25

CHAPTER 3
MATERIALS AND METHODS

3.1

SITE OF SOIL SAMPLE COLLECTION

Around 210 soil samples were collected from rhizosphere and non-

rhizosphereregions of different medicinal plants at Yercaud and Kolli hills

belonging to Eastern Ghats of Tamil Nadu, southern India from a depth of 6-10
cm as described by Lee and Hwang (2005). The study was carried out for a
period of seven months from June 2011 to December 2011. The collected soil
samples were subsequently allowed for shade dried for further analysis.

Figure 3.1

Sampling locations of Yercaud hills (A) and Kolli hills (B) of

Eastern Ghats of Tamil Nadu, southern India

26

3.2

EDAPHIC AND ENVIRONMENTAL ANALYSIS OF SOIL

SAMPLES
All the collected soil samples were subjected to analyse various soil

edaphic parameters such as soil pH, Ec, water holding capacity, organic
carbon (Walkley and Black 1934), nitrogen, available phosphorous,
exchangeable potassium, exchangeable calcium, sodium and magnesium
contents (AOAC 1990). Moreover, the same soil samples were subjected to
enumerate and isolate potential actinomycetes by using starch casein nitrate
agar (SCN) by following serial dilution technique.
3.2.1

Determination of Various Soil Edaphic Parameters (AOAC

1990)
Thirty grams of soil samples was mixed with 60 mL of distilled

water (1:2 ratio) and measured their pH using a digital pH meter (Elico). The
electrical conductivity (Ec) was measured in the same samples after settling the
suspension using an Ec meter (Elico). Soil organic matter content was
estimated by dry oxidation method in which the difference between initial and
final weight was arrived. Estimation of water holding capacity of the soil
samples was studied by following the method of filter paper containing soil
holding moisture at given time. A known amount of soil was kept to submerge
the bottom of container in water up to inch depth. The containers were
further placed in hot air oven at 105C and dried to get a constant weight in
which moisture absorbed by the filter paper and dried soil samples were
recorded.
3.2.2

Estimation of Total Organic Carbon (Walkley and Black 1934)

Total organic carbon content was determined by oxidation of

sulphuric acid and chromic acid mixture in soil samples in which 10 mL of

27

1 N potassium dichromate and 20

added. One

mL of concentrated sulphuric acid were

mL of ferroin indicator was added and titrated against 0.5 N

ferrous ammonium sulphates till the colour changes was observed from green
to red.
3.2.3

Estimation of Total Nitrogen (AOAC 1990)

One gram of soil sample was added with 3 mL of salicylic acid and a

pinch of sodium thiosulphate. Digestion was carried on a hot plate after adding
5 mL of hydrogen peroxide and the content was neutralized with a mixture
containing 2% sodium hydroxide, 10% sodium potassium tartarate and 1 mL of
Nesslers reagent. The solution was finally measured by taking optical density
at 420 nm using an UV-VIS Spectrophotometer (Hitachi, Japan) against
ammonium sulphate as a standard.
3.2.4

Estimation

of

Available

Phosphorus

and

Exchangeable

Potassium (AOAC 1990)

Around 100 mg of soil sample was digested with 2 mL of
concentrated nitric acid and 2 mL of perchloric acid in a heating mantle to near
dryness. After digestion, the solution was added with 0.4 mL of ammonium
molybdate solution and 5 drops of stannous chloride. The optical density was
measured at 690 nm against rock phosphorus as a standard. Potassium present
in the soil sample was analyzed using a Flame photometer. A standard
calibration curve was made using potassium chloride. The amount of K present
in the sample was calculated and expressed as per cent dry matter.

28

3.2.5

Determination

of

Exchangeable

Calcium,

Sodium

and

Magnesium (AOAC 1990)

Calcium, sodium and magnesium contents present in the soil samples
were analyzed using a Flame photometer. A standard calibration curve was
made using calcium chloride, sodium chloride and magnesium chloride to
measure calcium, sodium and magnesium; respectively. Using the various
standard graphs, the amount of unknown contents present in the samples were
calculated and expressed as per cent dry matter basis.
3.3

