Chapter 4 Materials and

Methods

MATERIALS
INSTRUMENTS

No. EQUIPMENT COMPANY / SUPPLIER

Refrigerated orbital shaker

1 SAKOVA SCIENTIFIC Com. Bombay
incubator

2 Refrigerated centrifuge SUPER SPIN Pvt.Ltd.Bombay

3 Autoclave EMKAY Pvt.Ltd. Bangalore

4 Vortex shaker SPINEX Pvt. Ltd. Bombay

5 Stirred tank Bioreactor SARTORIUS B-LITE Bangalore

6 Electronic balance SARTORIUS Pvt. Ltd. Bangalore.

7 pH meter ELICO Pvt. Ltd. Hyderabad.

8 Micropipettes ACCUPIPIPET Capp, Bangalore

9 Rotary evaporator HEIDOLPH Mumbai.

10 Hot air oven PSM industries Mumbai.

11 Laminar air flow KLENZAIDES BIOCLEAN Bangalore

12 Microscope MIKROTECH Bangalore.

13 Separating funnel SCOTT DURAN Mumbai.

14 U.V. Spectrophotometer U.V. 1201, Shimadzu, Japan.

Table No. 3. List of instruments.

MEDIA AND CHEMICALS

No.
MEDIA AND CHEMICALS SUPPLIER

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Chapter 4 Materials and
Methods

1 Chrome Azurol S dye RFCL Ltd. New Delhi

2 HDTMA RFCL Ltd. New Delhi

3 Activated charcoal RESEARCH-LAB Ind. Bombay

4 Nutrient agar HIMEDIA Lab Pvt Ltd Bombay

5 Nutrient broth HIMEDIA Lab Pvt Ltd Bombay

6 Peptone HIMEDIA Lab Pvt Ltd Bombay

7 Yeast extract powder HIMEDIA Lab Pvt Ltd Bombay

8 Agar HIMEDIA Lab Pvt Ltd Bombay

9 Sabouraud dextrose agar HIMEDIA Lab Pvt Ltd Bombay

10 Lactophenol Blue HIMEDIA Lab Pvt Ltd Bombay

11 Mannitol S D Fine Pvt Ltd Mumbai

12 TLC Plates SILICA GEL 60 F254 MERCK

13 Ethanol E MERCK Ltd Mumbai

14 Glycerol RANKEM New Delhi

15 PIPES buffer HIMEDIA Lab Pvt Ltd Bombay

Di-potassium hydrogen
16 RANKEM New Delhi
phosphate

17 Casein HIMEDIA Lab Pvt Ltd Bombay

18 Magnesium sulphate RANKEM New Delhi

19 Potato dextrose broth HIMEDIA Lab Pvt Ltd Bombay

20 Hydroxamate RANKEM New Delhi

21 Potato dextrose agar HIMEDIA Lab Pvt Ltd Bombay

22 Chloroform RANKEM New Delhi

23 n-Butanol S D Fine Pvt Ltd Mumbai

24 Acetic acid S D Fine Pvt Ltd Mumbai

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Chapter 4 Materials and
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25 Sodium chloride S D Fine Pvt Ltd Mumbai

26 Hydrochloric acid S D Fine Pvt Ltd Mumbai

28 E MERCK Ltd Mumbai

Hydrogen peroxide

Table No. 4. List of media and chemicals.

METHODOLOGY

4.1 Screening of Fungal species

4.1.1 Collection of soil sample:

Soil samples were collected from the local areas of Belgaum, Karnataka. The
collected soil samples were air dried before its screening for fungal species.

4.1.2. Preparation of Potato Dextrose Agar medium19:

One of the handiest media for culturing Fungi in the laboratory is Potato
Dextrose Agar medium. The medium was prepared by taking 200 gms of potatoes
infusion, 20 gms of Dextrose and 15 gms of Agar. Then water was added to make the
final volume to 100ml. the flask was then kept for autoclave at 121 oC for 15 minutes.

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Chapter 4 Materials and
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After sterilization, media was poured onto culture plates and allowed for
solidification.

