Collection and identification of plant parts and dental caries Pathogens.
Plant Materials Collection
The small branches of locally available Z. zanthoxyloides and T. glaucescens were collected from local Ogbete main market Enugu and were authenticated by Prof. Okigbo R.N of the department of Botany of Nnamdi Azikiwe University, Awka. These plant materials were dried under the sun for two weeks and also cut into pieces of approximately 15cms and transferred to the oven set at 45°C for 20-30mins before it was reduced to fine powder with the aid of mechanical grinder. The powder plant materials were collected and stored in a tightly covered glass jar for further studies.
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The organism isolated was identified on the basis of morphological, cultural and biochemical characteristics according to standard procedures (Holding and Colee, 1971).
2.3 PREPARATION AND EXTRACTION OF PLANT MATERIALS.
20g of fine-powder stem of Z. zanthoxyloides and T. glaucescens was weighed and soaked in 200mls of ethanol in a conical flask and kept at room temperature (25°C) in a rotary shaker for 48 hours. After 48 hours, filtered through Whatman No1 filter paper; solvent was allowed to evaporate and stored at room temperature until when required for use.
2.5 IDENTIFICATION OF THE ISOLATES
The isolated organisms were identified using sub-culturing, gram staining technique and biochemical test like catalase test, indole test, coagualase test, methyl red test, oxalase test, sugar fermentation test.
2.6 TEST ORGANISMS
ISOLATION OF THE TEST ORGANISMS.
The dental specimen collected was streaked out on Nutrient agar and Sabouraud dextrose agar plates. The plates were incubated at 370C for 24 hours for bacterial isolates and 31°C for 48 hours for fungi. Colonies that developed were respectively sub-cultured into freshly prepared Nutrient agar and Sabouraud dextrose agar.
2.7 PREPARATION OF SENSITIVITY DISC:
Disc of 6mm in diameter was punched out using Whatman No1 filter paper. Placed in bijou bottles, then sterilized the disc by autoclaving at 121°C for 15mins, and allowed to cool.
• PREPARATION OF SENSITIVITY DISC WITH ETHANOL EXTRACTS OF Z. zanthoxyloides and T. glaucescens :
The stock solutions of the ethanolic crude extracts (i.e. that were recovered) of these two plants were prepared by dissolving 0.5g (i.e. 500mg) of each of the two plant extracts in 5ml Dimethyl sulphoxide (DMSO). Therefore, each stock solution had a concentration of 100mg/ml.
Different concentrations of each of the plant extract were prepared from this stock. These are 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, and 3.13mg/ml by serial double dilution, followed by introducing the disc in each concentration. The disc was allowed to absorb the solution for 10mins and kept for further analysis. Each paper disc is capable of absorbing 0.01ml.
2.8 DETERMINATION OF ANTIMICROBIAL ACTIVITIES OF EXTRACTS.
Disc Diffusion Assay: Antibacterial activity of the ethanolic extracts of the plant sample was evaluated by noting the zone of inhibition against the test organisms. (Schaeken et al., 1986).
Antimicrobial activity was carried out using disc-diffusion method.
Two colonies of 24-hour plate culture of each organism was transferred aseptically into 10ml sterile normal saline in a test tube and mixed thoroughly for uniform distribution. A sterile cotton swab was used to spread the resulting suspension uniformly...