube.
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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 about24mm 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.
Observation:
Following incubation, zone of clearance
around the disc was measured using
ruler to assess the degree of sensitivity to a particular antibiotic.
VII. Catalase test
1. Laminar air flow chamber was cleaned and wiped with 70% ethanol and UV light was
turned on for 15 minutes.
2. The UV was turned off, laminar air flow was turned on and then the chamber is opened.
3. The bacterial culture plate, inoculation was kept inside.
4. After Turning on the flame, hand was wiped with 70% ethanol.
5. Using a wooden pick a small amount of bacterial colony was picked up and inserted into
3% hydrogen peroxide solution in a small Effendorf tube.
Observation:
Effervescence (Bubbling) was observed in the bacterial biomass picked up in the wooden pick.
The bacterial strain is catalase positive.
VIII. OXIDASE TEST
Procedure:
1. Laminar air flow chamber was cleaned and
wiped with 70% ethanol and UV light was turned
on for 15 minutes.
2. The UV was turned off and Air was turned on
then the chamber is opened.
3. The Bacterial culture plate, sterile toothpick,
Oxidase discs were kept inside.
4. After Turning on the flame, hand was wiped with 70% ethanol.
Zone of inhibition in mm
Ampicillin
25 g
Gentamicin
10 g
Rifampicin
5 g
Tetracycline
30 g
R
17
26
33
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NR 115526 Bacillus cereus
NR 115714 Bacillus cereus
NR 112630 Bacillus cereus
NR 074540 Bacillus cereus
NR 113266 Bacillus cereus
NCT11
NR 115933 Bacillus coahuilensis
NR 043015 Metabacillus litoralis
NR 024689 Bacillus atrophaeus
NR 109443 Bacillus songklensis
NR 036766 Neobacillus bataviensis
NR 024570 Escherichia coli
74
36
49
100
0.02
5. Using sterile toothpick, small amount of bacterial colony was scrapped out without
disturbing the agar medium.
6. Oxidase discs are sensitive to light thus the bacterial culture in the toothpick was smeared
over the discs as soon as possible.
7. The results are observed within 10seconds.
Observation:
Positive reactions turned the disc in to purple colour
within 30 seconds.
IX. Motility test
Procedure:
1. Cover slip and cavity slide was made grease free.
2. Vaseline drops were placed on four corners of the
cover slip.
3. A loop full of culture from the 24hrs broth was transferred to the center of the cover slip.
4. The cavity slide was taken and placed inversely on the cover slip so that the cavity faces
the culture drop.
5. Then the cavity slide was
immediately inverted so that the
drop will hang inside the cavity in
the cavity slide.
6. The slide was observed under
microscope before the drop dries.
Result:
The bacterial strain is motile in nature.
Identification of 16s rRNA gene
sequences
The 16S rRNA gene sequence of this
study shared 99.73% sequence
similarity with
Bacillus cereus
(GenBank
acc. no.: NR_115714).
The evolutionary history was inferred
using the Neighbor-Joining method. The
optimal tree is shown. The percentage of
replicate trees in which the associated
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taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches.
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 12 nucleotide
sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion
option). There were a total of 1387 positions in the final dataset. The 16S rRNA gene sequence
produced in this study was represented by NCT11. The 16S rRNA gene sequence of
Escherichia coli
was used as an out group.
Inference
The strain was assigned with NCT accession number
NCT11 and GenBank accession number OK631804
.
The 16S rRNA gene sequence if the strain could be now
globally accessed through GenBank accession number
OK631804.
https://www.ncbi.nlm.nih.gov/nuccore/OK631804