4.1.3. Fungi Isolation:

4.1.3.1 Sterilization Procedures

The isolation of fungal species were conducted using sterilized tools e.g. Pincers,
scalpels, forceps.

4.1.3.2 Preparation of media for isolation of fungi

Isolation media was prepared by dissolving 39.0gm potato dextrose

agar consisting of diced potato, 200 g/l; dextrose, 20 g/l; agar, 15 g/l. The resultant
solution was sterilized in autoclave for about 20 min at 121˚C, 15 lbs/sq. inch. The
media was allowed to cool up to 40˚C; then 150mg/l solution of streptomycin
sulphate solution was added. 15 ml melted potato dextrose agar (PDA) medium was
poured into each petri plate and allowed to cool.

4.1.3.3 Isolation of fungal species

1. Sterilization of root materials

All necessary steps in the sterilization were carried out. Before
sterilization the samples of grass roots were cut into pieces about 2 cm
x 2 cm with a sterile scalpel. Between steps the roots were blotted on a
sterile filter paper to avoid dilution of the sterilizing agent.
Pretreatment was done by washing root material in running tap water
for 2-3 times.
Root material was treated with 5% tween 20 solution for 3 min. The
excess of soap was completely removed by washing under a jet of tap water
and then with distilled water.

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Chapter 4 Materials and
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Surface sterilization of root material was done by using sodium

hypochlorite. Root material was treated with 5% and 10% solution of sodium
hypochlorite for 10 min and 5 min. After the treatment root samples were
rinsed in sterile water for two times.
2. Culture of sterilized tissues on nutrient medium

Sterilized root material was cut so as to expose the interior surface to nutrient
media. Root materials were placed on potato dextrose agar medium amended with
streptomycin sulphate solution (150 mgl-1) contained in petri dishes. The petri dishes
were incubated for 7 days at 27˚C. The fungal hyphae while emerge after inoculation
were picked and transferred onto sabouraud dextrose agar (SDA) for identification
and preservation.

In positive control non sterilized root tissues were cultured and in negative

control only the impression of sterilized tissues were taken and incubate.

4.2. Optimization of growth conditions

In order to maximize hydroxamate-type siderophore production, various

growth conditions were tested for optimal siderophore production.

MKP-4 strain were grown in 50 ml medium for 24 hours at 30°C in a orbital

shaker, and the biomass and siderophore production were measured. Growth was
measured after 24 hours by dry weight method, using uninoculated media as a blank.
Siderophore production was measured after 24 hours by measuring absorbance at 400
nm of the culture supernatant.

4.2.1. Media:

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Chapter 4 Materials and
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The MKP-4 strain was grown on two different media for 24 hours in an orbital
shaker. The media used were Yeast Potato Dextrose Broth & Potato Dextrose Broth.

4.2.2. pH:

The effect of pH on siderophore production was studied by growing MKP-4

strain in Potato Dextrose Broth medium of different pH from 5 to 10. pH was
maintained by using PIPES buffer.

4.2.3. Temperature:

Optimal temperature for the MKP-4 strain was determined by keeping the
inoculated Potato Dextrose Broth media at 25 0C, 300C, 350C and 370C separately for
24 hours in the orbital shaker incubator at 150 rpm.

4.2.4. Carbon source:

The Potato Dextrose Broth medium contains 2 % dextrose as carbon source.

In this study optimization of carbon source was carried out using glycerol, dextrose
and mannitol in individual concentrations of 0.5%, 1.0%, 1.5% and 2%

4.2.5. Nitrogen source:

The Peptone as nitrogen source was used in Potato Dextrose Broth medium.
In this study optimization of nitrogen source was carried out using peptone, casein
and yeast extract in individual concentrations of 1.0%, 1.5%, 2% and 2.5%.

