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44 Materials and Methods
3. MATERIALS AND METHODS
The main focus of the current research is to study the aeromicroflora associated with
the deterioration of the mural paintings of selected archeological sites and to develop a
protective bio-based coating for the mural paintings. The methods used for the isolation of the
predominant aeromicroflora from the selected archeological sites, extraction of the pigments
used in paintings and its antimicrobial properties of the extracted pigments, their
characterization, antimicrobial property of the combinatorial effect of the pigment extracted
and method for developing the biobased coating, its efficiency in comparison with commercial
biocides and slab studies with field trails are presented in detail.
The first step was to isolate the predominant aeromicroflora from the selected
archeological sites by swab technique, open plate technique and air sampler from various
study locations with specific reference to mural paintings of South India.
3.1. Study location
From South India, 21 archeological sites were selected for the present study. Among the
21 sites, 39 different mural paintings at different status where considered for observation. In
Table-3.1, location and condition of the mural paintings were presented. All the above said
archeological sites were keenly observed between the periods of October 2008 - September
2009 for different sample collection (Figure-3.1).
Samples were checked by two methods for the isolation of microbial predominants.
The indoor and outdoor air floras from the archeological sites were collected by swabbing and
air sampling methods. The samples were taken with the help of air sampler and processed in
microbiology laboratory. Each isolated colony was identified by morphological and
biochemical methods.
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Table 3.1 Selected archeological sites and the condition of mural paintings
No. Sampling area-
name Place District State
Location of the
mural
paintings
Condition
of the mural
paintings
1. Sree Krishna temple Guruvayoor Trissur Kerala
Surrounding
sanctum of the
temple
Poor
2. Mattancherry palace Eranakulam Eranakulam Kerala
On the inner
walls of the
palace
Good
3. Sankulangara temple Panangad Trissur Kerala Surrounding
sanctum Better
4. Sree narayana Sree
Krishna temple S N Puram Trissur Kerala
Surrounding
sanctum Better
5. Kazhuvilangu temple Mathilakam Trissur Kerala Surrounding
sanctum Better
6. Arakkal temple Arakkal Trissur Kerala Surrounding
sanctum Better
7. Thennarambetta Sree
bhagavathi temple Karimpuzha Palakkad Kerala
Two figures on
sanctum Poor
8. Sekarapuram Vishnu
temple Adakkaputhoor Palakkad Kerala
Surrounding
the sanctum
Partially
spoiled
9. Kuruvarakkad siva
temple Vellinezhi Palakkad Kerala
Surrounding
sanctum
Partially
spoiled
10. Kanthalloor Vishnu
temple Vellinezhi Palakkad Kerala
Surrounding
the sanctum
and on roof
Better
11. Panjal Ayyappan
temple Panjal Palakkad Kerala
Surrounding
sanctum Good
12. Panjal lakshmi
narayana temple Panjal Palakkad Kerala
Surrounding
sanctum Better
13. Chuduvalathoor siva
temple Lakkidi Palakkad Kerala
Surrounding
sanctum Poor
14. Kunathoormedu sree
Krishna temple Palakkad Palakkad Kerala
Surrounding
sanctum
Good
15. Institute for mural
paintings Guruvayoor Trissur Kerala
On the inner
walls of the
institute
Good
16. Dakshinachitra Muttukad Kancheepuram Tamilnadu
On the inner
walls of the
institute
Good
17. Vaikunda perumal
temple Kancheepuram Kancheepuram Tamilnadu
Inner side of
the outer wall Almost lost
18. Varada raja perumal
temple Kancheepuram Kancheepuram Tamilnadu
Many walls of
the
temple
Good
19. Kailasanatha temple Kancheepuram Kancheepuram Tamilnadu Inner part of
the outer wall Almost lost
20. Lepakshi temple Lepakshi Anathapur Andhra
Pradesh
Many parts of
the temple Good
21. Virupakshai temple Hampi Bengaluru Karnataka Many parts of
the temple Better
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46 Materials and Methods
Figure-3.1: Sampling Sites involved in the microbiological analysis of mural
paintings
Figure-3.2: Mural paintings of Adakkaputhur Vishnu temple- Dasavataram
Figure-3.3: Mural paintings of Dakshinachitra- Palazhimadhanam and
Gurudakshina
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47 Materials and Methods
Figure-3.4: Mural paintings of Kanthaloor Vishnu temple, Vellinezhi-
Dasavataram
Figure-3.5: Mural paintings of Kuroor Kadu Siva temple- Nataraja and
Bhoothagana
Figure-3.6: Mural paintings on Chuduvalathoor temple- Nataraja
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Figure-3.7: Mural paintings on Kunnathormedu Sri Krishna temple- Lord
Krishna and Gopikas
Figure-3.8: Mural paintings of Mannur Siva temple- Lord Vishnu and Lord Shiva
Figure-3.9: Panjal Ayyappan temple
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49 Materials and Methods
Figure-3.10: Mural paintings of Panjal Ayyappan temple- Bhadrakali and Lord
Ganesha
Figure-3.11: Panjal Lakshmi Narayana temple and Mural painting of Nataraja
Figure-3.12: Mural paintings of Institute for Mural Painting- Guruvayurappan
and Gopika
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Figure-3.13: Mural paintings of Institute for Mural Painting- Geethopadesam