ISOLATION AND IDENTIFICATION OF ACTINOMYCETES

FROM EASTERN GHATS
For the isolation of actinomycetes, around one gram of the dried

rhizosphere soil samples was mixed well with 100 mL of distilled water in
Erlyenmeyer flask. Then one mL of the diluents was pipetted out and serially
diluted up from 10-1 to 10-7 dilutions using sterile distilled water. One mL of
the each diluent was transferred to the Starch casein nitrate agar (SCN) and
incubated at 282C for 2-3 days. Actinomycetes colonies like chalky with
powdery mass having different colour were selected for further study.
Distribution pattern of actinomycetes strains was also studied in
terms of various depth and distance from the rhizosphere soils of different
medicinal plants such as Asparagus racemosus, Andrographis paniculata,
Solanum nigrum, Zingiber officinale and Cissus quadrangularis growing at
Yercaud hills Acacia nilotica, Adhatoda zeylancia, Andrographis paniculata,
Cissus quadrangularis and Eclipta prostrate growing at Kolli hills.

29

3.4

CHARACTERIZATION OF ACTINOMYCETES STRAINS

Generic level identification of actinomycetes strains was carried out

by following different morphological, physiological and biochemical traits by

adopting various polyphasic taxonomic approaches (Collins and Lyne 1980,
William and Wilkins 1994).
3.4.1

Effect of Different pH, Temperature and NaCl Concentration on

the Growth of Streptomyces Strains (Cappuccino and Sherman
1992)
The growth of actinomycetes strains at different pH and temperature

regimes was tested using SCN broth medium prepared at pH of 5, 6, 7, 8 and 9.

The effect of various temperature regimes on the growth of actinomycetes
strains was studied by keeping the inoculated cultures at the temperature of 10,
20, 30, 40 and 50C. The effect of various NaCl concentrations on the growth
of Streptomyces strains was studied with 1, 2, 3 and 4%. The growth of
actinomycetes at different humidity 60, 70, 80 and 90% was determined. The
actinomycetes strains were inoculated, incubated and their population was
enumerated in basal medium by following serial dilution technique.
3.5

EFFECT OF BIOTIC AND ABIOTIC FACTORS ON THE

GROWTH OF STREPTOMYCES STRAINS

3.5.1

Standardization of Various Media for the Growth and Mass

Multiplication of Streptomyces Strains
The ability of Streptomyces strains in colonization and mass

multiplication themselves was tested through various growth media viz., SCN
agar, Yeast extract malt extract agar (ISP2), Oat meal agar (ISP3), Inorganic
salt agar (ISP4) and

Glycerol asparagines agar (ISP5). The Streptomyces

strains were inoculated in the respective media individually and incubated at

30

28C for 3 days. The growth of Streptomyces strains in terms of population

load was enumerated in SCN medium by following serial dilution technique
(Dubey and Maheshwari 2009).
3.5.2

Effect of Different Carbon, Nitrogen, Amino Acids and Vitamin

Sources on Growth of Streptomyces Strains (Dubey and
Maheshwari 2009)
Different carbon sources covering monosaccharides, disaccharides

and polysaccharides, nitrogen compounds covering organic, inorganic, nitrate

nitrogen and ammonical nitrogen each at 1% concentration was used in this
study. Similarly, amino acids such as alanine (simple amino acid), serine
(hydroxyl amino acid), methionine (sulphur containing amino acid), aspartic
acid, glutamic acid (acidic amino acid), asparagine (amino acid amides),
arginine (basic amino acid) and proline (heterocyclic amino acid) at 400 ppm
concentration and vitamins viz., Thiamine (B1), Riboflavin (B2), Pantothenic
acid (B3), Nicotinic acid (B5), Biotin (B7), Folic acid (B9), Ascorbic acid (C)
and Vit A at 200 ppm concentration were amended in SCN medium. Control
was also maintained suitably. The inoculated cultures were incubated at
72 hours subsequently and the population of Streptomyces strains was
enumerated using suitable medium by following serial dilution technique.
3.6

ENZYME ASSAY
The isolates selected were subjected for the primary and secondary

screening methods. All the active isolates were exposed to primary screening
and those which give positive results were screened and subjected for the
secondary screening.

31

3.6.1

Primary and Secondary Screening

For primary screening the active isolates were inoculated on suitable

medium by the spot inoculation technique for amylase, protease, urease,

cellulase, lipase and chitinase. The isolates which showed positive were
subjected for the secondary screening process. The cultures were grown in
glycerol asparagine broth at 30C for 7, 14 and 21 days. The broth cultures
were filtered and the partially purified enzyme was carried out for the further
assay.

