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44
Materials and Methods
Table 3.1 Selected archeological sites and the condition of mural paintings
No.
Sampling areaname
Place
District
State
1.
Guruvayoor
Trissur
Kerala
2.
Mattancherry palace
Eranakulam
Eranakulam
Kerala
3.
Sankulangara temple
Panangad
Trissur
Kerala
4.
S N Puram
Trissur
Kerala
5.
Kazhuvilangu temple
Mathilakam
Trissur
Kerala
6.
Arakkal temple
Arakkal
Trissur
Kerala
7.
8.
9.
10.
11.
12.
13.
Thennarambetta Sree
bhagavathi temple
Sekarapuram Vishnu
temple
Kuruvarakkad siva
temple
Karimpuzha
Palakkad
Kerala
Adakkaputhoor
Palakkad
Kerala
Vellinezhi
Palakkad
Kerala
Kanthalloor Vishnu
temple
Vellinezhi
Palakkad
Kerala
Panjal
Palakkad
Kerala
Panjal
Palakkad
Kerala
Lakkidi
Palakkad
Kerala
Panjal Ayyappan
temple
Panjal lakshmi
narayana temple
Chuduvalathoor siva
temple
14.
Kunathoormedu sree
Krishna temple
Palakkad
Palakkad
Kerala
15.
Guruvayoor
Trissur
Kerala
16.
Dakshinachitra
Muttukad
Kancheepuram
Tamilnadu
17.
Vaikunda perumal
temple
Kancheepuram
Kancheepuram
Tamilnadu
18.
Kancheepuram
Kancheepuram
Tamilnadu
19.
Kailasanatha temple
Kancheepuram
Kancheepuram
Tamilnadu
20.
Lepakshi temple
Lepakshi
Anathapur
Andhra
Pradesh
21.
Virupakshai temple
Hampi
Bengaluru
Karnataka
Good
Good
Good
Almost lost
Good
Almost lost
Good
Better
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Materials and Methods
46
Materials and Methods
47
Materials and Methods
Figure-3.8: Mural paintings of Mannur Siva temple- Lord Vishnu and Lord Shiva
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Materials and Methods
49
Materials and Methods
50
Materials and Methods
51
Materials and Methods
3.2. Isolation of microbial predominants from mural paintings (Agrawal and Pathak,
2001)
The microbial predominants were isolated and identified from the air samples and the
swab samples, collected from the selected archeological sites. The samples were processed to
identify the predominant microbial flora present on the surface of the mural paintings
associated with biodeterioration, which in later was used to ensure the effectiveness of the
developed biobased coating.
3.2.1. Isolation of aeromicroflora from the selected archeological sites (Dhawan and
Pathak, 1989)
The microflora present in the indoor and outdoor air samples of the archeological sites
were isolated by two different methods: Air sampler method and Culture plate exposure
method.
3.2.1.1. Isolation of aeromicroflora by using Air sampler method
The aero-microflora from the archeological sites were collected using an Air sampler
[System Mark II make from HiMedia]. The air sampler consists of a small controller unit
with a sling and a hand held metal centrifugal air-sampling probe. The air sampler operates on
the impaction principle. The air under examination was sucked by the impeller in a Tornadolike spiraling conical form and the particles contained in it are centrifugally impacted against
the inward facing peripheral agar medium strip as the spiraling return air escaped around the
outer surface of the tornado. The impeller speed of 4100 rpm is so adjusted that 280 litres of
air is sampled every minute. The sampling volume corresponds to 40 litres/minute of the
separation volume. The nutrient agar strip for bacteria and rose bengal agar strip for fungi
could be inserted peripherally into the slot in the metal cup carefully so that the agar surface
faces the impeller. The strip could be almost fully inserted so that only 2 cm of the strip tab
stays out.
After sampling, the strip tab was carefully pulled out and replaced in the original
wrapper so that the agar surface faces away from the sliding lid. The sealed wrapper was
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Materials and Methods
transported aseptically to the laboratory condition and incubated at 37 C for 24 hours for the
isolation of bacterial species at 26 C for 48 72 hours for fungal species.
3.2.1.2 Isolation of aeroflora by Culture plate exposure method (Agrawal and Pathak,
2001)
The aeromicroflora of indoor and outdoor air of the selected sampling sites were
studied. The sterile nutrient agar and potato dextrose agar plates were exposed at different
points at various sites for 10 minutes. After the exposure, the plates were closed, sealed and
transported aseptically to the laboratory and incubated at 37 C for 24 hours for the isolation of
bacterial species and at 26 C for 48 72 hours for fungal species.
