6. MICROBIOLOGICAL EVALUATION Microbial assay measures the activity of antibiotics (Extent of ability to inhibit the growth of micro organism) or vitamins and amino acids (Extent to support the growth of micro organism) where as chemical assays of such substances estimate only their potency i.e concentration or amount. Antibiotics can be assayed by both turbidimetry and diffusion assay methods, but vitamins can be assayed by turbidimetry. 73 6.1 Diffusion assay method A geometric series of dilution is prepared for the antibiotic under test and for the standard preparation. Plates are seeded with the test organism and the medium is allowed to set on a perfectly horizontal surface so that the agar is constant in depth throughout the petridish. The organism may be streaked over the medium after it has set. The plates may be petridish or large flat dishes up to 0.5 m 2. The solution contained in filter paper or cellulose discs, absorbs a fixed volume of solution. The test and standard solutions are placed in the containers in random order, to prevent the bias that could be caused by a regular order of plating. The volume is critical for the cup method but is not significant when cylinders with the lid of the plate. The plates are left at room temperature for two hours to allow diffusion of the antibiotic to get a head of growth of the organisms. Then they are incubated at the appropriate temperature, usually for about 12-16 hours. After incubation, inhibition of growth can be seen as a clear zone around each container. The diameter of this is proportional to the log concentration of antibiotic. As soon as possible each diameter is measured by using an optical system that projects an image of the plate on to a large grid. Two diameters at right angles are used as a check on ability of the zone.
A graph is plotted for log concentration of standard against zone diameter and results for the test preparation are plotted on the same graph. Provided the two lines are parallel, the relative potencies of the standard and the test are represented by the horizontal distance between the two lines. The parallelism between the two lines can be confirmed mathematically and the potency of the test obtained by calculation. 74 6.2 Antibacterial activity 6.2.1 Muller Hington (MH) agar medium preparation Beef extract : 3 g Peptone : 5 g Sodium chloride : 5 g Agar : 15-20 g Distilled water : 1000 ml ph : 7.4 ± 0.2 All the ingredients of culture medium are weighed and dissolved in water. Heat the solution with agitation and add agar with continuous shaking for proper mixing of constituents. 6.2.2 Sample Preparation 10 mg of extracts (Ficus benghalensis aerial roots and Ficus religiosa leaves extract) were weighed and dissolved in 10 ml of the solvent (DMSO). The stock concentration of extract was 1 mg/ml. sub dilutions were made upto that 50 to 100 µg/ml for Ficus benghalensis aerial roots and 100 µg/ml to 500 µg /ml for Ficus religiosa leaves. All six extracts were prepared in the same way. 6.2.2 Standard Preparation
10 mcg conc. of tetracycline was prepared used as a standard against gram +ve microorganism and 10 mcg conc. of erythromycin used as a standard against gram ve microorganism. 