The primary screening of amylase (Bernfeld 1985, Chiaki Imada

et al 1988) production was studied by inoculating them on starch agar plate

method. Protease determination was carried out using modified agar plate assay
technique by following the method of Chu (2007) and Bajaj and Sharma
(2011).
The cultures were inoculated in the test tubes containing
Christensens agar medium at 35oC and observed for the color change at 6, 24,
48 hours till 7th day of incubation. The appearance of pink colour showed the
conversion of urea into ammonia and carbon dioxide for the determination of
urease production (Christensen 1946). The isolates of actinomycetes were spot
inoculated on the Czepeck mineral salt agar media inoculated onto the media
and incubated at 28oC for 7days to express cellulose production (Andro et al
1984, Ghose 1987). After that, the plates were flooded with Iodine-Potassium
iodide solution to detect clearing zones against a dark-brown back ground
(Fernandes-Salomao et al 1996). Lipase and chitinase determinations were
carried out using modified agar plate assay techniques by following the method
of Savitha et al (2007) and Omura (1986); respectively.

32

3.7

RESISTANCE OF STREPTOMYCES STRAINS TO VARIOUS

PESTICIDES, FUNGICIDES AND WEEDICIDES (Dubey and
Maheshwari 2009)
The pesticides such as propargite, dicofol, deltamethrin, quinolphos

and fenpyroximate, the fungicides namely hexaconazole, propiconazole,

copperoxychloride, mancozeb and carbendazim and the weedicides such as
parquat dichloride, 2, 4dichlorophenoxy acetic acid and glyphosate were
tested with Streptomyces strains. They were prepared in three different
concentrations (100, 300 and 500 g/mL) using sterile water and filter
sterilized. They were added in SCN broth and thereby Streptomyces cultures
were inoculated. Inoculated cultures were incubated at room temperature for
72 hours wherein, resistance of Streptomyces strains was observed by
measuring OD at 600 nm using an UV-VIS Spectrophotometer.
3.8

MOLECULAR CHARACTERISATION OF STREPTOMYCES

SPP. STRAINS

3.8.1

Study of Genomics
The genomic DNA were isolated from Yer11, Yer28, Kol35, Kol44

and MTCC strainsof Streptomyces spp. using four different methods to obtain
high yield of DNA.
3.8.1.1

STE lysis method (Sambrook and Russell 2002)

Pelletized strains of Streptomyces spp.were added with 200

STE lysis buffer and 10

L of

L of lysozyme, incubated at 37C for 2hours. To

that 200 L of 10% SDS was added and tapped gently for 30 mins at room
temperature. 150 L of sodium acetate was added and centrifuged at
12,000 rpm for 20 mins. The aqueous phase was transferred to a fresh
eppendorf tube and mixed with 100% of ice cold ethanol, kept at -20C for

33

1 hour. The contents were centrifuged at 10,000 rpm for 15 mins and the pellet
obtained was washed twice with ethanol, dried and stored for the further use.
3.8.1.2

CTAB Method (Murray and Thompson 1980)

The cultures of Streptomyces spp.were grown in Trypticase soy

broth, in which 15
15

mL was transferred to the centrifuge tube containing

mL CTAB extraction buffer, preheated to 90C and suspend thoroughly.

The tubes were then incubated at 65C for 30 minutes with occasional mixing
and cooled to room temperature. Then added equal volume of chloroform:
isoamyl alcohol mixture (24:1) to get an emulsion and centrifuged at
12,000 rpm for 5 minutes to separate the aqueous layer containing the DNA.
The chromosomal DNA was precipitated by the addition of iso-propanol. The
DNA was transferred to a clear tube and air dried for 15 minutes.The DNA was
dissolved in minimal volume of TE buffer followed by incubation of the tubes
at 65C for 10 minutes. RNase was added to the tube, incubated for 30 minutes.
Then added equal amount of Chloroform: isoamylalcohol (24:1) mixed well
and centrifuged at 12,000 rpm for 10 minutes. The supernatant was discarded
and the pellet was air dried and used for the further analysis.
3.8.1.3

Phenol Chloroform Extraction (Sambrook and Russel 2002)

The cultures of Streptomyces spp.were grown in Trypticase soy broth

for 7 days at 37oC. The cell pellets obtained after centrifugation was suspended
in 400Pl of sucrose TE solution. To this 32 PL of lysozyme (10 mg/mL) was

added and incubated at 37oC for 30 minutes. Following this 100Pl of EDTA
and 60 PL of freshly prepared 10% SDS was added and mixed gently. To this

