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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.
Collection and Recovery of Caries Sample
The dental extracts from 20 (twenty) patients with clinical features of dental caries were collected from School of dental technology and therapy clinic, Trans-ekulu, Enugu. The patients comprised of 8 males and 12 females with ages ranging from 16-40 years.
The samples were collected under strict aseptic conditions. Prior to the collection of dental caries sampling, patient was made to rinse the tooth with water. The tooth and the surrounding field were cleaned with 3% hydrogen peroxide and then decontaminated with a 2.5% sodium hypochlorite solution. The food debris on the chewing surface was removed using a dental excavating instrument .The tooth was then extracted by a clinician and then introduced into the 20ml broth of Brain Heart Infusion (BHI) in appropriate sterile screw cap bottles. The dental caries sample was collected from the extracted tooth using an excavator under aseptic conditions. The clinical samples were mixed well using a magnetic stirrer before incubation. The samples were then inoculated using the streak plate technique on to nutrient and sabouraud dextrose agar under various culture conditions- aerobic, microaerophilic, and anaerobic culture conditions for each patient sample (Holding and Colee, 1971).
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.
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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 on the surface of oven-dried Nutrient agar and Sabouraud dextrose agar plates for bacteria and fungi respectively. The disc containing each concentration were impregnated on the culture plates and incubated at 37°C for 24hours and 31°C for 48hours for bacterial and fungal isolates respectively.
Conventional antibiotics were used as positive controls for bacteria and fungi respectively; distilled water was used as negative control. The plates were then incubated accordingly. The zones of inhibition were measured and recorded after incubation. The inhibition around the extracts indicated antimicrobial activity of the extracts against the test organisms. The diameters of these zones were measured diagonally in millimetre with a ruler and the mean value for each organism from the triplicate cultured plates was recorded. Using the disc diffusion technique, an already made gram positive and gram negative (Asodisks Atlas Diagnostics, Enugu, Nigeria) standard antibiotic sensitivity disc bought from a laboratory chemical equipment store in Enugu state was used as positive control for bacteria while ketoconanzole was used as positive control for fungi. Distilled water was used as negative control for all the test organisms.
2.9 Determination of Minimum Inhibitory Concentration (MIC) of the extracts:
The MIC for bacteria was determined as the lowest concentration of the extracts inhibiting the visual growth of the test cultures on the agar plate. The initial concentration of the plant extracts (100mg/ml) was diluted using double fold serial dilution by transferring 5ml of the sterile plant extract (stock solution) into 5ml of sterile normal saline to obtain 50mg/ml concentration. Different concentrations were 50, 25, 12.5, 6.25 and 3.13 mg/ml respectively. Each dilution was introduced into nutrient agar plates and Sabouraud dextrose agar plate already seeded with the respective test organism. All test plates were incubated at 37°C for 24hrs for bacteria and 31°C for 72hrs for fungi. The Minimum Inhibitory Concentration (MIC) of the extracts for each test organism was regarded as the agar plate with the lowest concentrations without growth.
2.10 Determination of Minimum Bactericidal Concentration (MBC) of the extracts:
The Minimum Bactericidal Concentration (MBC) of the plant extracts were determined by the method described by Holding and Colee (1971). Samples were taken from plates with no visible growth in the MIC assay and subcultured on freshly prepared nutrient agar plates and Sabouraud dextrose agar plate and later incubated at 37°C for 24 hrs and 31°C for 48hrs for bacteria and fungi respectively. The MBC was taken as the concentration of the extract that did not show any growth on a new set of agar plates.