CHAPTER-3

MATERIALS AND METHODS

The present investigation entitled Isolation and Characterization of

Halobacteria from Rhizospheric Soil of Chilli (Capsicum annum L.) was
undertaken to isolate and characterize Halobacterium strains from samples of saline
soils collected from different agro climatic regions of Uttar Pradesh and screening of
halophillic plant growth promoting bacteria from rhizospheric soil of chilli. The
details of the materials and methods used during the course of present investigation
have been presented in the following heading;
3.1 Experimental Site
The present investigation was undertaken at National Bureau of
Agriculturally Important Micro organisms (NBAIM), Mau Nath Bhanjan (U.P.)
Laboratory. The details of materials used and methods followed in the experiment are
as follows;
3.2 SOIL SAMPLING
Soil samples were collected from different agro climatic zones of U.P.
affected by salt using systematic sampling method. Soil samples collected were
screened for presence or absence of available Phosphate, Ammoniacal nitrogen,
Nitrate Nitrogen, Phosphorus and pH.
3.3 SOIL ANALYSIS
Soil from different samples was analysed for the test of pH, Available
Phosphate, Ammoniacal Nitrogen, Nitrate Nitrogen & Phosphorus. The latest soil
testing kit by Hi-Media Laboratories was used for this purpose and performed as
described in the kit.
3.4 ISOLATION OF HALOBACTERIA
Isolation of halobacteria was done from soil collected from salt affected and
unaffected areas of different ecosystem of eastern U.P. Isolation was carried out for
rhizospheric Halobacteria using different types of media- Halophillic Agar, Nutrient

MATERIALS AND METHODS

Agar, Luria Bertani Agar and antifungal cycloheaxamide was also used. The
composition of media is given below:
1. Halophillic agar
Agar

20 g

Casein Acid Hydrochloride

10 g

Yeast Extract

10 g

Protease Peptone

5g

Trisodium Citrate

3g

Potassium Chloride

2g

Magnesium Sulphate

25 g

Sodium Chloride

250 g

2. Nutrient Agar
Peptic Digest

10 g

Beef Extract

5g

Sodium Chloride

100 g

Agar

15 g

3. Luria Berttani Agar

Casein Enzyme Hydrolase

10 g

Yeast Extract

5g

Sodium Chloride

100 g

From the above composition the media was prepared and autoclaved at 121C
for 15 min. at 15 lbs pressure. Media was then poured onto plates under aseptic
condition. Spreading technique was used for the isolation of halobacteria. Serial
dilution is performed by the following way: one gram (1g) of soil sample was
suspended in 10 ml of sterile DW and 10 -1 dilution was obtained. serial dilution were
prepard by mixing 1 ml of suspension made into 9 m sterile water blanks, until 10 -3
dilution was obtained. Each dilution was plated in NA plates. Plates are incubated for
26

MATERIALS AND METHODS

3 days at 37C to observe the colonies of halobacteria. Halobacterial colonies were

streaked on the other NA plates and the plates were incubated at 37C for 3 days.
Typical halobacteria colonies were observed over the streak. Well isolated single
colony was picked up and re-streaked on fresh NA plates and incubated similarly to
make single colony type.
3.5 Plant Growth Promoting Activities
Selected isolates were characterized for their plant growth promoting activities3.5.1 Phosphate SolublisationThe isolates were examined for phosphate production using Pikovaskayas
agar medium(1948).
Pikovaskayas agar mediumYeast Extract

0.50g

Dextrose

10.0g

CuPO4

5.0g

(NH4)2 . SO4

0.50g

KCl

0.20g

MgSO4

0.10g

MnSO4

0.0001g

FeSO4

0.0001g

Agar

20g

Phosphate solublization test by Halobacteria was carried out by spotting of the

cultured isolates to the sterilized petri plate containing Pikovaskayas medium having
tricalcium phosphate and incubated at 30C for 3 6 days. The plates were observed
for clear zones around the colonies. On the basis of the presence or absence of clear
zones around the culture, it may be proved that the present bacterial cultures are either
a phosphate solubilise or not.

