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

2 Viruses n Intracellular parasites –very small ( 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 n nm –Nano = 1/1,000,000,000 –~ 3 to 6 atoms end to end constitute a nanometer

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

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

7Bacteria n microscopic, single-celled organisms – nm –procaryotic (DNA not enclosed in membrane) –most are not pathogens n perform valuable functions in environment, our bodies, & wastewater treatment n Proliferate in: –feces: X 10 6 / gram –wastewater: ~ 10,000 / ml n Pathogenic bacteria cause typhoid, cholera....

8 Vibrio cholerae (Microbe causing cholera) From

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

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

11 Giardia lamblia n 111 waterborne outbreaks between 1965 and 1990, >26,000 cases n Causes diarrhea

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

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

14 Protozoa Detection n Not recommended for routine analyses n 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) n Beyond our capability

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

16 Intestinal nematodes - from

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

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

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

20 General Types of Tests n Prescriptive tests –Positive result good indication of presence of indicator organism, but not definitive n Confirmatory –Positive result indicates definite presence of indicator organism

21 Specific Tests n Membrane Filtration n Presence/Absence n Fermentation tube –(confirmatory)

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

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

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26 Fermentation Tubes n (1) Presence/Absence –Inoculate tube containing special broth –Incubate (time / temperature) n gas production in tubes indicates presence

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

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29 WWW Resources n EPA Pathogen Document – n Germ Tutorial – n Pathogenic Bacteria Photo Gallery – n Cryptosporidium Newsletter – n Online Microbiology Textbook –