Microbiology
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Item A microbiological study of an Ayurvedic compound preparation Dasamoola Arista with a view to defining an acceptable microbial quality standard(Sri Lanka Association for the Advancement of Science, 2008) Nageeb, B.M.; Widanapathirana, S.; Amarasinghe, A.P.G.The Indigenous system of medicine has been practiced successfully over several thousand years. The basic ingredients of indigenous medicine are plant materials. These materials contain natural inherent microbial flora and also may become contaminate during processing. Considering these facts the World Health Assembly in its resolutions WHA-31:33, 40:33, and 42:43 has emphasized the need for the microbial quality standard of medicinal plant products. Dasamoola Arista, has been used in therapeutics for several centuries. The objectives of this study were to enumerate the total viable count of bacteria, fungi and specific microorganisms such as Coliforms and Salmonella in the market samples of this drug. Fourteen different market samples were subjected to this study. Nutrient agar and Potato Dextrose agar were used as culture media. Pour plate and Spread plate techniques were used to study the microbial load in dilution series up to 10-3. Microbial counts on Nutrient agar and Potato dextrose agar were taken after 24 hours and 72 hours. Tests for Coliforms and Salmonella were done according to International standards. Coliform test was performed by MPN method using single strength MacConkey broth. Salmonella was tested after an enrichment process in buffered peptone. 0.1ml of this peptone was transferred to test tubes of Tetrathionate and Selenite broth separately and Incubated at 37 0C for 48 hours. These broths were streaked on Bismuthsulphiteagar (B/S.Agar) and Brilliantgreenbile agar(BGB ) Black colonies on B/S-Agar and Pink colonies on BGB Agar were considered as positive for Salmonella. These colonies were bio chemically tested for salmonella . All tests were repeated thrice and results were confirmed. The microbial load observed in this study was with in the limits of the WHA. The Colony count for Bacteria was in between 10x10 to 10x68. Fungi Colony count was in between 1x10 to 36x 10. The biochemical tests revealed that the Bacteria present in this preparation was Bacillus firmus. None of the drug samples were positive for Coliforms or Salmonella. These results revealed that these tested samples were microbiologically safe and up to the microbial quality standard.Item A study of total viable count of microorganism and specific microorganisms in four selected Arista and Asawa preparations(Sri Lanka Association for the Advancement of Science, 2009) Nageeb, B.M.; Amarasinghe, A.P.G.; Widanapathirana, S.Item Preliminary study of anti-bacterial effects of an Ayurvedic recipe Sharkaradi kalka(Sri Lanka Association for the Advancement of Science, 2005) Roshana, B.; Amarasinghe, A.P.G.; Widanapathirana, S.Item A comparative preliminary study of anti-bacterial effect of ayurvedic compound preparations of Dathree choorna and Hinguastaka choorna(Sri Lanka Association for the Advancement of Science, 2006) Nageeb, B.M.; Amarasinghe, A.P.G.; Widanapathirana, S.Item Variation of heterotrophic bacterial distribution in Kandy lake(Sri Lanka Association for Fisheries and Aquatic Resources, 2004) Sharaff, F.F.; Silva, E.I.L.; Abeygunawardane, S.I.Kandy Lake, a man-made water body, located in the heart of Kandy adjoining a world famous Buddhist Temple in Sri Lanka was affected by a sudden outbreak of cyanobacteria bloom (Microcystis aeruginosa) in May 1999 with the onset of the southwest monsoonal winds. Many aspects of limnology of this water body have been examined since mid 1990’s. A study based on bacteriology was carried out from July 2001- 2002 July with an objective to understand the distribution pattern of heterotrophic bacteria and their role in eutrophication and pollution. The samples were collected monthly from different depths in the deep and shallow basins. Physicochemical and nutrient analyses of the samples were also carried out simultaneously. Some heterotrophic bacteria were isolated, enumerated and identified up to their generic and species levels using their morphological and biochemical characteristics. The heterotrophic bacteria identified belonged to the genera Bacillus, Pseudomonas and Actinomycetes. Their plate counts were in the range of 106 cfu ml-1. Planktonic bacteria were mainly Gram negative (75%) bacteria. There was no significant (p>0.05) intersite differences with respect to bacterial counts and physicochemical characteristics. However, there was a prominent gradient in the plate counts from top to bottom in the deep basin due to oxygen depletion in the hypolimnion. High coliform counts (1200+/100ml) and faecal coliform counts (550/100ml) were also recorded indicating a high degree of faecal contamination. The results also indicate that the heterotrophic bacteria in Kandy Lake did not show a spatial variation but showed seasonal variation with a marked diversity. Their role in eutrophication is probably through cycling of nutrients such as nitrogen and phosphorus.Item A Preliminary Study on Microbial Quality Standards of an Ayurvedic Compound Preparation "Thalisadee Choorana".