Isolation and characterization of Mesobacillus jeotgali strain NCT237 from the human skin microbiome

Abstract

Objective is to isolate and characterize a bacterial pure culture from human skin.

Full text

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Isolation and characterization of Mesobacillus jeotgali strain NCT237 from the human
skin microbiome
Objective is to isolate and characterize bacterial pure culture from human skin.
. 1. ISOLATION OF BACTERIA:
Media preparation
1. Half strength nutrient agar (HiMedia) was prepared and autoclaved at 121⁰C, 15 lbs pressure for 15
minutes.
2. Laminar air flow chamber’s floor was surface sterilized with 70% ethanol and UV sterilized for 15
minutes.
3. After sterilization, autoclaved glass wares and Petri plates were brought to Laminar Air Flow Chamber.
4. Nutrient Agar ( NA) media was allowed to cool down to hand bearable temperature (~55⁰C) and
poured in Petri plates for solidification.
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 rubbed on forehead
( several times).
3. This cotton swab was simple streaked from top to bottom in zigzag
manner in the nutrient agar plates.
4. Inoculated plates were incubated for 24 hours at 370C in incubator.
5. An un-inoculated plate act as a control.
Result
164 CFUs were observed along the streak lines following incubation.
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.
SUBCULTURING THE TARGETED BACTERIAL COLONY
1. Half strength nutrient agar medium was prepared in distilled water.
2. Glass wares, Petri plates were sterilized by autoclaving for 20 minutes
at 121⁰C and 15 lbs. pressure.
3. NA plates were prepared as mentioned above and a single colony was
quadrant streaked into fresh NA plate.
4. The plates were incubated for 24 hours at 370C in incubator.

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Gram’s 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. A
quarter loops of cells were taken from an
isolated colony and smeared from the centre of
the droplet.
3. The smear was heat fixed by quickly
passing over the Bunsen burners flame.
4. The smear was flooded with primary
stain crystal violet for one minute.
5. 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. 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 ROD.
2)
SPORE 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. A quarter loops
of cells were taken from an isolated colony and
smeared from the center of the droplet.
(3) The smear was heat fixed by quickly passing
over the Bunsen burners flame.
(4) Then smear was filled with the stain
Malachite green and incubated for 5 minutes in
boiling stage of water bath.
(5) Following incubation, washout the stain and

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add saffron for 30 seconds.
(6) The slide was washed gently and blot dried with absorbent paper.
(7) The slide was observed under oil immersion (100x) using a bright field microscope.
Result
The bacterial spores and vegetative cells were observed in microscope and identified as SPORE
FPORMING BACTERIA.
3)
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 (grown for 24hrs in nutrient broth) 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 (by 900 for four times) 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 and dried before picking up
antibiotic discs.
6. The antibiotic discs were placed at a distance of about 24mm from each
other (maximum of 5 discs (including blank at the centre) could be placed in
15mm Petri dish).
7. Each disc was slightly pressed with forceps to ensure that it is in good
contact to avoid misplacement.
8. The plates were incubated straightly (not upside down; from preventing the disc to fall off) 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.
Result
NCT 236
Zone of inhibition in millimeters
Amoxicillin
Gentamicin
Rifampin
Tetracycline
Ampicillin
Penicillin
Resistance
22
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16
Resistance
Resistance
The strain NCT237 was resistant to all lactum ringed antibiotics and most sensitive to Gentamycin.
4)
CATALASE TEST
1. Using a sterile inoculation loop or Pasteur pipette, few drops of bacterial culture was place on a clean, dry
glass slide.

