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3.MATERIALS AND METHODS

The present study was undertaken in the Department of Microbiology, College of

Basic Sciences, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur. It is located

in the mid-hills and sub-humid agro-climatic zone of Himachal Pradesh, India. This town

is located at 32º6’N, 76º18’E and 1300 m msl.

The chemicals and media components used were of analytical grade (AR)

obtained from Merck limited - India, Sigma-Aldrich Inc. USA, and Hi Media

Laboratories, Bombay, India. The materials used and the methods employed are

presented below under the following sub-headings:

3.1 Standard microorganisms

3.2 Collection of rhizosphere soil samples

3.3 Isolation of Nitrogen fixing and phosphate solubilizing microorganisms

3.4 Qualitative assay of phosphate solubilizing activity

3.5 Quantitative assay of phosphate solubilizing activity

3.6 Quantitative assay of Nitrogenase activity

3.7 Detection of Indole acetic acid (IAA) production

3.8 Detection of siderophore production

3.9 Detection of ammonia production

3.10 Characterization, identification and maintenance of isolated microbial strains

3.10.1 Morphological and Biochemical characterization

3.10.2 Molecular characterization of efficient strains

3.11 Development of liquid formulations

3.11.1 Liquid carriers for formulations

3.11.2 Effect of stress conditions on liquid formulation

3.12 Statistical analysis of data

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3.1 Standard microorganisms

Standard strains of Pseudomonas striata (PSB), Azotobacter chroococum

(Nitrogen Fixer) and Azospirillum brasilense (Nitrogen Fixer) were procured from the

Institute of Microbial Technology (IMTECH), Chandigarh. Pseudomonas striata MTCC

1259 strain was maintained on nutrient agar, Azotobacter chroococum MTCC 446 was

maintained on Jensen’s medium and Azospirillum brasilense MTCC 125 was maintained

on nitrogen free malate (NFb) medium (Appendix I).

3.2 Collection of rhizosphere soil samples

Soil samples were collected from the rhizosphere of five crop plants (Plate 3.1)

viz., Triticum aestivum (Wheat), Zea mays (Maize) and Solanum tuberosum (Potato) and

two medicinal plants Aloe barbadensis (Aloevera) and Bacopa monnieri (Brahmi) grown

in the Model Organic Farm, CSK HPKV, Palampur. For this purpose, the plants were

uprooted carefully, shoots were cut off and roots along with rhizosphere soils were

brought to the laboratory in polythene bags. The soil samples were processed

immediately or stored at 4-8 ºC for the isolation of microorganisms.

3.3 Isolation of Nitrogen fixing and Phosphate solubilizing microorganisms

3.3.1 Processing of rhizosphere soil samples for isolation of Azotobacter

Isolation of Azotobacter from the above mentioned soil samples were done on

Jensen’s medium (Norris and Chapman 1968) by dilution plate technique. One gram of

each soil sample was transferred to 9 ml sterile dilution blank under aseptic conditions

and serial dilutions were made accordingly. Appropriate dilutions were plated on

Jensen’s medium and incubated at 30 ± 1 ºC for 2-3 days. The isolated strains were

stocked on Jensen’s medium slant for further studies.

3.3.2 Processing of rhizosphere soil samples for isolation of Azospirillum

Azopirillum was isolated from the above mentioned soil samples in semi-solid

nitrogen free malate (NFb) medium. Serial dilutions of soil samples were made as

mentioned above and one ml of appropriate dilution was inoculated in semi-solid NFb

medium (Dobereiner 1992) and incubated at 30 ± 1 ºC for 2-3 days. Formation of white,

dense pellicle in semi-solid NFb medium and change of color of medium from green to

100

blue indicated the presence of Azospirillum. The culture was purified by streaking white

pellicle on NFb agar plate and stocked on NFb agar slant for further studies.

