• Sample Preparation &canal inoculation:
1. Any calculus or soft tissue remnants will be removed by using ultrasonic scaler .
2. Crowns of all teeth will be cut off at cemento-enamel junction using a high-speed hand piece and the root lengths were standardized to a (15 ±1 mm) length.
3. All the samples will be sterilized at 121°C for 30 minutes, one tooth will be randomly selected and incubated to ensure the negative culture and sterilization of samples.
4. Standard strain of Enterococcus faecalis (ATCC 29212) will be used and revived on blood agar at 35°C for 48 h. Inoculum will be prepared in sterile brain heart infusion broth (BHI) and turbidity will be adjusted to 0.5 McFarland corresponding to approximately (1.5× 108) colony forming units per milliliter (CFU/mL).16,17
5. The apical foramen will be sealed using a resin composite and the external surfaces of the teeth will be covered with two layers of colorless nail varnish to prevent liquid seepage then 5 ml of bacteria will be placed into each root canal, thereafter, each sample will be placed in a sterile Eppendorf tube containing BHI and will be stored at 37°C for 2 weeks.
6. During this period, BHI will be refreshed every 3 days.
7. The excess broth will be eliminated from the root canal with paper points at the end of incubation period and the first microbial sample (S1) is taken.
• Root canal preparation& medicament application:
1. Patency will be established using a #15 hand file, the working length (WL) will be established 1 mm shorter than the apical foramen.
2. All the root canals will be prepared with rotary files till size #35/06iii according to the manufacturer’s specifications.
3. After each instrument change, canals will be passively irrigated with 2 ml of 5% NaOCl solution using 30 Gauge side vented needle.
4. 5 ml of 5% NaOCl will be used as a final irrigant followed by 3 ml of 17% (EDTA) for 1 min then (S2) is taken.
• Medication preparation &insertion:
5. The samples will be divided randomly into 3 groups:
G1 (triple antibiotic paste): Equal weight proportions of (Ciprofloxacin, Metronidazole and Minocycline) tablets will be crushed to form a powder and will be mixed with equal proportions of 1:1:1 ratio with a concentration of 1 mg/ml. The powder will be mixed with distilled water in an equal ratio of (1:1 w/v) to obtain a homogenous paste and will be inserted using an endodontic plugger.
G2 (Diclofenac paste): Sachets of diclofenac potassium powderiv will be mixed with distilled water in equal ratio of (1:1 w/v) and will be inserted the same as the previous group.18
G3 (control group): no medicament is applied.
6. The teeth in both groups will be sealed temporarily and incubated at 37°C for 7 days.
7. After this period, each tooth will be irrigated with 5 mL saline to remove intracanal medication from the canal then S3 will be collected and cultured.