(2007) Nageeb, B.M.; Widanapathirana, S.; Amarasinghe, A.P.G.; Kasthuriarchi, K.A.H.Thalisadee choorana is a common Ayurvedic medical preparation widely used by all indigenous medical practitioners in Sri Lanka. It is used for respiratory tract ailments such as cough, common cold, bronchitis, asthmatic conditions and gastro intestinal disorders such as diarrhoea, vomiting, indigestion and loss of appetite. It contains mainly Pipemigram (Gammiris),Piperlongum(Thippili), Abies�wehhiuna (Thalispathra),Cinnamum zylanicum (Kurundupothu), Elettaria repens( Heenensal),Bamboo salt (Unakapuru) and sugar. All plant materials contain a large number of microorganisms.Some are inherent and some are contaminated during the process of harvesting and manufacturing process. Considering these facts, the World Health Assembly in its resolutions WHA-31 :33(1978) 40:33(1987), 42:43(1989) has emphasized the need of ensuring the quality in regard to microbial content of the plant products. Hence this study was carried out to determine the microbial load of this product and the possible sterilization methods of reducing the microbial load. The effect of the method on the drug which reduces the microbial load of the drug also studied. Ten different samples of Thalisadee choorana were subjected to this study. 0.1 gram of the drug sample was dissolved in 10 ml of sterile distilled water. (10�). Using this solution 10-1, 10-2 10-3 dilutions were prepared. Routine sterilization procedures were carried out in all steps.Nutrient agar and Potato dextrose agar were used as general culture media. Pour plate technique and spread plate technique were used to detect the microbial count respectively. 0.1 ml of above dilutions was used on culture plates. Each plate was controlled by using another duplicate culture plate. Plates were sealed and kept under normal room temperature. Colony counts were taken after 24 hours and 72 hours for bacteria and fungi respectively. It was assumed that each colony was formed by a single organism. Same procedure was repeated three times. According to the W.I-I.A standard, aerobic bacteria up to 105 I gram, yeast and moulds up to 103 I gram arc permitted The results ofthe above study indicate that the bacterial count was in between 3x106 to 4xl06 /gram. These results indicate that the limits were exceeding on every sample. The following methods were tried to reduce the microbial load. I 00 grams of the above samples were subjected to (a) Heat treatment in a hot air oven at 80� C for 10 minuets for three consecutive days. (b) Ultra violet radiation at 256 wave length continuously for 24 hours. (c) Steam treatment under atmospheric pressure in a closed container for 10 minuets for three consecutive days. The study of microbial load was thereafter repeated.The plates of the steamed samples were sterile up to 72 hours while the plate of other two methods does not show any reduction in microbial load. The volatile oil content by reflux method using Dead and Stark apparatus and the thin layer chromatographic (T.L.C) patterns of Ethanol extract and Water extracts using Silica gel GF 254 and G06 at the ratio of 1:3 with several solvent systems of both samples (Steamed and un steamed) were studied. The T.L.C. patterns and the volatile oil content of both samples were comparatively same .This preliminary study reveals that the steam treatment method is comparatively an effective method to reduce the microbial load of the above preparation. A detail study of the chemical compounds through other chromatography methods is needed to confirm this.Item A preliminary study of anti-bacterial effect of selected five Ayurvedic compound preparations.(2006) Nageeb, B.M.; Widanapathirana, S.; Amarasinghe, A.P.G.Plant based medicaments have been man?s prime therapeutic weapons to rescue him from disease. Plants are of relevance to pharmacology. Pharmacological properties of medicinal plants may be used as leads in developing modern therapeutic agents. Thalisadee, Thripala,Hinguastaka, Dathree, and Manibadra are common Ayurvedic formulae used in traditional system of medicine in Sri Lanka. Thalisadee and Thripala Choorna are being commonly used in respiratory disorders such as cough, cold bronchitis and fever. The Hinguastaka, Dathree and Manibadra Choorna are being commonly used in gastrointestinal disorders such as diarrhoea dysentery vomiting and indigestion. Most of these conditions may develop due to bacterial infections .The main objective of this study was to evaluate the antibacterial effect of these preparations. Minimum human single dose of these drugs (2.5 gram) was dissolved in sterile distilled water and kept in the shaker at 100 rpm continuously for 04 hours in order to get the maximum soluble liquid extract of these drugs. 