Figure-3.14: Sekharipuram Vishnu Temple mural paintings- Shree Krishna Leela
Figure-3.15: Varadharaja Perumal Temple, Kancheepuram
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Figure-3.16: Mural paintings of Varadharaja Perumal Temple- Dasavataram
Figure-3.17: Lepakshi Mural Paintings- Hair styles and Hunting
Figure-3.18: Mural paintings on the pillars of Kailasanath Temple,
Kancheepuram- Lord Shiva
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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 ‘Tornado-
like’ 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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53 Materials and Methods
transported aseptically to the laboratory condition and incubated at 37 ºC for 24 hours for the
isolation of bacterial species at 26 ºC 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 ºC for 24 hours for the isolation of
bacterial species and at 26 ºC 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 Identification of the predominant microbes from mural paintings
The identification of organisms were done based on basic microbiological and
biochemical test (Bergey’s manual of systemic bacteriology, 1994, Alexopoulos and Mims,
1985)
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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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Table 3.2.Details of natural raw materials used for wall preparation and dye extraction
S.No. Name of the raw material Area Use
1. Blue from Indigoferra Chennai (Tamil Nadu) Painting
2 Blue from China blue Chennai (Tamil Nadu) Painting
3. Yellow laterite stone Guruvayur (Kerala) Painting
4. White lime Guruvayur(Kerala) Painting
5. Red laterite stone Guruvayur (Kerala) Painting
6. Green from Gamboge Guruvayur (Kerala) Painting
7. Black from Wicks, sesame oil and
mud pot Chennai (Tamilnadu) Painting
8. Sand Guruvayur (Kerala) Wall preparation
9 Neem gum Palakkad (Kerala) Adhesive for painting
10 Coconut water Chennai (Tamil Nadu) Secondary wall
preparation
11 Kadukka Chennai (Tamil Nadu) Wall preparation
3.4.2 Preparation of wall materials (Krishnakumar, 2003)
The preparation of wall for traditional mural paintings of Kerala is an elaborate process
and it is done in three stages. The brick wall is plastered with the mixture of lime and clean
sand in the ratio 1:2. This mixture is ground well to a paste. Traditionally it is advised that this
mixture has to be kept for one week and at the time of plastering, crushed and water boiled
Terminalia chebula seeds were added to increase the viscosity. After cooling, it was diluted
with lime while applying on the walls. The plastering was rough at first and the plastered wall
was dried for one day and thereafter, the surface was strengthened by repeating the plastering
process so that the thickness was about ½ inch to 1 inch. The second coating was done with
the mixture of lime and sand in the ratio 1:2, but along with this mixture, cotton
(Gossypium herbaceum) was also added. The ratio of cotton mixture was 1 Kg to every 100
gm, respectively. Cotton fibers imparted gleaming whiteness to the surface and gave better
texture to the base. This mixture was thoroughly ground manually on a grinding stone to make
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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 cow’s 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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3.4.3.3. Extraction of Black dye pigment
Lamp black dye was prepared by immersing many wicks, made up of muslin cloth in
gingelly oil. The wicks were then burnt in a lamp and the smoke thus evolved was collected on
the inner surface of a new mud pot. The pot was allowed to cool and later it was scratched out
slowly and lamp black dye thus obtained was stored in sterile air tight bottle for further use.
3.4.3.4. Extraction of Blue dye pigment
The blue dye was prepared from the fresh Indigofera tinctoria leaves collected from in
and around Chennai. The dye was extracted using water by allowing the leaves to ferment for
7 days. After seven days, the fermented solution was extracted, filtered and dried in shade.
After drying, it was powdered and stored in sterile air tight bottle for future use.
3.4.3.5. Basic adhesive
Gum obtained from the bark of neem (Azadirachta indica) was used as basic adhesive.
It was a clear, bright, amber colored material that blackens with age. This gum was mixed
with colors to make these pigments stick to the base wall. The ratio of color powder to
adhesive was 6:1.
3.4.3.6. Preparation of brush
Pigments were applied on the base wall with the help of brushes prepared from the
long soft awns of Elephant grass (Aristada setaces). The panicles were collected and dipped in
boiling water for 5 – 10 minutes and then dried and stored. The trifurcated portions were
carefully assembled by cutting the basal thicker portions off and then threaded together for the
required thickness. Tips were cut off when thick brushes were needed or retained if required to
sketch narrow lines. Stems of Areca catechu and Bambusa arundinaceae were used as brush
handles.
