ANTIMICROBIAL ACTIVITY

Transcription

1 ANTIMICROBIAL ACTIVITY

2 ANTI-MICROBIAL ACTIVITY ' In troduction Anti-microbial activity o f tlie plants can be detected by observing tlie growth response o f various microorganisms to those plants extracts which are placed in contact with them. Many methods for detecting such actions of organism, are available but since they are not equally sensitive, the results obtained will be influenced by the method selected and the microorganism used for the test. In order to detect anti-microbial activity of the plant extracts, three conditions are required to be fulfilled. First, the plant extract must be brought in to contact with the cell wall of the microorganism that have been selected for the test. Second, conditions must be adjusted so that the microorganism are able to grow when no anti-niicrobial agent are present. Third, there must be some means of judging the amount o f the growth, if any made by the test organism during the period o f time chosen for the test. Minimal inhibitory concentration (MIC) of an antibiotic is the minimum concentration required to inliibitthe growth of the test organisms after hours of incubation. In other words, MIC is the highest dilution of an antibiotic, which can inhibit the growth of the test organisms. An antibiotic possesses difl erent minimal inhibitory concentration values for different micro-organisms. On the other hand, a micro-organism needs different inhibitory concentrations for different antibiotics. A n tib acterial Activity The minimum bacterial concentration (MBC) can also be determined by preparing subculture from each test culture and noting the lowest, concentration of antibiotic from which no growth is obtained in the subculture. The MIC was determined by the agar plate difilision method. The alcoholic extracts were tested for antimicrobial activity against the test organisms. The microbiological assay is based on the comparison o f inhibition of growth o f bacteria by a measured concentration of plant extracts to be examined with that produced by known concentration o f standard preparation of antibiotic having known activity. 123

3 The general methods o f antimicrobial assay arc, 1. Cylinder-plate or cup-plate method 2. Turbidimelric method 3. Disc diffusion technique (Kirby-Biir method) M aterials and M ethods Test O rganism s and Inoculum s: Escherichia coli (NCTC-6571) and Staphylococcus aureus (NCTC-10418) were obtained from the Department of Microbiology, Majeedia Hospital, New Delhi. Sfaiidard: I'etracycline solution with specific activity of 30.Lg/ml was obtained from the Department of Microbiology, Majedia Hospital, New Delhi, Media: Dehydrated nutrient agar media was used and was prepared in distilled deionized water. The composition of the media was as given under: S. No. Ingredients Q uantity R equired For 100 ml (in g) 1. Peptone 5,1 2. Sodium chloride 5.0 3, Beef extract Yeast extract, Agar 1.5 P reparation of m edia; Dehydrated nutrient agar medium (28 g) was accurately weighed and suspended in 1000 ml of distilled water in a conical flask. It was heated on a water bath to dissolve the medium completely. Direct heating was avoided as it may lead to charring o f the medium components and render it useless for the purpose. Sterilization of media: The conical flask containing the nutrient agar medium was plugged with the help of a non-absorbent cotton bung. The mouth o f the conical flask 124

4 C.hapter-15 ANTIMICROBIAL A C TIV ITY and the cotton bung were properly covered with aluminum foil. The medium was then sterilized by autoclaving at 15-lbs/in^ pressure for 20 minutes. Metliods of jm'cparation of Test O rganism s: The test organisms were maintained on slants of medium and transferred to a fresh slant once a week. The slants were incubated at 37'*C for 24 hours. Using 3 ml of saline solution, the organisms were washed from the agar slant on to a large agar surface (medium) and incubated for 24 hours at 37+2 *'C. The growth from the nutrient surface was washed using 50 ml o f distilled water. A dilution factor was determined which gave 25% light transmission at 530 nm. The amount of suspension to be added to each 100 ml agar or nutrient broth was determined by use of test plates or test broth, The test organisms were stored under refrigeration. T em perature Control: Thermostatic control is required in several stages of a microbial assay when culturing a microorganism and preparing its inoculums and during inoculation in a plate assay. E xperim ental M ethods; Cup and plate m ethod A Previously liquefied and sterilized medium was poured in to plastic petri-plates o f 100 mm size. Sixteen plates were prepared and kept for solidifying. Six holes were made in each plate with a stainless steel borer having 6 mm i.d. Different dilutions o f the different oils were made in the concentration o f 2.5 il/ml in DMSO. Tetracycline (paper strips) was used as standard. Micropipette was used to deliver the solutions into the holes. The volume o f solution added to each hole was kept uniform (0.1 ml in each hole). One strip of tetracycline (standard) was placed aseptically to the central hole o f each plate. One hole was kept for blank. The plates were then left for standing for 1 hr. for proper diffusion of the drug solutions, They were incubated for about 24 hours at 32 ± 2 C. After 24 hrs the plates were examined and the diameter of zones o f inhibition was accurately measured. Anti-fungal activity The antifungal activity can be evaluated using the following methods 1. Microbial dilution assay 2, Disc diffusion assay 125