A B

D C

101

Plate 3.1 Selected crop/medicinal plants which were used to isolate nitrogen fixing and

phosphate solubilizing bacterial (PSB) isolates (A) Triticum aestivum (Wheat), (B) Zea

mays (Maize), (C) Solanum tuberosum (Potato), (D) Aloe barbadensis (Aloevera) and (E)

Bacopa monnieri (Brahmi)

E

102

Processing of soil samples for isolation of Phosphate solubilizing microrganisms

Phosphate solubilizing microorganisms (PSM) were isolated from the above

mentioned soil samples by dilution plate technique using Pikovskaya’s medium

(Pikovskaya 1948) containing tri-calcium phosphate (TCP). Appropriate soil dilutions

were plated on Pikovskaya’s agar medium by spread plate technique and incubated at 30

± 1 ºC for 2-3 days. The colonies forming halo zone of clearance (Pikovskaya’s

medium) around them were counted as P-solubilizers. All the bacterial colonies

exhibiting halo zones were selected, purified and maintained on nutrient agar slants for

further studies.

3.4 Qualitative assay of phosphate solubilizing activity

Pure cultures of phosphate solubilizing bacteria were spot inoculated at the centre

of already prepared plates of Pikovskaya’s agar medium. The plates were incubated at 30

± 1 ºC for 7-10 days. The colonies forming more than 5.0 mm zone of solubilization were

stocked. The zone of phosphate solubilization (mm) formed around colonies was

recorded after every 24 hours for 10 days.

The solubilizing efficiency of the microorganisms was calculated using following

formula:

Z – C

Solubilizing efficiency (% S.E) = x 100

C

Z = Solubilization zone (mm)

C = Colony diameter (mm)

3.5 Quantitative assay of phosphate solubilizing activity

3.5.1 Estimation of solubilized P by bacterial isolates

The quantitative estimation of solubilized P by bacterial isolates was done by the

vanadomolybdophosphoric yellow color method (Subba Rao 1988) in NBRIP (National

Botanical Research Institute’s Phosphate growth medium) broth (Nautiyal 1999; Mehta

and Nautiyal 2001) containing 1000 µg/ml tri-calcium phosphate (TCP).

103

Method

NBRIP broth (Appendix I) in 100 ml aliquots containing 1000 µg P/ml in the

form of TCP was inoculated aseptically with 1 ml of culture broth having OD of 1.5 at

600 nm. The aliquots were incubated with shaking at 28 ºC up to 11 days. Five ml of the

growth medium from each flask was taken out on 3rd

, 5th

, 7th

and 11th

day, filtered

through Whatman No. 1 filter paper, and centrifuged (R-24 REMI) at 10,000 r.p.m for 20

minutes. Phosphorus in the cell free culture supernatant was determined by above

mentioned method. For this, 0.5 ml or 1 ml of the supernatant was taken, 2.5 ml of

Barton’s reagent was added and volume was made up to 50 ml with double distilled

water (ddw). After 10 minutes, the intensity of yellow color was read on

spectrophotometer (UV-VIS Spectrophotometer- SL-159, Elico, India) at 430 nm and the

amount of P-solubilized was extrapolated from the standard curve.

3.5.2 Standard curve

3.5.2.1 Standard stock P solution

Weigh 0.2195 g of potassium dihydrogen orthophosphate AR grade (dried in oven

at 60ºC for 1 hr and cooled in a dessicator) and dissolved in one liter of ddw. For

preparation of working solution, 150 ml of this solution was taken and volume was made

up to 250 ml with ddw.

3.5.2.2 Preparation of the standard curve

Standard curve was prepared by taking 2, 3, 5, 8, 10, 15, 20, 25 and 30 ml of the

working solution in each 50 ml volumetric flask, and 2.5 ml of Barton’s reagent was

added to each flask and the volume was made up to 50 ml with ddw. After 10 minutes,

the intensity of yellow color developed was read at 430 nm spectrophotometrically.

Standard curve was prepared by plotting absorbance at 430 nm Vs concentration of P

(Figure 3.1).

3.5.3 Determination of pH

The pH of the growth medium was determined at regular intervals by using digital

pH meter (Decibel India Ltd).

104

0 250 500 750 10000.0

0.1

0.2

0.3

0.4

0.5

0.6

Concentration of P(g /ml)

Abso

rban

ce (

430nm

)

Figure 3.1 Standard curve for quantitative estimation of Phosphorus

3.6 Quantitative assay of Nitrogenase activity

Nitrogen fixation capacity of nitrogen fixers were quantified indirectly by

Acetylene reduction assay (ARA) measuring the reduction of acetylene to ethylene.

