Clinical Isolates Of H. Pylori Lab Report

Materials and Methods
Growth condition of clinical isolates and determination of MIC:
Twenty clinical isolates of H. pylori were obtained from patients suffering gastritis and duodenal ulcer who referred to Al- Zahra Hospital, Isfahan for gastrointestinal endoscopy in a year of 2011. The gastric biopsy specimens were homogenized and cultured on Brucella Blood Agar (Merck, Germany). Skirrow's supplement including polymyxin B, vancomycin and trimethoprim (Merck, Germany), 5% defibrinated Sheep's Blood (Bahar Afshan, Iran),7% Fetal calf serum (Sigma, USA), 4 % L-cystein (Merck, Germany) and amphotericin B (Merck, Germany) for preventing fungal contamination added to medium culture . Plates were incubated at 37°C for 3-5 days in a microaerophilic
pylori were assessed by gram staining to be assuring as a spiral form. Then, helical bacteria were suspended in Brucella broth (Merck, Germany) supplemented with 10% fetal calf serum and 2% L-cystein, and incubated overnight at 37°C in a microaerophilic rotary shaker (100 rpm). When the density of bacteria reached to a suitable concentration, amoxicillin solution was prepared in phosphate buffer and added to each tube at concentrations of MIC, 1/2 ,1/4 and 1/8 MIC. The tubes were examined regularly each 12 h until 144 h in order to obtain coccoid forms. Bacterial morphology was determined by gram staining via light microscopy (Camera Digital DP 72-BX 51, Soft imaging system, Olympus, Japan). When coccoid forms of H. pylori isolates reached to more than 90%, bacteria went for other processing as well as evaluating their viability. The same approach was repeated for reference strain as control group with and without antibiotic. Coccoid form suspensions of H. pylori were centrifuged and washed by PBS. Then, bacterial sediments were stained with propidium iodide as a fluorescent dye (PI; Sigma, USA) and incubated 30 min at 4°C. Afterwards, cell suspension analyzed by flow cytometer (FACS Calibur, Becton Dickinson). PI is easily able to distinguish populations of dead cells from viable ones. Meanwhile, impaired cell membrane let more propidium iodide to enter through the cell; fluorescent dye are not absorbed by viable cell due to its
pylori at the various time intervals was prepared using a commercially total RNA-extraction kit (RNeasy Mini, Qiagen, Germany) according to the manufacturer’s protocol. In order to prevent from DNA contamination, bacterial suspension treated with DNA-free DNase set I (Qiagen, Germany). Then, density of RNA sample and its integrity were checked by Nanodrop spectrophotometer (Biochrom, England) and gel electrophoresis, respectively. According to manufacturer’s protocol, 100 ng of RNA sample was transcribed by using available random primer and RevertAid Reverse Transcriptase enzyme (RevertAid™ First Strand cDNA Synthesis Kit, Fermentas) and supplied inhibitor efficiently keeps RNA templates from degradation. All purified RNA and synthesis cDNA were aliquted and placed at -70 ºC until