Ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples.
This study focused on ultrafiltration as a technique for simultaneously concentrating and recovering viruses, bacteria and parasites in 100-L drinking water samples. A chemical dispersant, sodium polyphosphate, and Tween 80 were used to increase microbial recovery efficiencies. Secondary concentration was performed to reduce sample volumes to 3-5 mL for analysis using tissue culture, microscopy, and real-time PCR and RT-PCR. At seeding levels of 100-1000 (CFU, PFU, oocysts, or particles), a "high-flux" ultrafiltration procedure was found to achieve mean recoveries of 51-94% of simultaneously seeded MS2 bacteriophage, echovirus 1, Salmonella enterica subsp. enterica serovar Typhimurium, Bacillus atrophaeus subsp. globigii endospores, Cryptosporidium parvum oocysts, and 4.5-mum microspheres. When 4-7% of the final sample concentrate volume was assayed using real-time PCR and RT-PCR, overall method sensitivities were <100 C. parvum oocysts, <240 PFU echovirus 1, <100 CFU Salmonella and approximately 160 CFU B. atrophaeus spores in 100-L drinking water samples. The "high-flux" ultrafiltration procedure required approximately 2 h, including time required for backflushing. Secondary concentration procedures required an additional 1-3 h, while nucleic acid extraction and real-time PCR procedures required an additional 2-2.5 h. Thus, this study demonstrated that efficient recovery and sensitive detection of diverse microbes in 100-L drinking water samples could be achieved within 5-8 h using ultrafiltration, rapid secondary processing techniques, and real-time PCR.
Polaczyk
Amy L
AL
University of North Carolina at Charlotte, USA.
Narayanan
Jothikumar
J
Cromeans
Theresa L
TL
Hahn
Donghyun
D
Roberts
Jacqueline M
JM
Amburgey
James E
JE
Hill
Vincent R
VR
D000818:Animals; D001419:Bacteria / Q000302:isolation & purification*; D001435:Bacteriophages / Q000302:isolation & purification*; D003902:Detergents / Q000494:pharmacology; D008828:Microbiological Techniques / Q000379:methods*; D008829:Microbiology; D008853:Microscopy; D010271:Parasites / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011122:Polyphosphates / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013997:Time Factors; D046509:Tissue Culture Techniques; D014462:Ultrafiltration / Q000379:methods*; D014780:Viruses / Q000302:isolation & purification*; D014871:Water Microbiology
D003902:Detergents; D011122:Polyphosphates; D011136:Polysorbates