3.6.1 Acetylene reduction assay (ARA)

Nitrogen fixation of the isolates was determined in nitrogen free medium by the

acetylene reduction assay (Hardy et al. 1968). Pure cultures of all the isolates were

inoculated to 100 ml of nitrogen free medium in 250 ml Erlenmeyer flask and were

grown to the mid exponential phase at appropriate growth temperature for 48 h on a

rotatory shaker (100 rev min-1

). The vials containing the same medium were inoculated

with the above aliquots (0.1 of O.D.600) and incubated till exponential phase. Following

incubation, the gas phase in the headspace was replaced with acetylene (10% v/v) and

again incubated at appropriate growth temperature for 18 h. Ethylene production was

measured using a Hewlett Packard gas chromatograph (Model HP Series 5890, USA)

fitted with flame ionization detector and a Porapak-N column. After completion of the

ARA, the cells were predigested by adding 10% SDS and sonicated briefly. Protein

concentration in the resulting distributed mixture of suspension was determined. The

nitrogenase activity was calculated by the formula:

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C x PS x V

Nitrogenase activity (nmole C2H4 h-1

mg-1

protein) =

PStd x T x P

C = conc. of ethylene in nmoles

PS = peak height of sample

V= volume of air space in the assay vial

PStd = peak height of standard

T = time of incubation in hrs

P = protein concentration of bacterial cell in mg

3.6.2 Protein Measurement of Bacterial cells

The protein concentration of the bacterial cell was determined by Lowry method

(Lowry et al. 1951). To 0.2 ml of predigested bacterial cell suspension, distilled water

was added to make the volume 1 ml. To this, 5 ml of reagent “C” was added with

immediate vortexing and kept for 10 minutes at room temperature. Finally, 0.5 ml of

reagent “D” was added and incubated in the dark for 30 minutes. The intensity of blue

color developed was read on spectrophotometer (UV-VIS Spectrophotometer-SL-159,

Elico, India) at 660 nm and the amount of protein was extrapolated from the standard

curve. The reagents used in Lowry Method are given in Appendix I.

3.6.3 Standard Curve

3.6.3.1 Standard stock Protein (BSA) solution

Standard stock solution of Bovine Serum Albumin (100 μg/ml) was prepared in

distilled water.

3.6.3.2 Preparation of the standard curve

Standard curve was prepared by taking 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and

1.0 ml of standard BSA solution in test tubes. The volume was made to 1 ml with

distilled water, and to this, 5 ml of reagent “C” was added with immediate vortexing and

kept for 10 minutes at room temperature. To this, 0.5 ml of reagent “D” was added and

incubated in the dark for 30 minutes. The intensity of blue color developed was read at

660 nm spectrophotometrically. Standard curve was prepared by plotting absorbance Vs

concentration of Protein (Figure 3.2).

106

0 25 50 75 100 1250.0

0.1

0.2

0.3

0.4

Concentration of BSA (g /ml)

Abs

orba

nce

(660

nm)

Figure 3.2 Standard curve for quantitative estimation of protein

3.7 Detection of Indole acetic acid (IAA) production

Indole acetic acid production was quantitatively measured by the method given by

Gordon and Weber (1951).

3.7.1 Method

Reagents:

a. Salkowski reagent: 1 ml of 0.5 M FeCl3 in 50 ml of 35% perchloric acid.

b. Orthophosphoric acid.

Bacterial cultures were grown in a Luria-Bertani broth amended with tryptophan

(5mM) for 3-4 days. Cultures were centrifuged at 10,000 rpm for 20 min. Two ml of

supernatant was mixed with two drops of orthophosphoric acid and 4 ml of Salkowski

reagent. Tubes were incubated at room temperature for 25 minutes. The intensity of pink

color was read at 530 nm spectrophotometrically and the amount of IAA produced was

extrapolated from the standard curve.

3.7.2 Standard Curve

3.7.2.1 Standard stock IAA solution

Prepare 100 μg/ml standard stock solution of IAA in 50% ethanol.

3.7.2.2 Preparation of the standard curve

107

Standard curve was prepared by taking 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and

1.0 ml of standard IAA solution in test tubes. The volume was made to 2 ml with distilled

water and then 4 ml of Salkowski reagent was added and tubes were incubated for 25

minutes at room temperature and optical density was measured at 530 nm. Standard curve

was prepared by plotting absorbance at 530 nm against concentration of IAA solution

(Figure 3.3).