0.7 gram of Nutrient broth was dissolved in 50 ml of distilled water and transferred in to five test tubes (10 ml. /tube) and sterilized by autoclaving at 121 C for 20 minuets. These Nutrient broth tubes were inoculated by using inoculating needle with already plated pure test cultures of Pseudomonas aeruginosa, ,Escherichia coli , Salmonella typhi.These tubes were incubated at 37 0 C for 18 to 20 hours. Sterilized Nutrient agar was transferred into ten sterilized Petri dishes at 40 0C and allowed to solidify on a horizontal plane. These plates were sealed and kept in incubator at 37 0C for 24 hours to exclude any contaminations and to reduce the moisture content. A known amount of (0.05 ml) each culture broth containing specific organisms was added to these solidified agar plates and spread evenly using a sterilized glass spreader. On these seeded agar plates sterilized metal cylinders were kept (03 Cylinders/plate) with gentle pressure. These cylinders were filled with 0.1 ml of above liquid extract of drug preparations.De ionized sterilized distilled water 0.1 ml and Chlorampenicol 0.025 mg/0.1 ml were used as controls. These plates were sealed and incubated at 37 0C .This same procedure was repeated for three times for each of the test organism. Chlorampenicol showed 1.0 cm -1.5 cm clear inhibition zones of the bacterial lawns on every test organisms. None of the drug preparations showed any effect on Escherichia coli culture plates. The Ayurvedic compound preparations of Hinguastaka Choorna and Manibadra Choorna extracts showed averagely 0.5cm and 0.3 cm clear inhibition zones of the bacterial lawns on Salmonella typhi plate respectively. Thripala Choorna showed averagely 1.0 cm clear inhibition zone of bacterial lawns on Pseudomonas aeruginosa plates. These zones were clear on every repetition. Theses results were statistically analyzed by using one sample student T-Test. All the means are in between accepted level and P value is <0.05. Comparison to the Chlorampenicol,Higuastka Choorna and Manibadra Choorna are active against Salmonella typhi. Thripala Choorna is active against Pseudomonas aeruginosa. Both, Dathree Choorna and Thalisadee Choorna are not active against any of tested microorganisms. This preliminary study scientifically justifies that the use of the powder preparations of Higuastka Choorna,Manibadra Choorna and Thripala Choorna for infective conditions such as diarrhea, dysentery, vomiting and indigestion.Item Microbiological investigations of Sigiriya Frescos(2006) Fernando, S.; Jayaratne, D.L.The archaeologically valued Frescoes at Sigiriya, a World Heritage Site in Sri Lanka,play a major role in remarkable historic interest. Microbial growth on fresco paintings has been identified worldwide as a significant factor which affects the quality of paintings. Microbiological investigation of fresco paintings therefore has become an important aspect of conservation strategy. The microbiological investigation of the Sigiriya frescoes was carried out in June 2005 by visual detection, microscopic investigations and using microbial culture techniques. The samples for microbial culture techniques were taken from each colour regions separately using sterile cotton swabs and cultured in Nutrient Agar (NA), Trypton Soy Agar (TSA) and Potato Dextrose Agar (PDA) media.The observations revealed that the fresco surface was free from any fungal, lichen or cyanobacterial growth but eleven bacterial cultures were isolated from a decayed patch,from non painted plaster and from a cavity. All the isolates belong to the Genus Bacillus having no conformity with any of the Bacillus spp either indicated in ?Bergy?s manual of systematic bacteriology vol I and II or included in the computer database developed by Trevor Bryant, University of Southampton, UK (2005). One isolate showed similar morphological characteristic features to the Bacillus decoloratiois sp. Nov. bacterium which has been proposed for the new isolate that was responsible for de-colorization of the fresco paintings in Spain and Austria. However, the biochemical characterization of the isolate showed that is a distinctive species having no conformity with Bacillus decoloratiois sp. Nov. Further characterizations using DNA based techniques are being carried out in order to determine whether the isolate is a new species or a similar strain of the Bacillus decoloratiois sp. Nov. In the process of conservation, periodic monitoring and further investigations are being carried out for the detection of microbial growth forms including the Bacillus species on the fresco paintings in order to protect them from any microbial de-colorization and degradation.