27

MATERIALS AND METHODS

3.5.2 Siderophore ProductionSiderophore production test was performed on the CAS medium. Orange
zones appeared around the spottings on the CAS plates which determine the
siderophore producing capability of the bacterial isolates.
CAS mediumSolution 1: 10ml of 1mM FeCl3.6H2O (1N 10 mM HCl)
+
50ml of aqueous solution of CAS (1.21mg/ml)
+
40ml of aqueous solution of hexadecyl trimethyl ammonium (HDTMA)
Bromide (1.82 mg/ml)
Solution 2: Nutrient Agar (L-1)
Peptone

5.0g

Beef Extract

3.0g

Sodium Chloride

5.0g

Agar

20.0g

pH

7.0 -7.2

Solution 1 and solution 2 was made, then autoclaved separately and these solutions
are mixed at time of pouring.

3.5.3 IAA ProductionA pink colour develops when a mineral acid is added to a solution containing
IAA, in the presence of ferric chloride. Different mineral acids HCl, phosphoric acid,
nitric acid, perchloric acid can be used for the development of colour.
IAA Reagent: Salkowskis Reagent
A.

0.5 solution of feCl3 in 5ml of DW , 0.41g feCl3 in 5ml of DW

B.

35% perchloric acid HClO4 (conc.)

[1ml of 0.5M feCl3 solution was added in 50ml of 35% HClO4].
28

MATERIALS AND METHODS

Procedure

IAA Production of test for the isolates was carried out by broth inoculation
bacterial culture, followed by centrifugation at 10,000rpm for 10mins at 4 C
in 2ml eppendrof tubes.

Following centrifugation, supernatant was taken out in dried test tubes and
pellets were discarded.

After addition of 2 3 drops of O-phosphoric acid (conc.) in each supernatant,

4ml of IAA solution was added (DW + HClO4 + 0.5M FeCl3) in each culture
and after 1hr incubation, O.D was taken at 530nm. The level of IAA produced
was estimated by a standard IAA graph.

3.6 PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION

Different biochemical tests were performed to establish various useful
activities of isolates thus obtained. The different biochemical tests that were
performed are as follows.
3.6.1 GRAM STAINING
Gram staining method was discovered by a Danish pathologist, Christian
Gram in 1884. In this method, a drop of sterile distilled water was placed in the center
of glass slide. A loopful of growth from young culture was taken, mixed with water,
and placed in the center of slide. The suspension was spread out on slide using
the tip of inoculation needle to make a thin smear. The smear was dried in air and
fixed through mild heating by passing the lower site of the slide 3 to 4 times over the
flame. The smear was then flooded with crystal violet solution for 1 min and washed
gently in flow of tap water. Then the slide was flooded with iodine solution,
immediately drained off, and flooded again with iodine solution. After incubation
at room temperature for 1 min, iodine solution was drained out followed by
washing with 95% ethanol. After that, it was washed with water within 15 to 30 s and
blot dried carefully. The smear was incubated with safranin solution for 1 min. The
slide was washed gently in flow of tap water and dried in air. The slide was observed
under microscope and data were recorded.

29

MATERIALS AND METHODS

3.6.2 SALT TOLERANCE

Isolates were inoculated to nutrient broth with 0%, 1%, 2%, 3%, 4% and 5%
6%, 7%, 8%, 9%, and 10% NaCl concentration to evaluate their salt tolerance
(Hayward, 1964). Inoculated salt free (0%) nutrient broth was used as positive control
and un inoculated broth of each salt concentration was used as negative control and
the presence or absence of growth was recorded.
3.6.3 CATALASE
The catalase test is used to detect the presence of catalase enzymes by the
decomposition of hydrogen peroxide to release oxygen and water.

Hydrogen

peroxide is formed by some bacteria as an oxidative end product of the aerobic

breakdown of sugars.
Procedure1. One colony of test organism was transfered with an inoculating loop to the glass
slide.
2. Placed one drop of catalase reagent onto area of a glass slide with test organism.
3. Bubbling (effervescence) was observed for positive test.
3.6.4 UREA HYDROLYSIS

Urea agar contains urea and phenol red

Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3

Ammonia combines with water to produce ammonium hydroxide, a strong

base which increases pH of the medium.

Increase in the pH causes phenol red to turn a deep pink. This is indicative of a
positive reaction for urease.

