A Comparsion of Immunofluorescence Microscopy and Flow Cytometry in Detection Limits and Viabilities of Giardia and Cryptosporidium in Various Environmental Conditions.

• Main Authors: Sau-Yu Liao, 廖少宇
• Other Authors: Bing-Mu Hsu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/91702019553809986556

碩士 === 國立中正大學 === 地震研究所暨應用地球物理研究所 === 96 === Giardia and Cryptosporidium are pathogenic protozoa, which can infect the gastrointestinal tract of human and many other animals. It may cause acute diarrhea for immunocompromised patients, and cause death for AIDS patients by giardiasis and cryptosporidiosis. The hosts can excrete the feces containing protozoa, and disseminate by water. These parasites could exist in water for a long time, therefore, they have the high possibility to cause massive waterborne outbreaks. In the study, we compared the difference between immunofluorescence microscopy and flow cytometry in detection limits and viabilities for the analysis of Giardia and Cryptosporidium. We calculated that using flow cytometry to detect Giardia and Cryptosporidium (oo)cysts can gain the more accurate count of (oo)cysts than immunofluorescence microscopy. In the high turbity condition, Cryptosporidium sample (4000 NTU), flow cytometry could detect the oocysts exactly. In order to determine the viability for these two pathogenic protozoa, 30 fecal samples of small cattles were collected from southern Taiwan. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) accompanied by microscopy were retained presumption test and confirm test, respectively. As for the polymerase chain reaction (PCR) accompanied by sequence analysis were defined as complete test in this study. The percentage of Giardia positive samples is 16.7％, and that for Cryptosporidium is 10.0％. In order to purify this stool samples, we used percoll-sucrose and immunomagnetic separation (IMS) methods. The (oo)cysts containing samples were stained by IFA and SYTO-9/PI and then observed by immunofluorescence microscopy. The result show that Giardia and Cryptosporidium (oo)cysts could keep viability in stod sample within 50 days. In the study, we chose aqueous sodium hypochlorite for disinfection test. While the contact time was 10 min and the concentration of sodium hypochlorite was 140 ppm for Giardia and 260 ppm for Cryptosporidium, both parasites would loss viability completely. From our predict diagram, we could indicate that disinfection in the condition of 18 ppm / 190 mins and 15 ppm / 170 mins for Cryptosporidium and Giardia, respectively, the inactivity would in creased to 99％. We concluded that in general disinfectant dosage used in the tap water treatment plant, the contact time of disinfectant should be increased to prevent protozoa infection.