6.2.3 Sub culturing Preparation of agar slants Place 6-8ml of melted agar medium in each test tube (15x125 mm). Apply the cotton plug for the tubes. Sterilize the tubes by autoclaving. Place the test tubes in an inclined position (at about 20 angle) and allow the melted agar to solidify at room temperature (25 ± 5 C) store in refrigerator at (2-8 C). By using this agar slants the following micro organisms are sub cultured from the stock culture. Sl no. Test organism NCIM number 1. Proteus vulgaris 2813 2. Staphylococcus aureus 2602 3. Bacillus subtilis 2480 4. E.Coli 2981 5. Staphylococcus epidermidis 2493 6. Lactobacillus acidophilus 2902 7. Staphylococcus griseus 2183 8. Salmonella typhimurium 2501 9. Corynebacterium diphtheriae 5212 6.2.4 Inoculating procedure Subculture tube were taken and then heat the inoculating loop in flame until nichrome wire was hot and then allows it cool for few min. The cotton plug has been removed at a time from the subculture tube and inoculating medium and simultaneously considering minimum time available for inoculums [to avoid exposure
to microbial contamination and heat the neck of the tubes with bumper]. The sterile inoculating loop was inserted into the stock culture and remove small amount of bacteria. This inoculum was transferred to inoculated slant. The necks of the tube were close with the respective caps. Heat the inoculating loop after inoculation to red hot. Slants were incubated for 24 hrs at 30-37 C. A small quantity of distilled water i.e., 5-10 ml was taken in a test tube. With the help of inoculating loop, the standard culture of micro organisms was transfered into distilled water by scrapping the culture. The entire process has been performed in aseptic condition. 6.2.5 Cup plate method Before perform the microbial assay, sterilized the glass ware, sterile the media, antibiotic solution (standard) and plant samples were kept ready. The first step of assay involves the preparation of inoculated plates. For this 2 % of microbial suspension is added to the quantity of medium per plate i.e., 0.5 ml of suspension per 25 ml of nutrient agar medium. Allow the petriplates for solidification for about 10 min. By using sterile glass (Pyrex) bores, cups were made by maintaining approximate distance between cups (cup diameter: 6 X 8 mm 2 ). Cups were labeled properly to enable the introduction of the test sample, standard and control precisely. The concerned samples were introduced into appropriate wells with the help of micropipette; all the cups were filled with equal volumes of sample. To minimize the effect of variants the petriplates were allowed to store at room temperature for 1-4 hrs, and then the plates were allowed to incubate for a time period of 18-24 hrs. The
Ethyl acetate Methanol zone of inhibition was examined and measured with the help of antibiotic zone reader 75-78. The results were represented in the Table: 6.1 & 6.2 and Fig: 6.1-6.38. (In figures the symbol denotes S Standard; L low concentration; H High concentration & C control) Table: 6.1. Microbiological assay of Ficus benghalensis aerial roots extracts Extract Name of the microorganism Gram ve P.vulgaris Low (50µg/ml) 9.0+0.3 *Zone of inhibition (mm) High (100µg/ml) 15.2±0.4 Standard (10µg/ml) 15.0±0.6 E.Coli 10.0±0.2 16.0±0.2 21.1±0.5 C.diphtheriae 8.2±0.2 14.1±0.5 22.2±0.4 S.typhimurium Gram +ve 10.0±0.6 15.0±0.1 23.0±0.5 S.aureus 7.2±0.1 13.1±0.3 17.1±0.5 B.subtilis 9.0±0.1 10.2±0.2 14.2±0.6 S.epidermidis 11.2±0.2 17.1±0.2 23.4±0.4 Lactobacillus 6.2±0.4 13.0±0.0 16.0±0.2 S.griseus Gram ve P.vulgaris 8.2±0.2 6.1±0.1 15.1±0.3 13.0±0.1 22.1±0.3 15.0±0.6 E.Coli 6.0±0.2 12.2±0.2 21.1±0.5 C.diphtheriae 7.1±0.4 15.0±0.0 22.2±0.4 S.typhimurium Gram +ve 8.4±0.2 10.2±0.4 23.0±0.5
Zone of inhibition in mm S.aureus 6.2±0.2 10.0±0.0 17.1±0.5 * B.subtilis 7.0±0.5 10.2±0.4 14.2±0.6 Avera S.epidermidis 9.5±0.5 16.1±0.3 23.4±0.4 ge of Lactobacillus S.griseus 6.02±0.3 6.2±0.4 13.2±0.5 12.0±0.2 16.0±0.2 22.1±0.3 three deter mina nts 25 20 15 10 5 0 Low 50 mcg High 100 mcg Standard10 mcg Fig: 6.1 Antibacterial activity of MEFB