1.5 PL of proteinase K and 3 PL of RNase was added mixed gently and

incubated at 55OC water bath for 2 hours. The tubes were then equilibrated

with 250 PL of phenol and 250 PL of chloroform, mixed well and spun at

34

12000 rpm for 10 minutes. The aqueous phase was twice extracted with
phenol: chloroform (1:1). Then added 2.5 volume of ice cold absolute alcohol
was added mixed well and kept at room temperature for 10 minutes which was
spun at 12000rpm. To the pellet added 100 PL of 70% ethanol and spun at

12000 rpm for 5 minutes. After the removal of supernatant, to the pellet 20 PL
of sterile distilled water or TE solution was added and mixed well and loaded
on to the 0.8% agarose gel.
3.8.1.4

Commercial kit Method

DNA was also extracted simultaneously from the isolates of

Streptomyces spp.using the commercial Mini Kit (Qiagen, USA) following the
instructions, and the DNA was eluted in 50l of TE buffer.
3.8.1.5

Assessment of quality and quantity of DNA

The genomic DNA obtained from Streptomyces spp. strains was

checked quantitatively and qualitatively for comparison of different methods

adopted. DNA was quantified following spectrophotometric method and
quality was checked by computing A 260/280 ratio. DNA quality was also
checked on 0.8% agarose gel.
3.8.1.6

16S rRNA gene amplification and RAPD

The 16SrRNA gene was amplified from genomic DNA obtained

from Streptomyces spp.cultures by PCR with Forward primer-F243 (5ATGAGCCCGCGGCCTA-3)

and

reverse

primer

R513GC-(5-

CGGCCGCGGCTGCTGGCACGTA-3). The reaction mixture contained 25 to

50 ng of DNA, Ex Taq PCR buffer, 1.5 Mm MgCl2, 10Mm deoxynucleoside
triphosphate mixture, 50 pmol of each primer, and 0.5 U of ExTaq polymerase.
PCR conditions consisted of an initial denaturation

at 94oC for 5mins;

35

30 cycles at 94C for 1mins, annealing 63oC for 1mins and 72o for 1 mins; and
final 5 mins extension at 72oC. The amplification products were examined by
agarose gel electrophoresis and purified by using a QIA quick PCR clean up kit
with the protocol suggested by Qiagen Inc. The complete 16Sr RNA gene was
sequenced by using the PCR products directly as sequencing template with
above mentioned primers. All sequencing reactions were carried out with an
ABI 377 automated DNA sequencer.The isolated DNA was subjected for the
RAPD analysis using 10 different operon primers.
3.8.1.7

Phylogenetic Analysis
Nucleotide sequences were compared to those in the Gene Bank

database with the Basic Local Alignment Search Tool (BLAST) algorithm to
identify known closely related sequences. Sequences were analyzed CHROMO
SOFTWARE. Trees were generated by the neighbour- joining (Saitou and Nei
1987) algorithm (Kluge and Farris 1969) implemented in Phydit.. The
assemblage of 16S rRNA gene sequences in each library was analyzed by
rarefaction analysis using EcoSim (Gotelli and Entsminger 2006) to assess the
extent to which the diversity of microbial communities was represented by the
library at the class and species level. The number of species in each clone
library was determined by comparing closely related sequences using bl2seq
(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) (Tatusova and Madden
1999). 16S rRNA sequences exhibiting a percentage of similarity of 97% or
lower (Devereux et al 1990) were considered for species authentication.

36

3.8.2

Study of Protein Separation

3.8.2.1

Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis

(SDS -PAGE) method (Laemmli 1970)
A separating solution was prepared by mixing of solution A

containing acrylamide and bis-acrylamide, solution B containing Tris-HCl.

Ammonium persulphate solution was prepared freshly and TEMED was added
for polymerization of the gel. Sample loading buffer was set up by mixing of
0.25 M Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, 20% -mercaptoethanol and
0.5% bromophenol blue. The protein samples obtained from the isolates of
Yer11, Yer28, Kol35, Kol44 and MTCC strains of Streptomyces spp.were
subjected to electrophoresis carried out in a vertical mini-gel unit in a
discontinuous buffer system using 7% acrylamide gel. After the electrophoretic
run, the gel was stained with coomassie brilliant blue overnight and destained
with a solution containing acetic acid, methanol and water in the ratio of
10:40:50. Protein molecular marker having molecular weight ranges from 14.4
to 66 (Kda) was used for the determination of molecular weight of the
unknown sample.
3.8.2.2

