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Chapter 4 Microbiological Contaminants

Chapter 4 Microbiological Contaminants. Waterworks Operations WQT 111 Lecture 4. E. coli are bad?. True False. Objectives. Compare virus (small), bacteria (medium), and protozoa (large) Review Total Coliform Rule. Go Over Total Coliform Analytical Procedures.

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Chapter 4 Microbiological Contaminants

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  1. Chapter 4 Microbiological Contaminants Waterworks Operations WQT 111 Lecture 4

  2. E. coli are bad? • True • False

  3. Objectives • Compare virus (small), bacteria (medium), and protozoa (large) • Review Total Coliform Rule. • Go Over Total Coliform Analytical Procedures. • Perform Colilert Test of Unknown sample.

  4. Viruses • Smallest living entities 10 - 25 nm .  Viruses can be observed only with the aid of an electron microscope. • Viruses are obligate intracellular parasites - they cannot live or multiply outside of a host cell.  • More resistant to chlorine relative to bacteria • Viruses are not cellular organisms.  Many consist of a protein coat or capsid and internal nucleic acid, either DNA or RNA.  • Viruses do not produce enzymes for metabolism. • In the process of multiplying, viruses kill the infected host cells.   Important viral waterborne infections include hepatitis A and gastroenteritis (norwalk and rotavirus)

  5. Virus

  6. You can see a virus with the naked eye? • True • False

  7. Viruses need a host? • True • False

  8. In the processes of viral metabolism enzymes are exuded which attack the host cell? • True • False

  9. Viruses are the smallest cellular organisms with a true nucleus? • True • False

  10. Bacteria • Smallest cellular organisms.  A typical bacterium is about 1 micrometer in size.  Bacteria have a unique cell construction that sets them apart from all other living organisms. • The vast majority of bacteria are free living soil and water microorganisms.  • Bacteria can exist in conditions that no other living organism can tolerate.  Conan the bacterium • Bacteria cause many diseases in animals and man.  Important waterborne infectious diseases include campylobacterosis, legionnaires disease, shigellosis, typhoid, enterovirulent E. coli 0157, and cholera.

  11. E. coli http://pathport.vbi.vt.edu/pathinfo/pathogens/E.coli_O157H7.html http://www.onr.navy.mil/media/view_image.asp?ID=63&SubID=64 • E.coli are rod shaped, facultative anaerobic, gram-negative bacillus • Waste processing, Vitamin B and K production, and food absorption • They are indicators of fecal coliform • 10 billion-100 trillion per day per person

  12. E. coli are necessary for human survival? • True • False

  13. E.coli are rod shaped, facultative anaerobic, gram-negative bacillus? • True • False

  14. Escherichia coli type 0157:H7 (bacteria, enteric pathogen) • The illness is characterized by severe cramping (abdominal pain) and diarrhea which is initially watery but becomes grossly bloody. Occasionally vomiting occurs. • Fever is either low-grade or absent. • The illness is usually self-limited and lasts for an average of 8 days. Some individuals exhibit watery diarrhea only. • Infectious dose is a few as 10 bacteria cells. http://en.wikipedia.org/wiki/2006_United_States_E._coli_outbreak

  15. "E Coli H0157" is the bacterium that is the best known serotype of "Enterohemorrhagic E. Coli"and known by its more formal scientific name of Escherichia coli 0157:H7. Transmitted from undercooked hamburger meat and raw milk, it releases verotoxin, which causes inflammation of the lower gastrointestinal tract, often resulting in bloody bowel movements, potentially several other illnesses and/or death. • True • False

  16. Bacteria

  17. Bacteria rod coccus spirillum

  18. Most total coliforms are rod shaped? • True • False

  19. Protozoa • Protozoa are mostly single celled organisms that have a complex cell construction (5-100 mm). • Protozoa are classified into groups according to their method of motility: • flagellates, move with the aid of one or more whip-like flagella. • ciliates, move with the aid of a group of short hair-like cilia. • amoeba, move with the aid of temporary cell projections called pseudopods (false feet). • Most protozoa are free living water and soil microorganisms. • Protozoa produce important diseases in animals and man.  Important waterborne diseases include giardiasis, cryptosporidiosis and amoebic dysentery.

  20. Protozoa like to eat bacteria? • True • False

  21. A cilia is a whip like tail structure in a protozoa? • True • False

  22. Protozoa Intestinal Parasites & Water Borne DiseasesGiardia lambliaCryptosporidium parvum

  23. Giardiasis (intestinal parasite) • Giardia lamblia (intestinalis) is a single celled animal, i.e., a protozoa, that moves with the aid of five flagella. • Giardia found in domestic animals (dogs and cats) and wild animals (beavers, ducks, and bears) • Most frequent cause of non-bacterial diarrhea in North America. ~ 25% of the cases of gastrointestinal disease • Giardiasis is most frequently associated with the consumption of contaminated water . 2% of US population. Oregon second most reported cases 1980s • Ingestion of one or more cysts may cause disease • Illness lasts for 1 to 2 weeks (chronic infections can last months to years). Treat with Flagyl=antibiotic http://www.cfsan.fda.gov/~mow/chap22.html