4.2.6. Optimal duration and Biomass measurement:

Optimal duration for the siderophore production was measured by using the
above optimized parameters. MKP4 strain was inoculated in optimized Potato
Dextrose Broth and incubated for 48 hours in a 2L fermentor. Samples were
withdrawn at every hour interval. The optimal duration, the time at which it showed
higher siderophore production, was determined by measuring the absorbance of
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Chapter 4 Materials and
Methods

culture supernatant at 400 nm. Biomass production was measured by dry weight
method.

4.3. Production and Purification of Siderophore

4.3.1. Production of Siderophore:

After optimization of fermentation parameters, production of siderophore was

carried out in 2L laboratory scale fermentor. The fermentor vessel containing the
optimized media was sterilized in autoclave at 121 oC for 15 minutes. After
sterilization it was allowed to cool at room temperature. Then the inoculum (100 ml)
was added containing MKP4 strain Fermentation was continued for 48 hrs and
subjected for downstream processing.

4.3.2. Isolation and Purification of Siderophore:

All the broth liquid was centrifuged at 15000 rpm for 10 minutes and the
supernatant was collected. The supernatant was divided into two bottles for
purification. The supernatant of the first bottle was treated with chloroform:phenol
(1:1) with vigorous shaking for 30 minutes followed by centrifugation at 10000 rpm
for 10 min. The organic layer was thrown as it contains the impurities and the
aqueous layer was taken for its spectral activity. Similarly the supernatant of second
bottle was treated with activated charcoal for deproteinization and stirred for 30
minutes at room temperature. Then it was filtered to remove the charcoal along with
the adsorbed impurities and the filtrate was collected.

4.4. Antimicrobial screening of Siderophore

To study the antimicrobial activity of siderophore, following media and

materials are required.

1) Nutrient broth / Sabouraud dextrose broth (SDB)

2) Nutrient agar / Sabouraud dextrose agar (SDA)

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3) Assay tubes
4) Standardized culture of test organism
5) Sterile pipettes and Petri dishes
6) Cork borer

4.4.1. Antibacterial Activity:

The compounds were tested in-vitro for their antibacterial activity against
following microorganisms by cup plate method44. The antibacterial activity was
studied using three Gram positive and two Gram negative bacteria.

Gram positive:-

1. Bacillus subtilis
2. Staphylococcus aureus
Gram negative:-

1. Escherichia coli
Antibacterial activity was carried out by cup plate method using nutrient agar
plates. The plates were incubated for 24 hrs at 28oC

a) Preparation of Nutrient Agar:

NA was dissolved in distilled water and pH was adjusted to 6.5 – 7.0. A total
of four conical flasks were prepared each having 25ml of water. These solutions were
sterilized by autoclaving at 121C for 15 min. after sterilization; pour the inoculums
of above four bacterial strains in their respective conical flask containing the NA
medium.

b) Method of testing:

NA plates were prepared by pouring 20-25 ml of the medium into each

sterilized petridish and were allowed to set at room temperature. Cups were scooped

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in each plate using a sterile borer. The 50 l solution of siderophore was added in the
cups by using micropipettes and these plates were subsequently incubated at 37C for
48 hr. The zone of inhibition was measured in mm for each organism.

4.4.2. Antifungal Activity:

The Antifungal activity was assayed against following fungi using Sabouraud
Dextrose Agar (SDA) media by cup plate method. The plates of each fungus were
incubated at 37oC for 48 hrs.

1. Neurospora crassa
2. Aspergillus niger
3. Candida albicans
4. Candida tropicalis

a) Preparation of Sabouraud-Dextrose Agar:

Components of SDA were dissolved in distilled water and pH was adjusted to

5.5 – 6.0. a total of four conical flask was prepared each having 25 ml of water. These
solutions were sterilized by autoclaving at 121C for 15 min. after sterilization; pour
the inoculums of above four fungal strains in their respective conical flask containing
the SDA medium.

b) Method of testing:

SDA plates were prepared by pouring 20-25 ml of the medium into each
sterilized petridish and were allowed to set at room temperature. Cups were scooped
in each plate using a sterile borer. The 50 l solution of siderophore was added in the
cups by using micropipettes and these plates were subsequently incubated at 37C for
48 hr. The zone of inhibition was measured in mm for each organism.