Urea Extraction protocol (Sambrook and Russell 2002)

Around 25 mL of the broth was mixed with 5 mL of ice-cold urea

dissolved HCl (2% pH = 8.8), 3.5 L of SDS, 12%glycerol and 2 L of

2-mercaptoethanol. Cell debris was removed by centrifuging at 500 g at 4 C
for 15 min. The supernatant was transferred to a new tube, and proteins were
precipitated by adding four volumes of ice-cold acetone containing
trichloracetic acid and 2 L of 2-mercaptoethanol. Pellets were obtained by
centrifugation at 15000 g at 4C for 45 min, and washed three times with an
ice-cold solution of 2-mercaptoethanol in water-acetone. Pellets were

37

centrifuged at 15000 g for 15 min between washes. Supernatants were

discarded, and pellets were dried under air at room temperature.
3.8.2.3

TCA Extraction Protocol (Caruso et al 2009)

The protocol was modified from a published TCA protocol. 10 mL

of the broth was mixed with 5 mL of ice-cold extraction buffer consisting

of Tris HCl (pH = 8.8), 3 L of SDS, 15% glycerol and 2 L of
2-mercaptoethanol. Cell debris was removed by centrifuging at 500 g at 4C
for 15 min. The supernatant was transferred to a new tube, and proteins were
precipitated by adding four volumes of ice-cold acetone containing
trichloracetic acid and 2 L of 2-mercaptoethanol. Samples were stored at
20C for at least 1 h. Pellets were obtained by centrifugation at 15000 g at
4C for 45 min, and washed three times with an ice-cold solution of
2-mercaptoethanol in water - acetone. Pellets were centrifuged at 15000 g for
15 min between washes. Supernatants were discarded, and pellets were dried
under air at room temperature.
3.9

ANTAGONISTIC ACTIVITY OF STREPTOMYCES SPP.

3.9.1

Primary Screening
The antagonistic activity of chosen actinomycetes from Yercaud and

Kolli hills were tested by following cross streak method (Ellaiah et al 1997).
Single streak of actinomycetes strains such as Yer11, Yer28, Kol35, Kol44 and
MTCC strains were streaked on the surface of modified nutrient agar medium
plates and incubated at room temperature 27oC for 5-7 days. On obtaining
ribbon like growth , the overnight culture of human pathogenic bacteria
Escherichia coli MTCC 1687, Serratia marcescens, Bacillus subtilis MTCC
2387, Proteus vulgaris MTCC 426, Salmonella paratyphi (A) MTCC 735,
Salmonella paratyphi (B) MTCC 733, Klebsiella pneumonia MTCC 618,

38

Staphylococcus aureus MTCC 7405, Pseudomonas aeruginosaMTCC 8485,

Clostridium sp.MTCC 1349 were streaked at perpendicular to the original
streak of actinomycetes and incubated at 282oC and the inhibition was
measured after 24 hours.
3.9.2

Secondary Screening

3.9.2.1

Mass cultivation of antagonistic actinomycetes for antimicrobial

activity (Ponmurugan et al 2011)
The selected actinomycetes were subjected for the mass cultivation

using fermentor. Fermentor was thoroughly washed with soap solution and
autoclaved without media for 15 minutes at 15 lbs. After sterilizing the whole
fermentor, the substrate (Bennett media) was transferred into the fermentor
under laminar air flow chamber followed by autoclaving the fermentor for
15minutes at 15 lbs at 121oC. Once the sterilization process gets completed, the
media was allowed to cool (thawed) to room temperature. A loopful inoculum
of morphologically different actinomycetes strains viz., Yer11, Yer28, Kol35,
Kol44 and MTCC were further inoculated into 500 mL conical flask containing
100 mL of Yeast extract-Malt extract broth and kept at 28oC for 72 hours with
continuous shaking. Then twenty millilitre of grown culture was transferred
into the fermentor containing 1000

mL of Bennett medium using sterile

syringe and incubated for 7 days under continuous shaking. The parameters for
fermentation process were properly set using external controlling device. The
temperature was maintained at 24oC, dissolved oxygen about 3, the agitator at
200 rpm and pH about 70.2. The fermentation process was carried out for 7
days. Continues checking was carried out so as to find out the contamination
i.e. other than desired organism.

