Chapter 4 Microbiological Contaminants

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