5 M icrobial D ilution Assay (MDA) The antifungal susceptibility of the tiingi to various oils were assayed by the microbial dilution method. Spores of Aspergillus were harvested from 96 h cultures and their numbers adjusted to 1 x 10^ /ml. The sabourand dextrose medium was dissolved in double distilled water and autoclaved at 10 psi for 10 min, A volume of 90.d of medium was added into the wells o f cell culture plates. The different dilutions of oils were prepared in duplicate wells and then the wells were inoculated with 10 jxl o f sore suspension. The plates were incubated at 37 C and examined microscopically after 48 h for the growth of Aspergillus mycelia. The activity was represented as negative if growth is there and positive if the medium appears clear without any growth o f Aspergillus. Disc Diffusion Assay The disc diffusion assay was performed in radiation-sterilized petri plates of 10 cm diameter (Tarsons) as per the method described in Indian Phamiacopoeia. Sabouraud Dextrose agar medium was dissolved in double distilled water and autoclaved at 10 psi for 15 min. it wa.s cooled to 45 C and 20 ml of it was poured into each petri plates. A total of 1 X 10^ conidia (fungal spores) in 1 ml of conidial suspension was prepared in Sabouraud maltose broth and overlaid on the surface of agar plate. Different dilutions of the oils were impregnated on the sterilized discs (5 mm in diameter) o f whatman filter paper number 1, The discs were placed on the surface o f agar plates already inoculated with Aspergillus conidia. The plates were incubated at 37 C and examined after 48 h for zone of inhibition, if any, around the disc. The diameter o f zone of inhibition was measured with the help of a scale. The concentration, which will develop the zone of inhibition of 6 mm diameter, was considered as minimum inhibitory concentration. Anti-fungal drug Amphotericin B was used as standard. An additional control disc without any sample but impregnated with equivalent amount of solvent was also used. The test was done in triplicate. Results The results have been tabulated in tables 7.1 and

6 Table 7.1; A iiti-m icrobiai activily of volatile oils. S. No. Oil Samples S. typhii P. aem ginosa Level of No. of Level of No. of Inhibiiioii Colonies Inhibition Colonies 1. Cinnamonmm camphor a Complete Nil Complete Nil 2. Cymhopogon ciiratus Complete Nil Complete Nil 3. Cymhopogon nardiis Complete Nil Complete Nil 4. Eucalyptus citriodora Complete Nil Complete Nil 5. Jasm im m grandiflorum Complete Nil Complete Nil 6. Jasminum sambac Partial 156 Partial Lavandula ojfwinalis Incomplete 210 Partial 35 8, Melaleuca auernifolia Partial 49 Complete Nil 9. Rosa rugosa Partial 124 Incomplete Santalim album Incomplete 292 Partial Syzygiim aromaticum Partial 121 Complete Nil 12, Vetivaria zizanioides Partial 126 Incomplete , Standard Tetracycline... Method, Dilution Method Complete Nil Complete Nil Standard Sample Organism : Tetracycline (5 al/plale) : 5 )al/plate ; Staphylococcus typhii. Pseudomonas aeruginosa \27

7 T able 7.2; Anti-fungal activity o f volatile oils. S. No, Oil Samples Activity Zone of Inhibition (in cm) 1. Ciniicmonium camphoru Cymbopogon cilratiis + 4,8 j. Cymhopogon nardus -f+ 5.4 Eucalyptus citriodora - - Jasminum grandiflorum 4-4,3 6. Jasmimim sanibac + 4,1 7. Lavandula officinalw - - 8, Melaleuca aucrnifolia + 5,1 9. Rosa ntgosa + 4,6! 0. Santalum album Syzygiurn aromaticum -h+ 5,4 12. Ve i iveria zizanio ides Standard Amphotericin B + 5,1 + Effective; ++ Efiective up to second dilution; - Ineffective M ethod Standard Sample Organism : Disc Diffusion Method : Amphotericin B (5 [.il/disc o f 5mra) : 5 il/disc o f 5mm : Aspergillus fum igatus 128

8 ANTIMICROBIAL AC TIV ITY 1.Gupta KC, Viswanathan R Anti-tuberculous substances from plants, Antihiol Chemother, 6, Indian Pharmacopoeia Controller of Publication, Delhi, II, P-A lo l. S.Mukherjcc PK. 2002, Oualily Control o f Herbal Drugs, T''' edn. Business Horizons Pharmaceutical Publisher, Delhi, p