39

3.9.2.2

Extraction of bioactive secondary metabolites from fermented

broth
The mass cultivated broth (pH 7.2) was adjusted to pH 5.0 using 1N

Hydrochloric acid and filtered by cheese cloth to remove the mycelia biomass.
One litre of the filtrate was mixed with 500 mL of ethyl acetate in a separating
funnel to extract the bioactive compound. After removing the lower aqueous
phase, the upper solvent phase was concentrated to obtain the crude extract.
This process was repeated for three times to obtain complete extraction of
active principles. This crude extract was used for further screening.
3.9.2.3

Minimum Inhibitory Concentration (MIC) (Hammond and

Lambert 1978)
Minimum inhibitory concentration is defined as the lowest

concentration of antibiotic that inhibit the growth of a particular microorganism

by broth dilution method. 0.1 mL of 24 hours microbial broth of human
bacterial pathogens Escherichia coli, Serratia marcescens, Bacillus subtilis,
Proteus vulgaris, Salmonella paratyphi (A), Salmonella paratyphi (B),
Klebsiella pneumonia, Staphylococcus aureus, Pseudomonas aeruginosa and
Clostridium sp. were added to the tubes containing 0.5

mL of nutrient broth

and various concentrations (10, 20, 30, 40 and 50 gmL-1) of bioactive

secondary metabolites from actinomycetes. Nutrient broth alone served as
negative control. Whole setup in duplicate was incubated at 37C for 48 hours
in a thermostat shaker. After incubation, optical density was measured at
620 nm by using UV-Visible spectrophotometer (Shimadzu, Japan).

40

3.9.2.4

Antibacterial sensitivity assay by agar well diffusion method

(Pandey et al 2004)
Antibiotic sensitivity test was performed by agar diffusion method

which was designed to determine the smallest amount of the bioactive

secondary metabolites needed to inhibit the growth of the pathogenic
microorganism. The medium of choice was Muller-Hinton agar with a pH of
7.2 to 7.4, was poured into plates to form a uniform depth of 5mm and allowed
for solidification. Prior to use, the plates were transferred to an incubator at
37oC for 10 to 20 minutes to dry off the moisture that was developed on the
agar surface. Overnight growth of chosen bacterial broth culture was swabbed
on the surface of the agar media and further well was prepared by using well
cutter. To each well, 50 g of bioactive secondary metabolites were added and
incubated in a thermostat incubator for 24 hrs. After, incubation, the zone of
inhibition around the well was calculated and expressed as zone of inhibition in
millimeter.
3.9.3

Mass Cultivation of Antagonistic Actinomycetes for Antifungal

Activity
A loopful of the purified culture of the selected actinomycetes was

inoculated into autoclaved 500 mL of SCN broth and incubated at 28C in

fermenter at 150 rpm for 5 days. The pH was maintained at 5.5 and the
temperature at 28C. After 5 days of fermentation the medium was harvested
and centrifuged to remove cells and debris. The filtrate was collected in a screw
capped bottle and filtered. Then the filtrate was mixed with ethylacetate in the
ratio of 1:1 (v/v) and shaken vigorously for 1 h in a solvent extraction funnel.
The solvent phase that contains antifungal compound was separated from the
aqueous phase. Solvent phase was evaporated to dryness in water bath at 80 90C and the residue is used to check antifungal activity.

41

3.9.3.1

Antifungal activity of actinomycetes

Determination of antifungal activities of Streptomyces spp. strains

performed by using agar disc method. Potato dextrose agar plates were
prepared and mixed with the culture extract of different concentrations such as
3, 6 and 9 mL were used. Then the plates were inoculated with the agar disc of
test fungi in the centre of the Petri dish and incubated at 28C for 4 days. The
test fungi used are Aspergillus fumigates (MTCC 3376), Aspergillus flavus
(MTCC873), Stachybotrys chartarum (KSR144), Histoplasma capsulatum
(KSR432), Pneumocystis jirovecii (KSR52), Candida albicans (MTCC 227),
Scizophyllum commune (KSR211), Ustilago maydis (MTCC 1474).
3.10