Methanol zone of inhibition in mm 25 20 15 10 5 0 Low 50 mcg High 100 mcg Standard10 mcg Fig: 6.2 Antibacterial activity of EEFB Table: 6.2. Microbiological assay of Ficus religiosa leaves extracts Extract Name of the microorganism *Zone of inhibition (mm) Gram-ve Low (100µg/ml) High (500µg/ml) Std (10µg/ml) P.vulgaris 12.2±0.2 19.0±0.0 15.0±0.6 E.coli 9.1±0.3 18.2±0.2 21.1±0.5 C.diphtheriae 11.4±0.2 14.2±0.2 22.2±0.4 S.typhimurium Gram +ve 16.0±0.2 17.2±0.4 23.0±0.5
Zone of inhibition in mm Ethyl acetate S.aureus 9.3±0.1 17.0±0.3 17.1±0.5 B.subtilis 9.2±0.4 18.2±0.2 14.2±0.6 S.epidermidis 14.2±0.2 18.0±0.0 23.4±0.4 S.gresius Gram-ve P.vulgaris 10.8±0.2 7.0±0.2 14.2±0.2 15.4±0.2 22.1±0.3 15.0±0.6 E.coli 5.2±0.2 16.0±0.8 21.1±0.5 C.diphtheriae 11.2±0.4 13.2±0.4 22.2±0.4 S.typhimurium 11.1±0.1 20.2±0.0 23.0±0.5 Gram +ve S.aureus 9.2±0.1 16.2±0.4 17.1±0.5 B.subtilis 13.2±0.4 14.2±0.4 14.2±0.6 S.epidermidis 14.0±0.0 15.0±0.0 23.4±0.4 S.gresius 0.8±0.2 18.2±0.2 22.1±0.3 * Average of three determinants 25 20 15 10 5 0 Low 100 mcg High 500 mcg Standard10 mcg Fig: 6.3 Antibacterial activity of MEFR
Zone of inhibition in mm 25 20 15 10 5 0 Low 100 mcg High 500 mcg Standard10 mcg Fig: 6.4 Antibacterial activity of EEFR Zone of inhibition of Methanolic Extract Ficus benghalensis aerial roots against gram negative microorganism Fig: 6.5 Proteus vulgaris
Fig: 6.6 E. coli Fig: 6.7 Corynebacterium diphtheriae Fig: 6.8 Salmonella typhimurium
Fig: 6.9 Staphylococcus aureus Fig: 6.10 Bacillus substilis Fig: 6.11 Staphylococcus epidermidis
Fig: 6.12 Lacto bacillus Fig: 6.13 Staphylococcus griseus Zone of inhibition of ethylacetate Extract of Ficus benghalensis aerial roots
Fig: 6.14 Proteus vulgaris Fig: 6.15 E.coli Fig: 6.16 Corynebacterium diphtheriae
Fig: 6.17 Salmonella typhimurium Fig: 6.18 Staphylococcus aureus Fig: 6.19 Bacillus subtilis
Fig: 6.20 Staphylococcus epidermidis Fig: 6.21 Lacto bacillus Fig: 6.22 Staphylococcus griseus
Zone of inhibition of methanolic extract of Ficus religiosa Linn Fig: 6.23 Proteus vulgaris Fig: 6.24 E. coli Fig: 6.25 Cornybacterium diphtheriae
Fig: 6.26 Salmonella typhimurium Fig: 6.27 Staphylococcus aureus Fig: 6.28 Bacillus subtilis
Fig: 6.29 Staphylococus epidermidis Fig: 6.30 Staphylococcus griseus
Zone of inhibition of Ethyl acetate extract of Ficus religiosa Linn Fig: 6.31 Proteus vulgaris Fig: 6.32 E. coli Fig: 6.33 Cornebacterium diphtheria
Fig: 6.34 Salmonella typhimurium Fig: 6.35 Staphylococcus aureus Fig: 6.36 Bacillus substilis
Fig: 6.37 Staphylococcus epidermidis Fig: 6.38 Staphylococcus griseus
6.3 Antifungal activity Sabouraud dextrose agar medium 79 (SDA) was prepared and 25 ml of each was poured in to sterile universals. The universals with the broth were inoculated with different species of fungi and incubated at 25 C overnight. A total of 25 ml of medium was poured into each sterile petriplate. Each petriplate was inoculated with 200 μl of different fungal species spreaded well and allowed to set. Using a sterile cork borer 6 mm diameter, four wells per plate were made into the set medium containing fungal culture. A total of 0.2 ml of plant extracts were incorporated into the each well with various concentrations. The plates were incubated overnight for 36 to 48 hrs at 25 C and the zone diameter was then recorded if greater than 6 mm. 6.4 Result and discussion Three different fungal organisms were tested against the methanolic extract of Ficus benghalensis Linn and Ficus religiosa Linn. The results of the antifungal study were represented in Table 6.3 & 6.4 and Fig 6.39 to 6.47.
