Transcript Lec4ch6micro

Chapter 4 Microbiological
Contaminants
Waterworks Operations
WQT 111
Lecture 4
E. coli are bad?
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2. False
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Objectives
1. Compare virus (small), bacteria (medium),
and protozoa (large)
2. Review Total Coliform Rule.
3. Go Over Total Coliform Analytical
Procedures.
4. Perform Colilert Test of Unknown sample.
Viruses
1. Smallest living entities 10 - 25 nm . Viruses can be
observed only with the aid of an electron microscope.
2. Viruses are obligate intracellular parasites - they cannot
live or multiply outside of a host cell.
3. More resistant to chlorine relative to bacteria
4. Viruses are not cellular organisms. Many consist of a
protein coat or capsid and internal nucleic acid, either DNA
or RNA.
5. Viruses do not produce enzymes for metabolism.
6. In the process of multiplying, viruses kill the infected host
cells. Important viral waterborne infections include
hepatitis A and gastroenteritis (norwalk and rotavirus)
Virus
You can see a virus with the
naked eye?
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Viruses need a host?
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In the processes of viral
metabolism enzymes are exuded
which attack the host cell?
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Viruses are the smallest cellular
organisms with a true nucleus?
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Bacteria
1. 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.
2. The vast majority of bacteria are free living soil and water
microorganisms.
3. Bacteria can exist in conditions that no other living
organism can tolerate. Conan the bacterium
4. 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
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
E. coli are necessary for human
survival?
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E.coli are rod shaped, facultative
anaerobic, gram-negative
bacillus?
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2. False
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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
"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.
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Bacteria
Bacteria
coccus
rod
spirillum
Most total coliforms are rod
shaped?
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Protozoa
1. Protozoa are mostly single celled organisms that have
a complex cell construction (5-100 mm).
2. Protozoa are classified into groups according to their
method of motility:
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•
•
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).
3. Most protozoa are free living water and soil
microorganisms.
4. Protozoa produce important diseases in animals and
man. Important waterborne diseases include
giardiasis, cryptosporidiosis and amoebic dysentery.
Protozoa like to eat bacteria?
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A cilia is a whip like tail structure
in a protozoa?
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Protozoa Intestinal Parasites &
Water Borne Diseases
Giardia lamblia
Cryptosporidium 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?
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Giardia is found in domestic
animals (dogs and cats) and wild
animals (beavers, ducks, and
bears)?
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2. False
Ingestion of one or more giardia
cysts may cause disease?
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2. False
Giardia is the most frequent
cause of non-bacterial diarrhea
in North America?
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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.
http://www.cfsan.fda.gov/~mow/chap24.html
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
Cryptosporidium/Protozoa
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!
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.
Presence/Absence
• Incubate tubes or bottles
• YES OR NO?
• Fluorescent end product from E.Coli
fermentation
Positive
E.Coli
What is the most common
method used in labs to test for
total coliform and E. coli?
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1. DMA
2. Green
3. Colilert
4. Lamp
Total Coliforms
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
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)
Fecal Coliform
MI-Media: Pure Culture of Escherichia coli ATCC
25922 with UV Light
(http://www.whatman.com/products/?pageID=7.61.409.297)
• Blue colony
within 24±2 hours
at 44.5±0.5oC
In the membrane filter method,
the number of coliforms is
estimated by the:
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fc
1. Number of colonies grown
2. Number of negative tubes
3. Number of positive tubes
4. Sum of positive and negative
tubes
Fecal Coliforms are incubated at
44.5oC for 24 hours
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When conducting the fecal
coliform membrane test, count
all the colonies that are _______
in color.
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1. Red
2. Yellow
3. Blue
4. Orange
Fecal coliforms are incubated in
a water bath at this temperature
for 24 hours.
oC
0%
37
.5
oC
3
10
.0
35
.5
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oC
0%
oC
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44
1. 44.5 oC
2. 35.0 oC
3. 103 oC
4. 37.5 oC
The recommended holding time
for a fecal coliform sample prior
to analysis is 24 hours or less at
4 deg. C.?
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2. False
Multiple Tube Fermentation Method
Inoculate lauryl tryptose broth and incubate for 24h at 35oC
No gas or acidity
Incubate
24 h at 35oC
Gas or acidity
Transfer to BGBB
incubate 48 h at 35oC
Gas produced
Coliform group
confirmed
Re-inoculate in
fresh BGBB
Positive completed
phase
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
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
Habitats of Fecal and NonFecal
Coliforms
There are 16 different species of
coliforms in nature?
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2. False
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E. coli results must always be
higher or lower than fecal
coliform results?
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1. Higher
2. Lower
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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
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, rodshaped 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.
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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
Total and fecal coliform are all
pathogenic?
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2. False
Coliform bacteria are:
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.
1. More resistant to chlorination than
pathogenic bacteria
2. Less resistant to chlorination than
0% 0%
pathogenic bacteria
3. A group of pathogens causing
hepatitis
4. A group of pathogens causing cholera
Which of the following are true
about indicator organisms?
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Be present when pathogen is present
Be at lower numbers then the pathogen
0% 0%
Have a shorter lifespan then a pathogen
Be able to kill a pathogen and a lab
technician in the same day
B
1.
2.
3.
4.
Fecal coliforms are a subset of
total coliform bacteria?
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2. False
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Coliforms are used as indicator bacteria
to verify the effectiveness of disinfection
techniques because ______.
0%
0%
0%
ha
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1. They are the most numerous of all
wastewater pathogens
2. They cause the worst diseases of any of
the wastewater pathogens
3. They are easier to detect and are harder
to destroy than most pathogenic
0%
organisms
4. They have a symbiotic relationship with
pathogens; one cannot survive without
the other.
Coliform bacteria are:?
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1. Commonly found throughout
distribution systems
2. Desirable in storage tanks for iron
digestion
0%
3. Highly resistant to chlorine
4. Indicator organisms
5. Deadly
.
# of organisms
Time
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1. A comparison of pathogens to indicator
organism
2. Indicator organisms should outlive a
pathogen
3. An indicator organism can coexist with 0%
a pathogen
4. All of the above
This is the special sugar that
fecal coliform bacteria can
metabolize.
ac
id
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Fr
uc
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0%
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0%
G
1. Glucose
2. Fructose
3. Lactose
4. Rosalic acid
Coliform bacteria and the
procedures used to test for them
are divided into the two
categories of _______.
a.
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0%
B
M
TF
0%
M
1. MF and MTF
2. LTB and BGB
3. Total and fecal
coliforms
4. 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?
60%
1. Strongly Agree
2. Agree
3. Disagree
4. Strongly Disagree
40%
0%
ag
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ly
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