Urea Basal Broth Media1% urea

10g urea was added to 100ml sterile DW water

Urea was filtered through membrane filter (0.20l)

After that sterilized urea was added to basal broth

30

MATERIALS AND METHODS

Basal broth compositionGlucose

1g

Casein Peptone

1g

Na2HPO4.2H2O

1.9g

KH2PO4

1.5g

MgSO4.H2O

0.5g

NaCl

5g

Phenol red

0.012g

Water

900ml

The isolates were inoculated by stabbing and incubated at 28C for 48-72
hours. The presence of pink coloration was recorded as positive for urease hydrolysis
3.6.5 TWEEN 80 HYDROLYSIS
It is a non-ionic surfactant and emulsifier derived from polyethoxylated
sorbitan and oleic acid, and is often used in foods. Some bacteria contain a type of
lipase which when added to a mixture of Tween 80 and phenol red, they cause the
solution to change colour, so this is used as a test to identify the phenotype of a strain
or isolate. The plates of tween 80 was made and spotted isolates on them and
incubated for 2-3 days. The presence of ring around the isolate was said to be
positive.
Tween 80 Agar MediaAgar

- 12g

Peptone

- 10g

Tween 8

- 10g

NaCl

- 5g

CaCl2

- 0.1g

pH

- 7.2 - 7.4 at 25C

31

MATERIALS AND METHODS

Procedure

All components were added to DW and bring volume to 1 lit. Mixed gently
and boiled. Autoclaved and poured into Petriplates.

The isolates were inoculated to plate and incubated at 37C for 2-3 days.

Bacteria that hydrolyse tween 80 appeared as colonies surrounded by an

opaque zone.

3.6.6 CASEIN HYDROLYSIS

The ability of the isolates to degrade the protein casein by producing
proteolytic exo-enzymes was tested by growing the isolates on milk agar plates. Clear
zone around the growth of the isolates was recorded as positive for casein hydrolysis
(Aneja, 1996).
Skim Milk Agar MediaSkim milk powder

100g

Peptone

5g

Agar

15g

Distilled water

1lit

PH

7.2

Media was autoclaved at 121 C for 15 minutes.

Procedure

Skim milk agar plate was made.

Single line streak was made from each culture into its labeled

Petriplates.

Incubated for 24 - 48 hrs at 37C

Any clear zone around the bacterial growth showed positive result.

3.6.7 HYDROGEN SULFIDE PRODUCTION

Sulphate serves as an inorganic source of energy (chemolithotroph). Medium
contains ferrous (iron) sulphate to detect the production of H2S (which is colourless
gas). The isolates were evaluated for H2S production using Sulphide Indole Motility
(SIM) agar medium.
32

MATERIALS AND METHODS

SIM Agar MediaPeptone

30g

Beef extract

3g

Ferrous ammonium sulfate

0.2g

Sodium thiosulphate

0.025g

Agar

3g

Distilled water

1lit

Media was autoclaved at 121C for 15 minutes.

The isolates were inoculated by stabbing and incubated at 28C for 48-72
hours. The presence of black coloration was recorded as positive for H2S production
(Aneja, 1996).
3.5.8 STARCH HYDROLYSIS
The isolates were streaked on starch agar medium to evaluate their ability to
hydrolyze starch (amylase production).
Starch Agar MediumStarch (soluble)

20g

Peptone

5g

Beef Extract

3g

Agar

15g

DW

1 lit

The plates were incubated at 28C and for 2-3 days starch hydrolysis was
observed by flooding the plates with Gram's iodine solution for 30 seconds. The
appearance of clear zone around the line of growth of each isolate indicated starch
hydrolysis (Aneja, 1996).

33

MATERIALS AND METHODS

3.5.9 GELATIN LIQUEFACTION

Ability to hydrolyze gelatin by isolates indicated the presence of enzyme
protease in these isolates. The isolates were inoculated in gelatin medium to evaluate
their ability to hydrolyze gelatin (protease production).
Gelatin MediumBeef extract

3g

Peptone

5g

Gelatin

120g

DW

1 lit

Procedure

All components were mixed and poured in to test tubes.

Autoclaved at 121C for 15 minutes and cooled without slanting.

The media were stab inoculated with each isolate grown for 48 hours on
gelatin medium and incubated at 28C.