4.5 Characterization of Siderophore

4.5.1. UV spectroscopy:
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Chapter 4 Materials and
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The siderophores are characterized by UV spectroscopy at different

wavelength ranging from 350 to 700 nm. Different siderophores have different
maximum absorption (λmax). Hydroxamate type of siderophore is measured at 480nm
employing Iron Perchlorate assay. Catechol type of siderophore shows maximum
absorbance at 500nm using Arnow’s reagent.

4.5.2. Iron-Perchlorate Assay for Detection of Hydroxamic Acids45:

If siderophore is detected with the CAS assay, then further assays are
employed to determine what type of siderophore is being produced. The iron-
perchlorate assay is a colorimetric assay used for detection and estimation of
hydroxamate-type siderophores. Because this assay is done under acidic conditions,
it does not detect the presence of a catechol-type siderophore, which react at alkaline
pH. Culture supernatants were collected as described previously and 0.5 ml
supernatant was added to 2.5 ml 5 mM Fe(ClO 4)3 in 0.1M HClO4 solution and
allowed to incubate at room temperature for approximately five minutes. If a
hydroxamate-type siderophore is being produced, an orange-red color will form,
which varies in intensity based on how much siderophore is produced. Absorbance is
measured at 480 nm, with uninoculated media mixed with reagent used as a blank.

4.5.3. Assay for Catechol-type Siderophores45:

To determine whether a culture is producing a siderophore that contains

catechol groups, Arnow’s method is used. This is also a colorimetric assay and can
be used to estimate catechol concentration using a known catechol as a standard. The
assay is performed by mixing the following in order: 1 ml culture supernatant, 1 ml
0.5 M HCl, 1 ml nitrite-molybdate reagent (prepared by dissolving 10 g sodium
nitrite and 10 g sodium molybdate in 100 ml ddH 2O), and 1 ml 1M NaOH. These are
allowed to incubate for 5 minutes for the reaction to complete. Absorbance is
measured at 500 nm with uninoculated media instead of supernatant used as blank.

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Chapter 4 Materials and
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Catechol groups can be detected because they form a yellow color in nitrous
acid, which turns pink-red when excess sodium hydroxide is present. A control
(either a culture grown in high iron or uninoculated media) will remain colorless with
the addition of reagents.

4.5.4 Csaky’s assay for Hydroxamate type Siderophores46

To determine whether a culture is producing a siderophore that contains hydroxamate

groups, Csaky’s assay is used. This is also a colorimetric assay and can be used to
estimate hydroxamate concentration using a known standard hydroxamate as
ferrichrome. The assay is performed by mixing the following in order: 1 ml
supernatant of culture was hydrolysed with 1 ml of 6N H2SO4 in a boiling water
bath/6hrs or 130°C/30mins. To this 3ml of sodium acetate was added for buffering;
Now 1 ml of sulfanilic acid & then 0.5 ml of iodine solution was added. After 3-
5mins, excess iodine is destroyed with 1 ml of Na-arsenite soln. 1 ml of alpha
naphthylamine was then added and water was used to make up vol to 10 ml. Color
was allowed to develop for 20-30mins.

Absorbance was measured with the help of UV-Visible spectrophotometer at 526nm

with uninoculated media instead of supernatant used as blank.
Hydroxamate groups can be detected by observing change in colour. A control
(either a culture grown in high iron or uninoculated media) will remain colorless with
the addition of reagents.

4.5.5. Siderophore characterization using Thin Layer Chromatography (TLC):

Siderophore can also be detected by using thin-layer chromatography.

Culture supernatants are spotted on silica gel plates and spots are allowed to dry. The
plates are then run in an n-butanol:acetic acid:dH2O (12:3:5) solvent system. Plates
are then dried and sprayed with 0.1 M FeCl 3 in 0.1N HCl. The formation of a wine-

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Chapter 4 Materials and
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colored spot indicates a hydroxamate-type siderophore, while a dark gray spot

indicates production of a catechol-type siderophore.

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