The micro flora present in the indoor and outdoor air samples were isolated and
compared over a period of one year from October 2008 September 2009. The isolated
organisms were pure cultured, subcultured and identified by standard microbiological and
biochemical reactions.
3.2.2 Isolation of microflora from painting surface by swab technique (Agrawal and
Pathak, 2001)
The microbial flora present in the mural paintings were isolated by collecting the swab
samples from different mural paintings surface of the selected archeological sites. The swab
samples from different mural paintings were collected aseptically, transported to the
laboratory under aseptical condition. The collected swabs were processed and each isolated
colonies were identified by morphological and biochemical tests.
The isolated bacterial and fungal strains were sub-cultured on nutrient agar and potato
dextrose agar respectively to obtain pure culture and stored at refrigerated condition till further
use.
3.3
biochemical test (Bergeys manual of systemic bacteriology, 1994, Alexopoulos and Mims,
1985)
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Materials and Methods
3.4
Natural raw materials used for wall preparation and dye extraction
(Krishnakumar, 2003)
The raw materials used in the mural paintings and wall preparation were collected,
processed and subjected for its antimicrobial and chemical properties. Dye pigments, mineral
salts, stones, gums and other natural compounds used for mural paintings were collected and
stored for further analysis.
3.4.1 Collection of natural raw material for wall preparation and dye extraction
Several raw materials were collected from various locations in Kerala and Tamil Nadu.
The raw materials for dye preparation used in mural painting collected from different location
have been extracted by traditional method. Details of the samples collected for dye extraction
was tabulated (Table 3.2).The materials used in the preparation of mural paintings were
collected for studying their antimicrobial activity and physico-chemical characterization. The
various raw materials used in the mural paintings are as follows:
1. Laterite red stone
2. Laterite yellow stone
3. Wicks & sesame oil
4. True indigo (Indigofera tinctoria) leaves
5. Kadukka (Terminalia chebula) seeds
6. Lime
7. Sand
8. Neem gum
9. Coconut water
10. Gambouge (Garcinia morella - Eravikkara) gum
11. China blue
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Materials and Methods
Table 3.2.Details of natural raw materials used for wall preparation and dye extraction
S.No.
Area
Use
1.
Painting
Painting
3.
Guruvayur (Kerala)
Painting
4.
White lime
Guruvayur(Kerala)
Painting
5.
Guruvayur (Kerala)
Painting
6.
Guruvayur (Kerala)
Painting
Chennai (Tamilnadu)
Painting
7.
8.
Sand
Guruvayur (Kerala)
Wall preparation
Neem gum
Palakkad (Kerala)
10
Coconut water
11
Kadukka
Secondary wall
preparation
Wall preparation
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Materials and Methods
it as smooth. It was applied on the rough coating and this smooth layer was plastered for a
thickness of about 1 mm. This layer was also allowed to get dry for one day.
3.4.2.1. Application of white color
The third layer of coating was done with the mixture of quick lime and the juice of
very tender coconut (Cocos nucifera). This mixture with the density of cows milk was
applied on the wall both length wise and breadth wise repeatedly for about 25 30 times. The
thickness of the coating was about 0.5 mm. The wall would gradually attain a bright white
background which also served as the white colours for mural paintings. For coating the wall
with this milky solution, a flat brush made by crushing a portion of the bark from
Sterculia foetida (Java-olive) was generally preferred. But nowadays, sophisticated brushes
available in the market are also in use.
3.4.3 Extraction of dye pigments (Krishnakumar, 2003)
3.4.3.1. Extraction of Red dye pigment
Red laterite stones were collected. The stones were washed thoroughly, powdered
manually (traditional manual grinder) and mixed with water. The process of decantation was
done repeatedly to make sure that there was no unwanted residue along with the pigment. The
resultant solution was kept undisturbed for 3 days to get separated as water on the top and
pigment at the bottom. Without disturbing the residual pigment, the upper water was poured
out. This process was repeated 3 times. The residual pigment was dried under shade and stored
in sterile containers.
3.4.3.2. Extraction of Yellow dye pigment
Yellow laterite stone were collected. The stones were washed thoroughly, powdered
manually (traditional manual grinder) and mixed with water. Decantation was done repeatedly
to make sure that there was no unwanted residue along with the pigment. The solution was
kept undisturbed for 3 days to separate pigments. Without disturbing the residual pigment, the
water was discarded and residual pigment was dried under shade and stored in sterile
containers.