  24. Giardia lamblia is a bacteria that causes waterborne infection? • True • False

  25. Giardia is found in domestic animals (dogs and cats) and wild animals (beavers, ducks, and bears)? • True • False

  26. Ingestion of one or more giardia cysts may cause disease? • True • False

  27. Giardia is the most frequent cause of non-bacterial diarrhea in North America? • True • False

  28. Giardia Lamblia/Protozoa

  29. Cryptosporidiosis (protozoa) • Cryptosporidium parvum, a single-celled animal, i.e., a protozoa, is an obligate intracellular parasite. • Infects many herd animals (cows, goats, sheep among domesticated animals, deer and elk among wild animals) • The infective stage (oocyst) is 3 µm in diameter or about half the size of a red blood cell. • Severe watery diarrhea lasting 2-4 days (no reliable treatment for cryptosporidiosis) • Serological surveys indicate that 80% of the population has had cryptosporidiosis. http://www.cfsan.fda.gov/~mow/chap24.html

  30. Cryptosporidiosis • The new “superbug” Cryptosporidium parvum is first identified as an important waterborne infection in the 1990's. • Resistant to chlorination • Oregon reports waterborne outbreaks in 1992 Talent Oregon communities. • 1993 largest waterborne disease outbreak in US • ~400,000 Milwaukee Wisconsin http://www.cfsan.fda.gov/~mow/chap24.html

  31. Cryptosporidium/Protozoa

  32. Total Coliform Rule • Total Coliform Rule (TCR) on June 29, 1989 • monitor for total coliforms at a frequency proportional to the number of people served • Further test that culture for the presence of either fecal coliforms or Escherichia coli; • Take samples at end of distribution systems • If positive occurs: • Re-collect and analyze at least 3-4 repeat samples within 24 hours : • 1 at the same tap as the positive • The others at sites located within 5 or fewer service connection adjacent (upstream and downstream) to the location of the routine positive sample; and • Take at least 5 routine samples the next month of operation. • 16 different species of Total Coliforms!

  33. EPA APPROVED METHODSTotal Coliforms • Presence–absence (P-A) The P-A test is a qualitative procedure that was developed as a sensitive, economical, and efficient means of analyzing drinking water samples • Membrane filter (MF) The MF procedure was introduced to bacteriological water analysis in 1951, after its capacity to produce results equivalent to those obtained by the MTF procedure was demonstrated • Multiple tube fermentation (MTF) procedures - The MTF procedure, in comparison with the MF procedure, lacks precision, is more difficult to perform, and takes longer to produce results; because of this, the latter has largely replaced it for routine examinations of drinking water.

  34. Presence/Absence • Incubate tubes or bottles • YES OR NO? • Fluorescent end product from E.Coli fermentation Positive E.Coli

  35. What is the most common method used in labs to test for total coliform and E. coli? • DMA • Green • Colilert • Lamp

  36. Total Coliforms

  37. Membrane Filtration Total Coliforms (m-Endo broth) Total Coliform Total Coliform Total Coliforms (http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm) • Red colony with a metallic sheen within 24±2 hours at 35±0.2oC

  38. Fecal Coliforms (m-FC broth) • Blue colonies for fecal coliforms. Gray to cream colored are non fecal coliforms Fecal Coliform Fecal Coliforms (http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm) • Blue colony within 24±2 hours at 44.5±0.5oC Fecal Coliform MI-Media: Pure Culture of Escherichia coli ATCC 25922 with UV Light (http://www.whatman.com/products/?pageID=7.61.409.297)

  39. In the membrane filter method, the number of coliforms is estimated by the: • Number of colonies grown • Number of negative tubes • Number of positive tubes • Sum of positive and negative tubes

  40. Fecal Coliforms are incubated at 44.5oC for 24 hours • True • False

  41. When conducting the fecal coliform membrane test, count all the colonies that are _______ in color. • Red • Yellow • Blue • Orange

  42. Fecal coliforms are incubated in a water bath at this temperature for 24 hours. • 44.5 oC • 35.0 oC • 103 oC • 37.5 oC

  43. The recommended holding time for a fecal coliform sample prior to analysis is 24 hours or less at 4 deg. C.? • True • False

  44. Multiple Tube Fermentation Method Inoculate lauryl tryptose broth and incubate for 24h at 35oC Gas or acidity Transfer to BGBB incubate 48 h at 35oC No gas or acidity Incubate 24 h at 35oC No gas produced - test. Coliform group absent Gas produced Confirm as in (1) Acidic growth produced Confirm as in (1) • No gas or acidic • growth produced • test. • Coliform • group absent Gas produced Coliform group confirmed Re-inoculate in fresh BGBB Positive completed phase

  45. Multiple Tube Fermentation Method • Step 1 presumptive phase • Use lauryl tryptose broth • Grow “stressed” organisms • Confirm positives • Calculate MPN Gas Growth http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=55&theme=printer

  46. Habitats of Fecal and NonFecal Coliforms

  47. There are 16 different species of coliforms in nature? • True • False

  48. E. coli results must always be higher or lower than fecal coliform results? • Higher • Lower

  49. What is an IDEAL INDICATOR? • Be applicable in all water types. • ALWAYS be present in water when pathogenic bacteria of fecal contamination are present. • Density or numbers should relate to amount/degree of pollution • Greater survival time in water • Disappears rapidly following the disappearance of pathogens

  50. Coliform bacteria- Microorganisms predominantly inhabiting the intestines of humans and other animals, but also occasionally found elsewhere. They include all aerobic and facultative anaerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with the production of gas. Also included are all bacteria that produce a dark, purplish-green metallic sheen by the membrane filter technique used for coliform identification. • True • False

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