0 25 50 75 100 1250.0

0.1

0.2

Concentration of Indole Acetic Acid (g /ml)

Abso

rban

ce (

530nm

)

Figure 3.3 Standard curve for quantitative estimation of IAA

3.8 Detection of siderophore production

The isolated microorganisms were screened for siderophore production by the

universal chemical assay (chrome azurol S assay) as described by Schwyn and Neilands

(1987).

3.8.1 Deferration

All the glasswares were washed in 6 M hydrochloric acid to remove traces of

contaminating iron and then rinsed thoroughly with distilled water.

3.8.2 Chrome azurol S assay

108

The chrome azurol sulfonate (CAS) assay was used for the detection of

siderophores. This assay uses an iron-dye complex which changes color on loss of iron.

Siderophores, which have more affinity for the iron than the dye, remove the iron,

resulting in a change in color of the dye to orange. The iron-dye complex used to detect

the siderophore thus can be incorporated into King’s B medium

CAS-HDTMA solution

CAS-HDTMA solution was prepared by dissolving 121 mg chrome azurol sulfate

(CAS) in 100 ml of distilled water, and to this, 20 ml of 1 mM FeCl3.6H2O solution (1

mM FeCl3.6H2O solution prepared in 10 mM HCl) was added. It was slowly added to 20

ml hexadecyltrimethylammonium bromide (HDTMA) solution (729 mg HDTMA in 400

ml distilled water) and autoclaved at 121ºC for 15 minutes.

To 900 ml sterilized King’s B medium, 100 ml CAS-HDTMA solution was added

drop wise and mixed gently to avoid foaming. Plates were poured and incubated for 24

hrs at 30ºC for checking any contamination. All the isolates were spot inoculated on these

prepared plates of CAS agar and incubated at optimum growth temperature for 3-4 days.

The isolates producing orange color in the form of halo zone around the colonies were

considered as siderophore producers.

3.9 Detection of ammonia production

Qualitative detection of ammonia production was done by the method given by

Bakker and Schippers, (1987).

3.9.1 Method

Nessler’s reagent: 100 g Mercuric iodide and 70 g potassium iodide were

dissolved in a small quantity of distilled water and this mixture was added slowly, with

stirring, to a 500 ml cooled solution of sodium hyroxide (160 g sodium hyroxide

dissolved in 500 ml distilled water) and finally, this solution was diluted to 1 liter with

distilled water. Reagent was stored in rubber-stoppered borosilicate glassware in darkness

to maintain its stability for up to a year under normal laboratory conditions.

Bacterial isolates were grown in peptone water for 2-3 days at optimum growth

temperature. After incubation, 1ml of Nessler’s reagent was added in each tube. Tubes

109

showing faint yellow color indicated small amount of ammonia, and deep yellow to

brownish color indicated maximum amount of ammonia.

3.10 Characterization, identification and maintenance of isolated microbial

strains

3.10.1 Morphological and Biochemical characterization

The efficient phosphate solubilizing and nitrogen fixing bacteria were identified

on the basis of morphological, physiological and biochemical characteristics according to

the standard methods described in Bergey’s manual of Systematic Bacteriology (Kreig

and Holf 1984) and Laboratory Manual of Basic Microbiology (Kanwar et al. 1997).

All the isolated and identified cultures were stocked in appropriate agar slants

(nutrient agar, Jensen’s medium and NFb medium or soft agar (0.5% agar) in tight screw

capped sterile vials and incubated for 24-48 h at optimum growth temperature. Vials

having microbial cultures were then preserved at 4 0C.

3.10.2 Molecular characterization of efficient strains

Molecular characterization of most efficient bacterial isolates was done by

sequencing of their 16S rRNA gene.

3.10.2.1 Extraction of genomic DNA

Total genomic DNA of each isolate was extracted using commercial DNA

isolation kit (Real Biogenomics) by following the instruction manual of the manufacturer.

The amount of DNA was quantified by recording the absorbance at 260 nm wavelength

using UV/VIS spectrophotometer (Bio Rad, SmartSpec 3000). DNA was stored at -20oC

for further use.