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Materials and Methods
Direct sketching of outline was done in yellow pigment. Yellow wash was done
wherever it was needed to get satisfactory color. Then the outline sketch alone was repeated in
red. Shading was done by dot method. Wherever red was needed, it was done in the same way.
If the figures were in red for which shading was needed, it was done with the red pigment in
dotted method. All the outlines done in yellow and red were again drawn in black pigment. If
the black color was needed, it was done according to the method suitable (dotted or wash) for
getting the required intensity. White was the blank wall itself and its shading was done in
suitable color according to the compositional color balance of the picture. The order of
coloring is firstly yellow followed by red, green, blue. White by no means used, except for the
prevailing white spaces which are retained during the initial coating. On shading, black is used
to delineate and bring life to the portrayal. The painting is over-coated with pine resin and oil
for sheen and protection. Coloring of the character goes by virtue or characteristics as defined
in the Bhagavat gita. The spiritual, divine and dharmic characters (Satvika) are depicted in
shades of green. Those influenced towards power and materialistic wealth (Rajas) are painted
in shades of red to golden yellow. The evil, wicked and mean characters (Tamas) are generally
painted in white or black. Green and blue colors are to be painted only after applying two or
three coatings of copper sulphate solution on the place where they are to be applied. The
themes in temples were either iconic single figures of gods and goddesses or some episodes
narrated in puranas. For most of the figures, there were verses for meditation in Sanskrit. The
traditional artists working on temple walls would do all kinds of rituals prescribed for it.
According to the sequence Unmeelanam (The ritual opening of the eye of the iconic picture)
was the last item after which the picture was no more a space of line and color but a space
sanctified by the presence of god or goddess.
3.5
Antimicrobial assessment of natural raw materials used for wall preparation and
selected as representative organism from the genus of Aspergillus; Tricoderma was selected as
the representative of dermatophytes.
3.5.1 Determination of antimicrobial activity by well diffusion method (Rojas et al.,
2006)
The inhibitory effects of dyes against the test organism were determined by well
diffusion method. Muller Hinton agar was prepared, sterilized and poured in petriplates. Wells
of 6 mm diameter were punctured using a sterile well borer. The test microbes were inoculated
on the prepared medium by spreading the culture using sterile cotton swab. The well were
filled with dye extract (50 l prepared with concentration of 1 g/ ml) and incubated at 37C for
determining the antibacterial activity and at 28C for antifungal activity. After incubation, the
plates were observed for the zone of bacteriostasis and zone of mycostasis (mm) by measuring
the clearance zone around each well against each of the test organisms.
3.5.2 Minimal inhibitory concentration of extracted pigments (Rojas et al., 2006)
The minimum inhibitory concentrations of 4 main dye extract extensively used in
mural painting against the selected organisms were determined. A stock solution of 1000
mg/ml of dye extract was prepared in sterile water. This was serially diluted to obtain various
range of concentration in 2 ml of nutrient broth with 0.5 ml of test organism of concentration
105 cell/ml. A set of test tube containing broth alone were kept as control, and incubated at 37
C for 24 hours. After incubation, the turbidity of growth was measured by spectrophotometer
analysis at 560 nm.
3.6
2006)
Since mural paintings were done on layers and different shades of colours were made
by combining basic colours there are different combinations of dyes formed; hence it is
essential to know the combinatorial effect of dyes. The dyes were combined in 1:1 proportion
and the antimicrobial activity was tested against the selected bacterial and fungal isolates. The
antimicrobial activity was assessed by well diffusion method against the selected strains
according to the test procedure given above.
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Materials and Methods
3.7
Electron
Microscope
coupled
with
energy
dispersive
X-ray
used was Electron impact (EI) with Chemical ionization (CI). GC conditions: Capillary
column- DB-5ms Agilent (30 m x 0.25 mm i.d.) coated with 0.25 microm film 5%. Carrier
Gas was Helium, Column Oven Temperature 70C, Injector Temperature 250C, Injection
volume was 1 microlitre, Column Flow, 1.5 ml/min. MS conditions: Ion source temp: 230 C,
Interface temp: 240C, Scan range: 40 700 m/z, Solvent cut time: 3 mins, MS start time: 3
(min), MS end time: 35 (min), Ionization: EI 70ev), Scan speed: 2000.
Hence, in the present study, the dye samples such as Kadukka, Indigo, China blue and
Gamboge were analyzed for the presence of organic constituents using GC-MS.
