Chapter 4 microbiological contaminants
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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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Chapter 4 Microbiological Contaminants

Waterworks Operations

WQT 111

Lecture 4

E. coli are bad?

  • True

  • False


  • Compare virus (small), bacteria (medium), and protozoa (large)

  • Review Total Coliform Rule.

  • Go Over Total Coliform Analytical Procedures.

  • Perform Colilert Test of Unknown sample.


  • 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)


You can see a virus with the naked eye?

  • True

  • False

Viruses need a host?

  • True

  • False

In the processes of viral metabolism enzymes are exuded which attack the host cell?

  • True

  • False

Viruses are the smallest cellular organisms with a true nucleus?

  • True

  • False


  • 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.

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

E. coli are necessary for human survival?

  • True

  • False

E.coli are rod shaped, facultative anaerobic, gram-negative bacillus?

  • True

  • False

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.

"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






Most total coliforms are rod shaped?

  • True

  • False


  • 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.

Protozoa like to eat bacteria?

  • True

  • False

A cilia is a whip like tail structure in a protozoa?

  • True

  • False

Protozoa Intestinal Parasites & Water Borne DiseasesGiardia lambliaCryptosporidium parvum

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

Giardia lamblia is a bacteria that causes waterborne infection?

  • True

  • False

Giardia is found in domestic animals (dogs and cats) and wild animals (beavers, ducks, and bears)?

  • True

  • False

Ingestion of one or more giardia cysts may cause disease?

  • True

  • False

Giardia is the most frequent cause of non-bacterial diarrhea in North America?

  • True

  • False

Giardia Lamblia/Protozoa

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.


  • 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


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!


  • 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.


  • Incubate tubes or bottles

  • YES OR NO?

  • Fluorescent end product from E.Coli fermentation



What is the most common method used in labs to test for total coliform and E. coli?

  • DMA

  • Green

  • Colilert

  • Lamp

Total Coliforms

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

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 25922 with UV Light (

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

Fecal Coliforms are incubated at 44.5oC for 24 hours

  • True

  • False

When conducting the fecal coliform membrane test, count all the colonies that are _______ in color.

  • Red

  • Yellow

  • Blue

  • Orange

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

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

  • True

  • False

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


24 h at 35oC

No gas produced

- test. Coliform group absent

Gas produced

Confirm as in (1)


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

Multiple Tube Fermentation Method

  • Step 1 presumptive phase

  • Use lauryl tryptose broth

  • Grow “stressed” organisms

  • Confirm positives

  • Calculate MPN



Habitats of Fecal and NonFecal Coliforms

There are 16 different species of coliforms in nature?

  • True

  • False

E. coli results must always be higher or lower than fecal coliform results?

  • Higher

  • Lower


  • 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

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


  • 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

Total and fecal coliform are all pathogenic?

  • True

  • False

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

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

Fecal coliforms are a subset of total coliform bacteria?

  • True

  • False

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.

Coliform bacteria are:?

  • Commonly found throughout distribution systems

  • Desirable in storage tanks for iron digestion

  • Highly resistant to chlorine

  • Indicator organisms

  • Deadly


# of organisms

  • 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


This is the special sugar that fecal coliform bacteria can metabolize.

  • Glucose

  • Fructose

  • Lactose

  • Rosalic acid

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

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

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