3.10.2.2 PCR amplification of 16S rRNA gene

The 16S rRNA gene of the target bacterial isolates was amplified by using

universal eubacterial primers as reported by Heddi et al. (1998). The base sequences of

the primers used are presented in Table 3.1. These were custom synthesized (Sigma, Pvt.

Ltd.).

110

Table 3.1: Base sequences of 16S rRNA gene primers

Name of the Primer Sequence (5’ to 3’)

16S rRNA gene F

R

AGA GTT TGA TCA TGG CTC AG

TAC CTT GTT ACG ACT TCA CC

The PCR amplification was carried out in 0.2ml PCR tubes with 25 μl reaction

volume consisting of following components:

Reaction Mixture Quantity(µl)

Buffer 10 X 2.5

MgCl2 (25 mM) 1.5

dNTPs mix (10 mM each) 2.0

Taq DNA polymerase (5U/µl),

(Life Technologies India, Pvt. Ltd)

0.2

Primer forward 20 pmol 1.0

Primer reverse 20 pmol 1.0

Water (SDW) 14.8

DNA (10ng/ µl) 2.0

Total Volume 25.0

Reaction mixture was vortexed and centrifuged in a microfuge (Bangalore Genei,

India). Amplifications were performed using thermal cycler (GeneAmp PCR system

9700, Applied Biosystems, USA) with following temperature transitions:

Steps Temperature (oC) Time

111

1. Initial denaturation 94 5.00 min

2. Denaturation 94 45 sec

3. Annealing 53 45 sec

4. Elongation 72 30 sec

The thermal cycler was programmed for 30 cycles with one cycle of initial

denaturation and steps 2-4 were repeated 30 times and a final extension at 72oC for 30 sec

using fastest ramp time between the temperature transitions.

3.10.2.3 Agarose gel electrophoresis of PCR products

The amplified PCR products (Plate 3.2) were resolved by electrophoresis using 1

per cent agarose gel in 1X Tris acetate EDTA buffer (2 M Tris base, 57.10 ml acetic acid

and 0.5 M EDTA, pH 8.0, 50 X). Agarose gel was mixed with ethidium bromide (0.5 μg/

ml) before pouring. 1 kb DNA ladder (Bangalore Genei) was used as a marker. The gel

was run at 120V for 45 minutes using Bangalore Genei Power Pac system. The ethidium

bromide stained gel was viewed and image captured using gel documentation system

(Alphalmager 2200, Alpha Infotech Corporation, USA).

3.10.2.4 Sequencing and Data Analysis

PCR products of 16S rRNA gene of six efficient bacterial isolates obtained

through amplification with specific primer (section 3.10.2.3) were freeze dried in a

lyophilizer (CHRIST ALPHA I-2LD) and sent for custom sequencing using same

upstream and downstream primers used for the amplification of 16S rRNA gene

(Ocimum Biosolutions, Pvt. Ltd.).

3.10.2.5 Nucleotide sequence analysis

The 16S rDNA sequences of different bacterial isolates were BLAST (Basic

local alignment search tool) searched against the sequences of 16S rRNA of bacterial

isolates available in the Genbank Nucleotide Database (http://www.ncbi.nih.gov/blast;

112

Base P airs

500

1,000

1,500

6 5 4 3 2 1 M

Plate 3.2 PCR amplification of 16S rRNA gene of the test isolates using bacterial

universal primers. Lane 1-6 corresponds to isolate WT-A2, PT-A1, MZ-AS2, WT-AS3,

MZ-P4 and PT-P2 respectively; M is 1kb DNA ladder (Bangalore Genei)

Altschul et al. 1997) for sequence comparison. The sequences were aligned by Clustal W

program using website http://www.ebi.ac.uk/clustalw/ (Higgins et al. 1994) and pair wise

per cent nucleotide sequence similarities between WT-A2, PT-A1, MZ-AS2, WT-AS3,

MZ-P4 and PT-P2 isolates and other selected bacterial sequences were determined.

Phylogenetic analysis was performed with neighbor-joining method using

program in Molecular Evolutionary Genetics Analysis (MEGA) version 4 (Tamura et al.

113

2007). Confidence limits on grouping were done by the bootstrapping technique (1,000

repeats).