Table: 6.3 Antifungal study of methanolic extract of Ficus benghalensis Linn.aerial roots *Zone of inhibition in mm S.No Micoorganisms 1000 500 250 125 62.5 A* μg/ml μg/ml μg/ml μg/ml μg/ml 1. 2. 3. Candida albicans (NCIM 3484) Monilinia fruticola (NCIM 1011) Auricularia polytricha (NCIM 1303) 18 10 09 0 0 28 20 13 05 0 0 24 26 18 03 0 0 28 A* - Standard Amphotericin-B (1000 μg/ml) * Average of three determinants Table: 6.4 Antifungal study of methanolic extract of Ficus religiosa Linn.leaves *Zone of inhibition in mm S.No Micoorganisms 1000 500 250 125 62.5 A* μg/ml μg/ml μg/ml μg/ml μg/ml 1. 2. 3. Candida albicans (NCIM 3484) Monilinia fruticola (NCIM 1011) Auricularia polytricha (NCIM 1303) 22 19 12 07 06 30 21 18 15 15 11 28 23 22 19 0 0 25 A* - Standard Amphotericin-B (1000 μg/ml) * Average of three determinants
Fig: 6.39 Antifungal activity of methanolic extract of Ficus benghalensis against Candida albicans Fig: 6.40 Antifungal activity of methanolic extract of Ficus benghalensis against Monilinia fruticola Fig: 6.41 Antifungal activity of methanolic extract of Ficus benghalensis against Auricularia polytricha
Fig: 6.42 Antifungal activity of methanolic extract of Ficus religiosa against Candida albicans Fig: 6.43 Antifungal activity of methanolic extract of Ficus religiosa against Candida albicans Fig: 6.44 Antifungal activity of methanolic extract of Ficus religiosa against Monilinia fruticola
Fig: 6.45 Antifungal activity of methanolic extract of Ficus religiosa against Monilinia fruticola Fig: 6.46 Antifungal activity of methanolic extract of Ficus religiosa against Auricularia polytricha
Fig: 6.47 Antifungal activity of methanolic extract of Ficus religiosa against Auricularia polytricha Antibacterial activity was performed by cup plate method using two standards (Tetracycline and Erythromycin), and all the extracts has been used as sample out of which two had shown positive results. The zone of inhibition of methanolic extracts shows very high inhibition compare with ethyl acetate extracts. While comparing zone of inhibition of the methanolic extract of Ficus religiosa leaves and Ficus benghalensis aerial roots, it was concluded that Ficus benghalensis linn was highly suitable as it excerts better antimicrobial activity in low concentration (50 µg/ml). Antifungal activity was performed by cup plate method using amphotericine used as a standard (1 mg/ml). Methanolic extract of Ficus benghalensis aerial roots shows inhibition against candida albicans (NCIM 3484), Monilinia fruticola (NCIM 1011), Auricularia politricha (NCIM 1303). The effective concentration was found to be 1000 µg/ml. The zone of inhibition of methanolic extract of Ficus religiosa leaves shows very high inhibition compared to the methanolic extracts of Ficus benghalensis aerial roots, which concluded that methanolic extract of Ficus religiosa leaves was highly suitable as it excerts better antifungal activity in low concentration (62.5 µg/ml). From the results obtained in both the studies, methanolic extract of either plant materials excerts better activity at lower concentration hence methanolic exctract of Ficus benghalensis aerial roots and Ficus religiosa leaves was selected for the isolation of active constituent by column chromatography.