After 3 days of incubation, each isolate was evaluated for gelatin liquefaction.
The isolates in test tubes were kept at 4C for 30 minutes and gently tipped
immediately.

Medium that flows readily as the tube is gently tipped was taken as positive
for gelatin liquefaction (Dickey and Kelman, 1988).

3.5.10 ACID PRODUCTION FROM GLUCOSE

Acid production from glucose was done by preparation of Glucose agar medium
Glucose agar mediumPeptone

2g

NaCl

5g

K2 HPO4

0.3g

Carbohydrate solution

100ml (10%)

Bromothymol blue solution

15 ml (0.2%)

Agar

3g,

Distill water

1000ml

34

MATERIALS AND METHODS

Procedure

All constituents were dissolved in distilled water and autoclaved.

Then using a sterilized filter bromothymol blue was added under sterile
condition.

1ml of sterile glucose solution was added (10%) to the medium to produce
final concentration of 1%.

Each tube was inoculated with single isolate by stabbing and liquid paraffin
was poured in tubes to form a layer of 1cm.

Tubes were then incubated at 35C for 2-3days and change of color was
observed.

3.6 GLYCEROL STOCK PREPARATION

The pure cultures isolated were stored in 20% glycerol stocks at -80C. The
bacterial lawn was grown on petriplates and cultures were scrapped using sterile
inoculation loop and transferred onto 20% glycerol vials. The cultures were vortexed
and were transferred successfully at 4C for 16 hour, -20C for 24 hours and finally at
-80C in ultra deep freezer were the cultures remain viable for 1-2 years.
3.7

MOLECULAR CHARACTERIZATION

3.7.1 ISOLATION OF GENOMIC DNA OF HALOPHILIC BACTERIAL

STRAINS
Cultures obtained from different sites under study were selected for the
molecular characterization. The subsequent genomic DNA extraction method was
used as described.
Halobacterial cultures were grown in Nutrient Broth for 7days and genomic DNA was
extracted.

Inoculated broth of pure cultures were centrifuged at 10,000rpm at 20C for

10 min and the pellets were collected by discarding the supernatant.

The pellet cells were washed twice with TE buffer.

The cells were resuspended in 0.5 ml SET buffer (75mM NaCl, 25mM
EDTA and 20 mM Tris).

35

MATERIALS AND METHODS

To the above cell suspension, 10 l (10 mg/ml) of lysozyme solution was

added and incubated at 55C for 1hr.

After incubation 0.1 volumes of sodium dodecyl sulfate (10%), and 10 l of

proteinase K (10 mg/ml) was added to above suspension and incubated at
55C for overnight in water bath to lyse the cells.

To the mixture, 0.3 volume of NaCl (5M) and equal volume of water.
saturated phenol and Chloroform: Isoamyl alcohol (25:24:1) was added and
incubated at room temperature for 30 min followed by gentle vortexing and
centrifuged at 5000 rpm for 5 min at 4C temperature.

Then aqueous layer was transferred to fresh tube. To the aqueous layer 0.1
volumes of 3M Sodium acetate and 2 volumes of chilled 95% ethanol was
added and kept at 40C for 30 minutes.

The mixture was centrifuged at 10,000 rpm for 5 min or at 8000 rpm for 8
min at 40C. The DNA pellet was washed with 70% ethanol and again
centrifuged at 10,000 rpm at 40C for 5 min.

The pellet was dried by keeping at 37oC for 10 min. The DNA was then
dissolved in 50l of milli Q water and stored at 4oC for further analysis.

3.8 Quantification of genomic DNA by Agarose Gel Electrophoresis

The quality and quantity of the genomic DNA was checked on 0.8% agarose gel
electrophoresis.
Reagent Required
3.8.1 TAE (TRIS ACETATE EDTA) BUFFER - 50X STOCK
TAE buffer was prepared by dissolving

242 g tris base

57.1 ml glacial acetic acid

100ml of 0.5 M EDTA (pH 8)

Sterile water was added to above components to make volume 1000ml.

3.8.2 Ethidium Bromide: stock 10 mg/ml
10mg of EtBr was dissolved in 1ml of DW and volume made up to 1 ml. the solution
was stored in an amber colored bottle.
36

MATERIALS AND METHODS

3.8.3 6X loading dye

30% glycerol

60mM EDTA

The volume was made up to 20 ml with milipore water.