3.11 Development of liquid formulations

3.11.1 Liquid carriers for formulations

The following types of materials were used as liquid carriers for formulation

development with efficient isolates.

i) Biogas slurry

ii) Vermiwash

iii) Compost wash or Compost Tea

iv) Matka Khaad

v) Minimal Growth Medium (Peptone water)

Biogas slurry was obtained from Department of Agricultural Engineering, College

of Agricullture, CSK HPKV, Palampur. Vermiwash, Compost Tea and Matka Khaad

were obtained from “Model Organic Farm”, CSK HPKV, Palampur.

Biogas slurry was centrifuged at 10,000 rpm for 15 minutes to remove the

suspended particles and the supernatant was used for formulation development.

Vermiwash, Compost Tea and Matka Khaad were filter through muslin cloth to remove

any suspended particles, and used for formulation development.

The above liquid carriers were tried in combination with different chemicals to

study the survivability of each efficient isolates. To develop liquid formulations the

amendments viz., trehalose (10mM), polyvinylpyrollidone (PVP) (2%), glycerol (10mM)

and polyethyleneglycol (PEG) (1 %) were added separately to different liquid carriers.

These liquid carriers were then sterilized at 121°C for 15 minutes in an autoclave.

3.11.1.1 Mass production of efficient strains

For mass production of inoculant, 500 ml of respective growth medium was

inoculated with 5ml of efficient microbial cultures and incubated for 48-72 h at their

optimum growth temperatures under shaking (120 rev/ min.) conditions. Following

incubation, the cells were harvested by centrifugation (10000 rpm) for 10 min at 4 °C,

washed twice with 100 mM potassium phosphate buffer solution and resuspended in 100

114

mM potassium phosphate buffer, so as to achieve the final concentration of 109 cfu/ml.

This served as mother culture for inoculating various liquid carriers.

3.11.1.2 Mixing of mother culture with the carrier material

The above prepared bacterial suspension was mixed thoroughly with 100 ml of

liquid carrier at the rate of 1 per cent (%). The inoculated flasks were kept at room

temperature for different intervals of time. The average room temperature of Palampur is

shown in Figure 3.4.

Figure 3.4 Average (Max. & Min.) room temperature of Palampur for different months

of year 2009

3.11.1.3 Enumeration of viable cell population

The population of inoculated bacteria in liquid carriers was enumerated at monthly

intervals up to decline phase by serial dilution technique by taking 1 ml of the carrier

material and aseptically transferring it to 9 ml distilled water and shaking it vigoursly, so

as to form homogeneous suspension. The enumeration was carried out using spread plate

technique using appropriate growth medium. These plates were incubated at appropriate

Month

s

Tempe

rature

in °C

Tem

per

atu

re i

n °

C

115

growth temperature for 24-48 h. Plates containing 30-300 colonies were recorded and the

counts were expressed as log cfu per ml of carrier material.

3.11.2 Effect of stress conditions on liquid formulation

The best formulation was subjected to various stress conditions viz., temperature

(15, 25, 40 and 50 °C), pH (4.5, 5.5, 6.5 and 7.5) and desiccation. The best formulation

was amended with different chemicals and inoculated as described in 3.11.1 and 3.11.1.2.

The viable populations of bacterial cultures were enumerated as described in 3.11.1.3 at

regular intervals of time.

3.11.2.1 Effect of temperature stress on Survivability

The inoculated formulation was incubated at different stress temperatures and

was enumerated for viable cell population at regular intervals of time.

3.11.2.2 Effect of pH stress on Survivability

The best formulation prepared in 3.11.2 was subjected to varying pH, and

incubated at their optimum growth temperature. The population of viable cells was

enumerated at regular intervals of time.

3.11.2.3 Desiccation tolerance of efficient strains

One ml of six fold diluted formulation was taken in sterile 1.5 ml eppendorf

tubes. The tubes were kept open in a sterile box and incubated at 37 °C in an incubator

till these were dried up. The dried cells further incubated and viable cells were

enumerated at regular intervals of time by resuspended in 100 μl of sterile distilled water

with vigorous agitation.

3.12 Statistical analysis of data

Results of the measurements were subjected to analysis of variance (ANOVA) by

Least Significant Difference (LSD) using Windowstat package, Version 8.0 (Windowstat

2008).