microbial pathogens n.
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Microbial Pathogens

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  1. Microbial Pathogens • Living organisms that cause disease • Can be • Viruses • Bacteria • Protozoa • Helminths • But not all are pathogens

  2. Viruses • Intracellular parasites • very small (20-100 nm), very simple • not composed of cells • need host cells to replicate • infection usually person-to-person, not through water • hepatitis, gastroenteritis....

  3. Aside - Units • nm • Nano = 1/1,000,000,000 • ~ 3 to 6 atoms end to end constitute a nanometer

  4. Detection of Viruses • Not recommended for routine analyses • Should be done only by competent and specially trained water virologists • Three Steps • Collect representative sample • Concentrate viruses in sample • Identify and quantify • Beyond our capability

  5. Problems, Virus Methods • Very small (20 to 100 nm) • Generally present at low concentration, but variable in amount and type • Unstable as biological entities • Other compounds interfere • Current methods are limited

  6. Bacteria • microscopic, single-celled organisms • 500-5000 nm • procaryotic (DNA not enclosed in membrane) • most are not pathogens • perform valuable functions in environment, our bodies, & wastewater treatment • Proliferate in: • feces: 1 - 1000 X 106 / gram • wastewater: ~ 10,000 / ml • Pathogenic bacteria cause typhoid, cholera....

  7. Vibrio cholerae (Microbe causing cholera) From www.bact.wisc.edu/microtextbook/TOC.html

  8. Detection Methods - Specific Species • Not recommended for routine analyses • Three Steps • Collect representative sample • Concentrate bacteria in sample / Grow bacteria colonies • Identify and quantify • Stains, size, shape, growth patters, what they grow on... • Beyond our capability

  9. Protozoa • Microscopic, single-celled "animals", more complex and larger than bacteria • 10000-15000 nm • eucaryotic (DNA in nucleus within cell) • Most not pathogenic • Form Cysts / Oocysts • Resistant forms which allows Protozoa to survive under adverse conditions • Pathogenic protozoans cause diarrhea (Cryptosporidium), dysentery, gastrointestinal infection (Giardia lamblia)...

  10. Giardia lamblia • 111 waterborne outbreaks between 1965 and 1990, >26,000 cases • Causes diarrhea

  11. Cryptosporidium parvum • Of increasing concern • Causes cholera-like diarrhea • can be life-threatening to immunodeficient persons 1993, Milwaukee - 400,000 sick - 50 dead

  12. Entamoeba histolytica • Causes amebic dysentery • Averages 28 deaths / year • Has not been a frequent cause of waterborne outbreaks in recent times

  13. Protozoa Detection • Not recommended for routine analyses • Crypto and Giardia • concentrate, purify and distribute organisms in monolayer on membrane filter • label with fluorescent antibody reagents • identify cysts and oocysts by specific criteria (immunoflorescence, size, shape, internal morphological characteristics) • Beyond our capability

  14. Helminths (worms) • Humans can ingest worm eggs in contaminated water • Worm can grow inside body, causing disease • Some (e.g., Hookworms) can infect by penetrating skin • Worms can cause joint arthritis, damage lymph nodes, damage tissue and organs • Not of Concern in US

  15. Intestinal nematodes - from www.life.sci.qut.edu.au/LIFESCI/darben/paramast.htm

  16. Drinking Water Standards & Pathogens • Maximum Contaminant Level Goal • zero pathogens • Maximum Contaminant Level • We will accept a limited number of positive samples (indicator organism) • to account for inadvertent contamination • re-check water

  17. Indicator organisms • Too difficult to identify all pathogens, so we use indicator organisms • May not be pathogens themselves • Find indicator organisms? • sample might be pathogen contaminated • Don't find indicator organisms? • very unlikely sample is contaminated

  18. Common Indicator Organisms • Total Coliform • Fecal Coliform • E. Coli • Common denominator is fecal coliform • found in intestines • evidence of fecal contamination Bacteria

  19. General Types of Tests • Prescriptive tests • Positive result good indication of presence of indicator organism, but not definitive • Confirmatory • Positive result indicates definite presence of indicator organism

  20. Specific Tests • Membrane Filtration • Presence/Absence • Fermentation tube • (confirmatory)

  21. Membrane Filtration • Filter known volume through sterile filter • with proper dilution, deposit isolated bacteria • Place filter in petri dish w/ sterile agar • promotes organism of interest, inhibits others • Incubate (time / temperature) • isolated bacteria grow into easily identified colonies • Count colonies • Concentration = Colonies / Volume of sample

  22. Presence/Absence • Add 100 mL sample to broth • Incubate (time / temperature) • yellow color indicates presence of coliforms • Determines presence or absence only

  23. Fermentation Tubes • (1) Presence/Absence • Inoculate tube containing special broth • Incubate (time / temperature) • gas production in tubes indicates presence

  24. Fermentation Tubes • (2) Concentration • Inoculate series of tubes with various amounts of sample • # of bacteria introduced proportional to sample amount • Incubate • Observe which tubes generate gas • Statistically relate to most likely concentration

  25. WWW Resources • EPA Pathogen Document • www.epa.gov/enviro/html/icr/gloss_path.html • Germ Tutorial • www.mwra.state.ma.us/germs/intro.htm • Pathogenic Bacteria Photo Gallery • www.geocities.com/CapeCanaveral/3504/ • Cryptosporidium Newsletter • www.fspubl.com/index.html • Online Microbiology Textbook • www.bact.wisc.edu/microtextbook/TOC.html