3.7.3 Fourier Transform Infrared Spectroscopy (Coates, 2000).
FTIR is the most powerful tool for identifying types of chemical bonds (functional
groups). The wavelength of light absorbed is characteristic of the chemical bond as can be
seen in this annotated spectrum. The samples were analyzed for their variations in chemical
groups using FTIR spectroscopy and the results were compared for further analysis.
The selected dye pigment extracts (yellow, china blue, black, white lime, red, green
(combination of dyes) and neem gum) were analyzed for their chemical groups representing
the antimicrobial properties and other chemical characteristics.
3.8 Development and formulation of biogel for coating traditional mural paintings
(Perera, 2003)
Gums and resins are used in many countries for preserving the paintings from various
types of damage from very olden days. In china peach gum and Arabic gum and in Sri Lanka
gum Arabic and wood apple gum were used as consolidation materials in mural paintings.
Traditionally in India a mixture of pine gum and sesame oil were used as a preservative
coating for mural paintings. Most of the gums possess both antimicrobial, medicinal and
consolidation properties. The biobased coating was developed based on the naturally available
gums and resins. The following plant gums were used for the development of biobased coating
for the mural paintings.
Mattipaal (mal) (Aliantus triphysa)
Kunthirikkam (mal) (Boswellia serrata)
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Materials and Methods
compounds were mixed in equal propositions at the rate of 5g/100 ml of distilled water. After
dissolving the solutions were mixed together to form biogel.
3.8.1 Antimicrobial activity of plant gums (Rojas et al., 2006)
The preliminary antimicrobial activity was tested by mixing 0.5 g of each plant gum in
1 ml of sterile distilled water. The prepared suspension was used for the antimicrobial assay.
The antimicrobial activity was measured in terms of zone of bacteriostasis and zone of
mycostasis for the antibacterial and antifungal assessment respectively. The compounds with
maximum antimicrobial activity were chosen for combinatorial studies. The selected
compounds were tested for their antimicrobial activity at various concentrations.
3.8.2 Minimal inhibitory concentration of selected gums (Rojas et al., 2006)
The minimum inhibitory concentration of the Fagopyrum esculentum and
Ferula asafoetida and Azadirachta indica were studied. Similarly, the minimum inhibitory
concentration of the selected combination was also analyzed by well diffusion method.
3.8.3 Combinatorial study of selected plant gums
The combined antimicrobial activities of the selected natural compounds
(Fagopyrum esculentum, Ferula asafoetida and Azadirachta indica) were analyzed by mixing
the compounds in various ratio (1:1:1, 1:1:2, 1:1:3, 1:2:1, 1:3:1, 2:1:1, 3:1:1, 2:2:1, 2:3:1,
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Materials and Methods
3:3:1) and the antimicrobial activity of the different proportions were analyzed by well
diffusion method against the selected strains.
3.8.4 Statistical analysis
The results were expressed as mean SE of three parallel measurements. The data was
analyzed by one way ANOVA by Duncans multiple comparison tests using SPSS-9 for
Windows 7 as a statistical tool to determine the antimicrobial activity of the different
proportions of selected natural compounds with P<0.05 considered significant. The hypothesis
selected (H0) was that There is significant effect of antimicrobial effect of different
proportions of selected natural compounds against the bacterial and fungal isolates.
3.8.5 Chemical properties of developed biogel: GC-MS analysis. (Shuya et al., 2011)
The combination with maximum antimicrobial activity was chosen and their physicochemical characteristics were determined by performing GC MS analysis.
3.9
available in the market. Bavastin (Methyl N (1H benzimidazol -2yl) carbonate, Benlate
(benzimidazole), Ketamine, Cetyl pyridinum bromide, Nystatin, Parachlorom Cerol,
Phenylmercuric acetate, Quaternary ammonium compound were commercially purchased and
antimicrobial activity was compared with the developed protective coating.
3.11
Azadirachta indica) was applied on mural paintings on slab. The ability to preserve the murals
from biodeterioration was studied by collecting the swab samples at various time intervals in
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Materials and Methods
the laboratory condition. The slab samples were kept at the laboratory at 27oC with medium
humidity and the microbial flora present on the slab was studied. The shelf life period of the
developed coating was determined by frequent sampling from the slabs and the antimicrobial
properties were determined based on the occurrence of the microbial flora.
3.12
Azadirachta indica) was applied on traditional mural paintings on prepared walls of Institute
of Mural Painting, Guruvayoor, Kerala .Aqueous and oil emulsion was used to coat the mural
paintings. The shelf life period of the developed coating was determined by frequent swab
sampling (30 days) from the painting and efficiency of the developed coating was determined.
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Materials and Methods