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2. Over the sample, 2-3 drops of 3% hydrogen peroxide
solution (H2O2) was added and mixed well.
3. The effervescence of oxygen bubbles was observed.
RESULT: CATALASE POSITIVE
9)
OXIDASE TEST
1.
A clean glass slide was taken and aseptically oxidase disk was placed
.
2.
Over the disc few drops of bacterial culture was aseptically placed.
3.
The change in the colour of the disc from white to violet was observed.
4.
The change in the colour indicate oxidase positive.
RESULT
The colour change was observed in the disc. OXIDASE POSITIVE.
10)
INDOLE TEST
1. 5ml of Tryptone broth was prepared and transferred to test tubes, sterilized by autoclaving at 121°C for
15 minutes.
2. After sterilization, tubes were cooled and then the test cultures were inoculated to the respective test
tubes except control tube.
3. All the tubes were incubated 24 hours at 37°C.
4. After incubation, the few drops of Kovac’s reagent were added and the formation of a red
layer at the top of the culture was observed indicating the positive reaction.
RESULT:
The red layer was observed in the tube culture. INDOLE POSITIVE.
11) METHYL RED TEST
1. 5 ml MR-VP medium was transferred to the test tubes and sterilized by autoclaving at 121°C for 15 minutes.
2. After sterilization, the tubes were inoculated with the test culture (except for the control
tube).
3. All tubes were incubated at 37°C for 24 hrs.
4. After incubation, 3-4 drops of methyl red indicator was added into each tube. A distinct red
colour indicates the positive test; yellow colour indicates a negative test.
RESULT:
The red ring was observed. Methyl red positive.
12) VOGES PROSKAUER TEST:
1. 5 ml MR VP medium was transferred to the test tubes and sterilized by autoclaving at 121°C for 15
minutes.
2. After sterilization, the tubes were inoculated with the test culture (except for the control tube).
3. All tubes were incubated at 37°C for 24 hrs.

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4. After incubation, 0.5 ml of Barrit’s solution A and equal volume of Barrit’s reagent B
was added into each tube. A pink colour formation indicates the positive test; yellow
colour indicates a negative test.
RESULT:
The pale yellow colour was observed. VOGES PROSKAUER NEGATIVE.
13) CITRATE UTILIZATION TEST:
1. Simmons Citrate agar was prepared and sterilized at 121.5°C for 15 minutes.
2. After sterilization, the media was cooled and transferred to test tube forming
slants.
3. After solidification, test tubes were inoculated with the bacterial culture except the
control tube.
4. The tubes were incubated at 37°c for 24-48 hrs.
5. After incubation, the colour change in the media from green to blue regarded as
positive.
RESULT:
The media’ colour were didn’t change. citrate negative.
14) TIPLE SUGAR IRON TEST (TSI):
1. TSI agar was prepared and sterilized by autoclaving at 121°C for 15 minutes.
2. After sterilization, the medium was cooled and transferred into tubes and slants
were prepared with butt and slant.
3. After solidification an under aseptic conditions the organisms were inoculated in
butt and slant region of medium using inoculation needle.
4. The tubes were incubated for 24-48 hrs at 37°C and then results were recorded.
RESULT:
The butt and slants are red. So it is an alkaline/alkaline reaction.
15) STARCH HYDROLYSIS:
1. Starch agar medium was prepared and transferred to the petri plates.
2. Under aseptic conditions, a simple streak was made on the agar plate using
inoculation loop.
3. The plates were incubated at 37°C for 24 hrs.
4. After incubation, the plates were flooded with gram’s iodine solution.
5. The plates were observed for the zone of transparency around the bacterial colony, an indication of

