Theses and Dissertations at Montana State University (MSU)
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Item Detection of cryptosporidium and E. coli using fluorescent in situ hybridization and solid phase laser cytometry(Montana State University - Bozeman, College of Letters & Science, 2013) Broadaway, Susan Cameron; Chairperson, Graduate Committee: Barry H. PyleCryptosporidium parvum is a protozoal pathogen transmitted through water by the fecal-oral route as oocysts. Because the oocysts are more resistant to environmental stresses than the bacteria conventionally used as indicators of fecal contamination, they can be present in water when indicator organisms, such as E. coli, are not found. In addition, because they are resistant to chlorine, they can pass from source water through water treatment into drinking water systems. The EPA method for detection of Cryptosporidium oocysts consists of identifying oocysts with fluorescently labeled antibodies, staining with 4',6-diamidino-2-phenylindole and examining slides with epifluorescent microscopy and differential interference contrast microscopy. This protocol is labor intensive and subject to technician error. A new method was developed for the rapid detection of Cryptosporidium parvum oocysts using fluorescent in situ hybridization (FISH) and the ScanRDI, a solid phase laser cytometer. Optimization of the FISH protocol for use with the ScanRDI was done with E. coli cells and known Cryptosporidium oocysts as a model. Source water and treated drinking water from the water treatment plant at Crow Agency on the Crow Indian Reservation in Montana was collected over the course of a year and concentrated using the EPA protocol for collection of oocysts. The samples were then examined for Cryptosporidium oocysts using both the ScanRDI method and the standard US EPA method. The combination of FISH for labeling Cryptosporidium and the ScanRDI for examination results in significantly higher numbers of Cryptosporidium detected as well as greater ease in identification. A statistical comparison was done that determined there was no correlation between the number of E. coli cells found in the water samples and the number of Cryptosporidium oocysts present. Additionally, although not tested on environmental samples, the FISH/ScanRDI method allowed for simultaneous detection of Cryptosporidium parvum oocysts and E. coli cells on the same membrane filter. Membranes were incubated before hybridization, hybridized concurrently with a Cryptosporidium specific probe and a probe specific for E. coli, followed by detection for both organisms with the ScanRDI.Item Development of fluorescent in situ hybridization for detection of L. pneumophila using solid phase laser cytometry(Montana State University - Bozeman, College of Letters & Science, 2013) Lim, Kien Seng; Chairperson, Graduate Committee: Barry H. PyleLegionella pneumophila was first identified and documented after a pneumonia outbreak during an American Legion convention held in Philadelphia in 1976, with 25 casualties. L. pneumophila is a gram negative bacillus found mostly in fresh water environments. It is known as a facultative intracellular parasite of protozoa in its natural habitat, and can also infect macrophages in humans when contaminated aerosols are inhaled. The current detection procedure for L. pneumophila is a culture based method, which is slow and may not detect viable but non-culturable cells; presence of L. pneumophila usually takes 3 to 10 days to confirm by this method, due to its slow growth. New more reliable, rapid and sensitive methods are needed. In this research, a method coupling fluorescent in situ hybridization (FISH) and ScanRDI solid phase laser cytometry was developed to detect L. pneumophila relying on growth with extended incubation to obtain cultures. Published FISH methods were optimized by addition of HCl and Tween 20 incubation, dextran sulfate, helper probes and tyramide signal amplification to increase fluorescence intensity of probed L. pneumophila. ScanRDI settings were also adjusted to be able to detect TSA-FISH probed L. pneumophila. The resulting TSA-FISH method coupled with ScanRDI was able to detect almost 90% of L. pneumophila cell compared to plate counts. This optimized TSA-FISH method was also used on Escherichia coli and Aeromonas hydrophila; the ScanRDI detected 79.5% and 39.7% of these organisms respectively compared to plate counts. The results suggested that L. pneumophila can be detected and enumerated using the method developed in this research. It can be completed within 12 hours, which is a significant improvement compared to at least 3 days for current detection methods. The TSA-FISH method can be used for detection and enumeration of at least 2 microorganisms, L. pneumophila and E. coli. For reliable detection and enumeration of other organisms such as A. hydrophila, procedures need to be further optimized and ScanRDI settings adjusted for each organism. It is anticipated that several microbes could be detected on one membrane filter using appropriate probes, procedures and ScanRDI settings.