10_chapter3

A Study on Microbial Deterioration of Ancient South Indian Mural Paintings and

Fabrication of Biobased Natural Protective Coating for its Conservation --------------------------------------------------------------------------------------------------------------------------------------------------------------------------

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.




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




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




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




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




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




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




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




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




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)




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




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




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.




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.




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




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.




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

http://en.wikipedia.org/wiki/Gas_chromatograph

http://en.wikipedia.org/wiki/Mass_spectrometer

http://en.wikipedia.org/wiki/Ion




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)




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,




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




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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