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starch hydrolysis.
RESULT:
When the gram’s iodine was added the zone of hydrolysis was observed. starch positive.
16) ISOLATION OF BACTERIAL GENOMIC DNA ISOLATION:
1. For bacterial isolates from solid media, a loop full of bacterial colony was dispensed into 1 ml of
phosphate buffer saline (PBS) (for 100ml volume: 300 mM NaCl (1.75g), 2.7 mM KCl (0.02g), 10mM Na 2HPO4
(0.14g), 1.7 mM KH2PO4 (0.022g), pH 7.4) in 1.5ml centrifuge tube under clean room condition.
2. The culture in PBS was well agitated or flash spun to dispense most of the bacterial cells into the PBS
solution.
3. The inoculation loop was then flame sterilized and kept aside.
4. 1.5ml tube was closed and inverted several times for dispensing the bacterial cells and centrifuged at
6000 g for 5 minutes.
5. For broth cultures, 1.5ml of culture broth was taken in the 1.5ml centrifuge tube and centrifuged at
6000 g for 5 minutes.
6. The supernatant was carefully discarded and 600 ml of lysis buffer (for 500 ml volume: 50mM Tris –
HCl (3.028g), 20mM EDTA (2.922g), 2% SDS (10g), pH 8) was added to the pellet.
7. The tube was incubated in the water bath at 65°C for 1 hour.
8. The mixture was vortexed for 30 seconds every 10 minutes throughout the water bath incubation. An
equal volume (600 ml) of Phenol-Chloroform-Isoamyl alcohol (PCI) (25:24:1) reagent was added and gently
mixed by inverting the tube.
9. The tubes were centrifuged at 6000g for 10 minutes and the supernatant was transferred to a fresh 1.5
ml tube.
10. Equal volume of 100% ethanol was added and DNA was precipitated (overnight incubation at -20°C
could be followed after ethanol addition for enhanced precipitation).
11. Tubes were centrifuged at 10,000g for 10 minutes and the pellets were air dried (until the ethanol smell
vanishes).
12. 20-50 ml (based on the pellet size) of TE buffer (for 100 ml TE buffer: 1M Tris HCl (0.121g), 0.5 M EDTA
(0.029g), pH 8) was added and the DNA was stored at -20°C.
17) Agarose gel electrophoresis:
1. 0.5g agarose was weighed and dissolved in 50mL of 1x Tris base Acetic acid Ethylenediaminetetraacetic
acid (TAE) Buffer (100 ml of 50X concentration; Tris Base (24.2g), Acetic acid (5.71 ml), 0.5M EDTA, pH 8). 250
ml conical flask was used for preparing 50 ml solution to avoid overflow of gel solution during heating in
microwave oven.
2. The solution was heated in microwave oven to boiling constituency (until the solution turns
transparent from translucent).
3. The solution was cooled down to hand bearable temperature and 2μl of Ethidium bromide (EtBr)
solution (10 mg /ml) was added and mixed well by gentle swirling.

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4. The solution was then poured in to the gel tray-comb set up (securely tapped gel plates containing well
combs).
5. The solution was allowed to cool to form a gel.
6. The gel was carefully transferred to the electrophoresis tank pre-filled with 1x TAE buffer (composition
as mentioned above).
7. DNA samples were prepared by mixing up to 8μl of DNA sample with 2μl of 5x gel loading dye (5x) in
the insulation tape placed in the floor near the tank.
8. The comb was removed from the gel and the samples were
loaded into the well.
9. Appropriate electrodes were connected to the power pack
and electrophoresis was conducted at 50-100volts for 20min.
10. The movement of the sample was monitored with
reference to tracking dye (Bromophenol blue) and electrophoresis
was terminated when the marker has run 3/4th of the gel's length.
11. The gel was placed in the UV-trans illuminator and examined for orange colored bands.
18) PCR and 16S rRNA gene sequencing
1. 16S rRNA genes of the bacterial isolates were amplified using a set of bacterial universal forward primer 8-
27F (5'-AGAGTTTGATCCTGGCTCAG-3’) and reverse primer 1492R (5'-TACACCTTGGCGACGACTT-3’).
2. For polymerase chain reaction (PCR), a total of 50 μl PCR reaction mixture was prepared having 5 μl of 10X
Taq polymerase buffer, 1 μl of 10 mM deoxynucleotides mix , 0.2 μM of each forward and reverse primer,
1.5U Taq DNA polymerase and 30 ng of genomic DNA. PCR amplification was carried out using Applied
Biosystem Veriti 96 well Thermal cycler.
3. The PCR program involved an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at
95°C for 1 min, annealing at 54°C for 45s, primer extension at 72°C for 1min for 35 cycles with a final
extension step at 72°C for 10 min.
4. PCR and DNA sequencing was commercially outsourced to Barcode Biotechnologies Pvt. Ltd., Goa, India.
19) Sequence analysis
BLAST analysis revealed that the 16S rRNA gene sequence of NCT237 shared 99.49% with Mesobacillus
jeotgali (Acc. No.: NR_025060).

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Neighbor-Joining tree computed using the Maximum Composite Likelihood method is shown. This analysis
involved 15 nucleotide sequences. There were a total of 1380 positions in the final dataset. All reference
sequences were obtained from Genbank and are indicated by the accession numbers “NR”. The 16S rRNA gene
sequences produced in the present study was indicated by NCT237.
Genbank accession number of 16S rRNA gene sequence produced in the present study could be accessed
through the accession number: OP351519. The Mesobacillus jeotgali strain NCT237 was deposited to
National College Culture Collection Centre. Users can obtain NCT237 through placing a culture request here
www.ncccc.in.