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 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?
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
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.

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 Diseases Giardia lamblia Cryptosporidium 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

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.

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

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 METHODS Total 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. Total Coliforms (m-Endo broth)
Membrane Filtration
Total Coliforms (m-Endo broth)
Total Coliform
Total Coliform
Total Coliforms (
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 (
Blue colony within 24±2 hours at 44.5±0.5oC
Fecal Coliform
MI-Media: Pure Culture of Escherichia coli ATCC with UV Light (

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
Gas produced
Coliform group confirmed
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
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

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?

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

51. What is an IDEAL INDICATOR?
Absent from a bacteriologically safe water
Easily analyzed for without any anomalies or false positives.
Greater survival time in water
Disappears rapidly following the disappearance of pathogens
Be harmless to man or other animals

52. Total and fecal coliform are all pathogenic?
True
False

53. Coliform bacteria are:
More resistant to chlorination than pathogenic bacteria
Less resistant to chlorination than pathogenic bacteria
A group of pathogens causing hepatitis
A group of pathogens causing cholera

54. Which of the following are true about indicator organisms?
Be present when pathogen is present
Be at lower numbers then the pathogen
Have a shorter lifespan then a pathogen
Be able to kill a pathogen and a lab technician in the same day

55. Fecal coliforms are a subset of total coliform bacteria?
True
False

56. Coliforms are used as indicator bacteria to verify the effectiveness of disinfection techniques because ______.
They are the most numerous of all wastewater pathogens
They cause the worst diseases of any of the wastewater pathogens
They are easier to detect and are harder to destroy than most pathogenic organisms
They have a symbiotic relationship with pathogens; one cannot survive without the other.

57. Coliform bacteria are:?
Commonly found throughout distribution systems
Desirable in storage tanks for iron digestion
Highly resistant to chlorine
Indicator organisms
Deadly

58. A comparison of pathogens to indicator organism .
# of organisms
Time
A comparison of pathogens to indicator organism
Indicator organisms should outlive a pathogen
An indicator organism can coexist with a pathogen
All of the above

59. This is the special sugar that fecal coliform bacteria can metabolize.
Glucose
Fructose
Lactose
Rosalic acid

60. Coliform bacteria and the procedures used to test for them are divided into the two categories of _______.
MF and MTF
LTB and BGB
Total and fecal coliforms
M-Endo and M-FC

61. Objective #4 on the syllabus (Understand the fundamentals of water microbiology, especially as it relates to waterborne infectious disease) has been met?
Strongly Agree
Agree
Disagree
Strongly Disagree