3.4.3.7. Painting on prepared wall (Mini, 2010)
The images were sketched on prepared wall. The outlines were originally done with
cow dung pencils, called Kittalekhini. The shading is adeptly colorized by symbolizing the
characteristics separately for each figure.
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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
extracted dyes (Rojas et al., 2006)
All the natural raw materials used for wall preparation and paintings procured from
different locations were subjected to antimicrobial activity against the representative
organisms. Among the 9 bacterial isolates and 6 fungal isolates the most predominant
organism has been selected as representative organism. E.coli was selected as Gram negative
representative, S.aureus was selected as Gram positive representative. Similarly, A.niger was
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59 Materials and Methods
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 µl prepared with concentration of 1 g/ ml) and incubated at 37°C for
determining the antibacterial activity and at 28°C 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 Assessment of antimicrobial activity of the combined dye pigments (Rojas et al.,
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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3.7 Characterization of the dye pigments (Singh, 2011)
The physico-chemical characteristics of the dye pigments were determined by
performing GC – MS and FTIR analysis. The characteristics of different fragments of
primitive mural painting (ground, middle and upper) were analyzed by SEM-EDAX.
3.7.1 Scanning Electron Microscope coupled with energy dispersive X-ray
Spectrometry (SEM – EDAX) (Singh, 2011)
SEM gives high resolution surface morphological images, and also has the analytical
capabilities such as detecting the presence of elements down to boron (B) on any solid
conducting materials through the energy dispersive X-ray spectrometry (EDX) providing
crystalline information from the few nanometer depth of the material surface via electron back
scattered detection (BSD) system attached with microscope and advanced technological PBS
(WDS) for elemental analysis.
Different fragments (ground, middle and upper) of wall with mural painting were
analyzed to confirm the presence of all three layers of the wall prepared for the mural
paintings and the elements or components of dyes used for coloring.
3.7.2 Gas Chromatography – Mass Spectrometric analysis
The GC-MS is composed of two major building blocks: The gas chromatography and
the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on
the column's dimensions (length, diameter, film thickness) as well as the phase properties (5%
phenyl polysiloxane). The difference in the chemical properties between different molecules in
a mixture will separate the molecules as the sample travels the length of the column. The
molecules take different amounts of time (called the retention time) to come out of (elute
from) the gas chromatograph, and this allows the mass spectrometer downstream to capture,
ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer
does this by breaking each molecule into ionized fragments and detecting these fragments
using their mass to charge ratio. The organic dyes and the pigment extracts were analyzed by
GC-MS (JEOL GCMATE-II) with Data system, a high resolution, double focusing instrument.
Maximum resolution was 6000; maximum calibrated mass was 1500 Daltons. Source options
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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 70˚C, Injector Temperature 250˚C, Injection
volume was 1 microlitre, Column Flow, 1.5 ml/min. MS conditions: Ion source temp: 230 °C,
Interface temp: 240°C, 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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Rochanum (mal) (Myristica fragrans)
Kaathu (mal) (Fagopyrum esculentum)
Neem gum (Azadirachta indica)
Kungulyam (mal) (Abrus precatorius)
Paalkayam (mal) (Ferula asafoetida)
These materials were procured from Herbal Medical shop, Palakkad, Kerala. The materials
were tested for their antimicrobial properties against the isolated microbial predominants from
mural paintings by well diffusion method. Among the 7 naturally available gums three natural
gums Fagopyrum esculentum, Azadirachta indica, Ferula asafoetida were selected based on
the effective antimicrobial activity exhibited for the preparation of biogel. The three
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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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 Duncan’s 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 physico-
chemical characteristics were determined by performing GC – MS analysis.
3.9 Antimicrobial property of developed bio-based protective coating against
microbial predominant of archaeological area (Rojas et al., 2006)
Antimicrobial property of the developed bio-based protective coating was tested
against the microbial predominant (9 isolated bacteria and 6 purified fungi) by well diffusion
method.
3.10 Comparative study of commercial biocides with developed biobased coating
The developed protective coating was also compared with the commercial biocides
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 Slab study (Ciferri, 1999)
The developed biobased coating (Fagopyrum esculentum, Ferula asafoetida and
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
A Study on Microbial Deterioration of Ancient South Indian Mural Paintings and
Fabrication of Biobased Natural Protective Coating for its Conservation --------------------------------------------------------------------------------------------------------------------------------------------------------------------------
64 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 Field study of developed biogel (Ciferri, 1999)
The developed biobased coating (Fagopyrum esculentum, Ferula asafoetida and
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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