EVALUATION AND COMPARISON OF CONCENTRATION AND CULTIVATION METHODS FOR DETECTION OF CLOSTRIDIUM PERFRINGENS SPOR ES IN WATER SAMPLES

The 17 th International and Iranian Congress of Microbiology

23 till 25 August 2016، Tehran - Iran

Presentation Type: Speech
Abstract:

Background and Aim: Since Cryptosporidium oocysts and Giardia cysts together with some enteric viruses are resi stant against routine chemical and mechanical drinking water treatment, C. perfringens spores could be good indicators to evaluate treatment processing of drinking water resources.
Methods: Serial dilutions of extracted spores were prepared in defined amoun ts of sterile PBS to give final concentrations: 101, 102, 104, 106 and 107 spores/mL. Filtration of diluted samples was done using 0.2μm cellulose nitrate filter membranes. After filtration two processes were performed. 1.
Concentration method: the membran e was soaked in 300 ml 0.5 mM H2so4 to facilitate the elution. Then, 50 ml elution buffer was added to the membrane and eluted spores were collected in sterile tubes. After centrifuge, supernatant was discarded and DNA extraction was performed on pellet. F inally, PCR was done using specific primers. 2. Cultivation method: The filtered membranes were placed on surface of egg yolk agar mediums and all mediums were incubated in anaerobic condition. The cultures results were followed to one week
Results: Cultiva tion method: C. prefringens specific dark colonies were growth in egg yolk medium supplemented by Neomycin after 48h. Limit of detection of C. perfringens spores for cultivation was 102 spores/ml. Concentration method: DNA fragment about 279 - bp of 16SrRNA gene was acquired for concentrations 102 - 107. The sample with 10 spores/ml was positive for neither cultivation method nor concentration method.
Conclusion: Concentration method is a fast and easy method that has same results compared with traditional cul tivation method.