ANTICANCEROUS ACTIVITY

3.10.1

Description about cell lines chosen for the study

There were three types of breast cancer cell lines such as SKBR3,

MCF7and MDA-MB231 were used for the present study. SKBR3 cell line was
maintained in Dulbecco Modified Eagle Medium (DMEM) supplemented with
4.5 g/L glucose, 2 mM L-glutamine and 10% fetal bovine serum.MCF7 cell
line is a competent hormone sensitive and non- invasive luminal breast cancer
lines that express ER+ and p53 and maintained in DMEM with 10% fetal
bovine serum and 1% Streptomycin+Penicillin solution using 5% CO2 at 37oC
as described by Soule et al (1973). MDA-MB231 breast cancer cell lines were
maintained in DMEM with 15% fetal bovine serum and 1.5% streptomycin +
penicillin solution using 5% CO2 at 37oC as described by Cailleau et al (1978).
3.10.2

Cell Line Maintenance

Cells were grown as monolayer in 25 cm2 or 72 cm2 ( depending on

the requirement) tissue culture flask and maintained in DMEM medium

supplement with 10% FBS and 1% antibiotic solution at 37oC with 5% CO2 in

42

humidified atmosphere. The culture flask containing the media and inoculum
were observed daily for pH change based on the colour. This may be due to
normal media utilization by cells or abnormal level of O2/CO2 tension in
incubator or contamination. Contamination or over growth of cells also results
in cloudiness of media. Cells were observed daily under inverted phase contrast
microscope for attained health and growth confluence, phenotype and possible
contamination or other abnormalities and recorded. Media was changed after
2-3 days as per requirement and noted in the flask with marker pen. Cells
grown to 70-80% or more confluence (number of cells over flask area) were
subcultured into new flask. Over confluence or other stress such as inadequate
media change, abnormal O2 /CO2 tension etc., were checked continuously to
find out the changes in cell characteristics which may interfere the experiments
under in vitro condition.
3.10.3

In vitro Cytotoxic Assay (MTT Assay)

The cytotoxicity activity of bioactive secondary metabolites from

Streptomyces spp. strains was evaluated by the MTT assay on human breast
cancer cell line (SKBR3, MCF7 and MDA-MB231). Around 200 L of cell
suspensions at a density of 5 104 cell mL-1 were plated in 96- well microtiter
plates and incubated for 2 hour above condition. Then 5 L, 10 L, 15 L
and 20 L of the bioactive secondary metabolites solutions at different
concentrations was added in triplicate to each well and further incubated for 48
hrs in the same conditions. 50 L of 80% Trichloroacetic acid (TCA) was
added to each well to fix the cells for 1.5 h in 4oC. 200 L of an old medium
containing TCA was washed repeatedly by water and stained with 100 L of
MTT and incubated for 4 hrs. Formazan products formed were solubilised by
adding 200 L DMSO per well including control. The plate with cover was left
for 15- 20 minutes in dark at room temperature. After that, plate cover was

43

removed and absorbance was measured at 540 nm by using microtiter plate

reader.
3.11

BIOSYNTHESIS

OF

NANOPARTICLES

AND

THEIR

EVALUATION
Efficient strain of Streptomyces spp. (Kol35) was subjected for the
biosynthesis of silver, gold and copper nanoparticles, after confirmation using
UV-VIS spectroscopy, Fourier Transform Infra-Red (FTIR), and Transmission
Electron Microscope (TEM). Moreover, the nanoparticles were evaluated
against the high resistant bacterial pathogens and the human breast cancer cell
lines isolates for their antagonistic activity and stability of efficacy for a period
of one year.
Kol35 strain was grown in SCN broth at a temperature of 253C.,
for 7 days. The biomass was separated and washed thrice with sterile distilled
water. Ten grams of microbial biomass was mixed with 1000

mL aqueous

solution of 1 mM silver nitrate, chloroauric acid and copper sulphate

separately. The mixture was placed in a 100 rpm rotating shaker at 28C for
120 hrs. The reduction of silver, gold and copper ions was routinely monitored
at different time intervals by collecting the aqueous layer and subsequently
subjected to read at continuous wavelength of 300 - 600 nm.
The culture filtrates containing silver, goldand copper nanoparticles
were tested against human bacterial pathogens and cancer cell lines at different
concentrations (10 L, 15 L and 20 L) to assess the antimicrobial and
anticancerous activities. The percent of growth inhibition was calculated.

44

3.12

STATISTICAL ANALYSIS
All the data were statistically evaluated using SPSS 14.0 statistical

package (SPSS, Inc. Chicago, IL). The signicant means were segregated by
critical difference (CD) at various levels of signicance. The standard error
(SE), standard deviations (SD) and covariance analysis (CV) were also
calculated (Gomez and Gomez 1984).