Overview of indicator organisms tủ tài liệu bách khoa

Overview of Indicator Organisms “indicators” of fecal contamination | Non-pathogenic organisms that can indicate the presence of pathogens z Bacteria • • • • • z Total coliforms Fecal coliforms Escherichia coli Enterococci Clostridium perfringens Viruses • coliphage Why not test directly for waterborne pathogens? | Safety z | Time and cost z | May require cultivation and direct manipulation of pathogenic organisms Each pathogen must be detected using a different test Requires processing of large volumes of sample z Pathogens usually are present in low concentrations Advantage of “Indicators” | Indicator organisms z z | NOT pathogenic Used to directly detect the presence of fecal contamination from warm-blooded animals which "indicates" the possible presence of pathogenic organisms Indicator chemicals z z Used to indirectly indicate the presence of fecal contamination from humans or animals caffeine, coprostanol, fragrances and other human-associated chemicals An Ideal Indicator Organism | | | | | | Has an easy testing procedure Is of human or animal origin Survives as long as, or longer, than pathogens Present at densities related to the severity of fecal contamination Is a "surrogate" for many different pathogens Useful in fresh and saline waters Weaknesses of Indicator Organisms | May be present when there is no fecal contamination z | Absent when pathogens are present z | Total coliforms and C perfringens are found in soil so they are poor indicators of fecal contamination E coli may die off faster than viral pathogens Density may not always relate well to the density of pathogens z E coli can reproduce in warm, tropical waters BACTERIAL INDICATORS E coli cells (from USEPA web site) Common Indicator Bacteria | Coliform group z z z | Streptococci z z | total coliforms fecal coliforms Escherichia coli Enterococci fecal streptococci Spore formers z Clostridium perfringens The Coliform Group of Bacteria Enterobacteriaceae Total coliforms Fecal coliforms E coli Coliform Characteristics Coliforms are facultative anaerobic, gram-negative, nonspore forming, and able to ferment lactose | Total coliforms z z | Fecal coliforms z z | Ubiquitous in the environment; because they include fecal coliforms and E coli they may indicate fecal contamination Ferment lactose at 35 °C May be of fecal origin Ferment lactose at 44.5 °C Escherichia coli z z Inhabits gastrointestinal tract of warm-blooded animals so is a specific indicator of fecal pollution Ferment lactose at 44.5 °C, not produce urease, and produce β-D-glucuronidase Other Equipment | | Incubator Magnification z z | | | | Hand lens Dissecting scope Portable incubators UV light box Light Hand counter Autoclave UV light Magnification Autoclave Basics of Membrane Filtration A specific volume of sample is passed through a membrane filter to separate out the bacteria The filter membrane is placed on a plate of nutrient agar medium that provides the growth needs of the target organism The plate is incubated at a specific temperature Target colonies are counted Step one: Filter the sample | | | | Filter 3-5 volumes (e.g 1, 3, 10, 30, 100 mL) The volumes selected depend on how “dirty” the sample is expected to be The goal is to select at least one volume that produces counts within the “ideal” range A very dirty sample may need to be diluted to produce a count in the ideal range Dilution Series 30 mL 10 mL mL mL Serial Dilutions—an Example mL of sample in | If a sample has 30,000 target bacteria per 100 mL; z For mL filtered, ~3,000 colonies are present—too many to count z But for 0.01 mL filtered, only 30 colonies are present—a count in the ideal range but a volume too small to easily measure z mL of a 1:100 sample dilution = 0.01 mL of original sample 99 mL of buffer makes 1:100 dilution 1:100 dil’n Sample 99-mL dilution buffer Step 2: Nutrient Agar Medium Place filter on a nutrient medium that promotes target organism growth z z z z z z Carbon source Energy source Water Inorganic ions Growth factors Selective and differential agents Media Contain Selective and Differential Agents mFC medium | Selective agents z z | inhibit growth of non-target bacteria enhance the growth of target bacteria mTEC medium Enriched lactose medium, rosolic acid favor fecal coliforms Differential agents z Exploit unique characteristics of the target bacteria that allows for its identification Sodium lauryl sulfate, sodium desoxycholate favor E coli; enzyme cleaves urea phenol MUG medium E coli has enzyme to cleave MUG and produce fluorogen Step 3: Incubation The plate is incubated at a specific temperature z z z z mENDO: 35 °C mFC: 44.5 °C Modified mTEC: 35 °C for hours then 44.5 °C mEI: 41.5 °C Step 4: Results Colonies that grow and meet the differential criteria for target organisms are counted mFC plate: Count blue colonies NOT gray or creamcolored Defined substrate technology (DST) sterile DI water mixing bottle sample pre-measured media quantitray How does DST work? | | | Nutrient medium for coliforms z ortho-nitrophenyl-β-Dgalactopyranoside (ONPG) and z 4-methylumbelliferyl-β-Dglucuronide (MUG) Total coliform enzyme metabolizes ONPG to form a yellow product E coli enzyme metabolizes MUG to form a fluorescent product How does DST work? | Nutrient medium for enterococci z | 4-methylumbelliferyl-βD-glucoside Enterococci enzyme metabolizes the reagent to form a fluorescent product DST Steps Seal, incubate Count the positive cells Mix media with sample; pour into Quantitray Preparing a sample for incubation Determining a final count | | | Compare color intensity to the “comparator” tray Count the number of large and small wells that are positive Look up the most probable number on the IDEXX table Blank Comparator Positive test ... manipulation of pathogenic organisms Each pathogen must be detected using a different test Requires processing of large volumes of sample z Pathogens usually are present in low concentrations Advantage of. .. Advantage of “Indicators” | Indicator organisms z z | NOT pathogenic Used to directly detect the presence of fecal contamination from warm-blooded animals which "indicates" the possible presence of pathogenic...“indicators” of fecal contamination | Non-pathogenic organisms that can indicate the presence of pathogens z Bacteria • • • • • z Total coliforms