3.8.4 AGAROSE GEL (0.8%)

First of all, 1X TAE was prepared by adding 2ml of TAE buffer to 98ml
sterile distilled water and 0.8g was added to the above buffer in a flask and
flask was boiled till agarose was mixed. The agarose was allowed to cool to
45C

Added with 2l Ethidium bromide stock per 100ml gel.

Casting tray was assembled with the combs and the cooled agarose was added
gently to the tray and gel was allowed to solidify, in the mean time 1X TAE
was prepared (1000ml) and poured in the Electrophoresis tank.

Cooled agarose gel along with the tray was assembled in the tank and combs
were gently removed without disturbing the wells.

5l of genomic DNA and 1l of gel loading buffer were mixed and loaded
into the wells gently.

After loading of the samples the lid was assembled and the gel was run at 70V
for 45 minutes. When the tracking dye migrated 3/4th gel was removed and
documented.

3.9

16S rRNA GENE AMPLIFICATION

3.9.1 DNA Quantification by UV spectrophotometer

Five l of the DNA sample was taken in a quartz cuvette. Volume was made
up to 1ml with distilled water (995l). Absorbance of the solution was measured at
wavelengths 260 and 280 nm and A260/A280 ratioswas calculated. A good DNA
concentration using the relationships for single stranded DNA, 1 OD at 260 nm = 50
g/ml. This estimate is influenced by contaminating substances like RNA and very
low molecular weight DNA in the solution. The presence of protein, phenol and
chloroform also influences the reading. A working stock of 100 l was prepared with
10 ng/l concentrations for PCR amplification.
37

MATERIALS AND METHODS

3.9.2 16S rDNA Amplification DNA Amplification is a very simple method for in vitro amplification of
specific nucleic acids using Taq DNA Polymerase and minimum two oligonucleotides
specific to the DNA to be amplified. The technique involves repeated rounds of DNA
synthesis which is based on three simple steps for any DNA amplification reaction.
The three steps involve1. Denaturation of the template into single strands;
2. Annealing of primers to each original strand for new strand synthesis;
3. Extension of the new DNA strands from the primers.
3.9.3 SOLUTION AND BUFFER FOR THE POLYMERASE CHAIN
REACTION (PCR)
Taq DNA polymerase (3 units/l)
Deoxyribonucleotide triphosphate mix (This mixture has a concentration of
2.5 mM of each dNTP.
10X Taq polymerase buffer: 10X Stock buffer contains 100mM Tris HCl (pH
9.0), 500 mM KCl, and 0.1 % gelatin. (without MgCl2)
25mM MgCl2
DNA of isolated cultured Halobacteria (apprx.20-25ng/ul)

1Kb DNA Ladder

16S rDNA primer set (25)

The melting temperature (Tm) of forward and reverse primers should be the

same. For the amplification of 16S rDNA, following forward and reverse primers
were used to obtain approximately 1500-bp fragent from the amplification of 16S
rDNA (EDWards et al., 1989)pA (5AGAGTTTGATCCTGGCTCAG3)
pH (5AAGGAGGTGATCCAGCCGCA3)
Thermal profiles DNA denaturation is the critical step in the DNA amplification reaction .The
38

MATERIALS AND METHODS

time specified for DNA depends on annealing temperature may result in non- specific
amplification. Primer extension, in most amplification occurs efficiently at a
temperature of 72 oC and seldom needs optimization. Time specified for extension
depends on the length of target sequence for example for 1kb target DNA usually 1
mint extension is recommended.
Procedure for control DNA amplification The reagents was added to make PCR mix (100l)10X buffer

10l

dNTPs

6l

Primer F

1.5l

Primer R

1.5l

Taq Polymerase

1.0l

DNA Template

4.0l

The solution was mixed gently.

The amplification was carried out using following reaction condition for at
least 40 cycles.

940C for 5 min. (Initial denaturation).

940C for 45 sec. (40 cycles).

500C for 40 sec. (annealing).

720C for 1min 30 sec. (extension).

720C for 7 min. (final extension).

After amplification the PCR product was resolved by electrophoresis in 1.2%

agarose gel stained with ethidium bromide and visualized on a gel documentation
system (Alpha-Imager) and gel images were digitalized.

39