Transcript coliforms - Elsevier

Indicator Microorganisms – Chapter 23
Objectives
• Be able to define what an indicator microorganism is and why they
are used
• Be able to list the criteria for an ideal indicator organism
• Be able to list at least three different types of indicators
• Be able to describe standard detection methods used to measure
indicators
• Understand the difference between Water Quality Standards and
Water Quality Guidelines
What Is an Indicator Microorganism?
•
•
A nonpathogenic microorganism whose presence suggests the
presence of enteric pathogens
Indicator organisms are used because pathogens themselves are
frequently difficult to detect in drinking water and wastewater
low numbers (but still above MID)
difficult, time consuming, or expensive to culture
•
U.S. Public Health Services adopted coliforms as indicators of
fecal contamination of drinking water in 1914



•
coliforms are bacteria that live in the intestines of warmblooded animals and are excreted in high numbers in feces
indicate fecal contamination of drinking water
presence demonstrates a breakdown of wastewater
treatment processes
The food industry uses other indicator microorganisms to
evaluate the efficiency of food processing
Criteria for an Ideal Indicator Organism
• Should be useful for all types of water (drinking water, wastewater,
recreational water, sea water)
• Should be present whenever enteric pathogens are present, and
absent when pathogens are absent
• Should survive longer in the environment than the toughest enteric
pathogen
• Should not grow in water
• Detection protocols should be easy and inexpensive
• Density of indicator microorganisms should correlate with the
degree of fecal pollution
• Should be a member of the normal intestinal microflora of warmblooded animals
Types of Indicators
• Total coliforms
• Fecal coliforms
• Fecal Streptococci
• Anaerobic bacteria
• Bacteriophage
Estimated levels of indicator organisms in raw sewage
Organism
CFU/100ml
Coliforms
107 - 109
Fecal coliforms
106 - 107
Fecal streptococci
105 - 106
Entercocci
104 - 105
Clostridium perfringens
104
Staphylococcus
103
Pseudomonas aeruginosa
105
Acid-fast bacteria
102
Coliphages
102 - 103
Bacteroides
107 - 1010
.
Total coliforms
Most commonly used indicator for: drinking water, wastewater treatment,
shellfish harvesting water, and recreational water
• Aerobic or facultatively anaerobic
• Gram negative
• non-spore-forming
• rod-shaped
• gas production during lactose fermentation within 48 hours at 35°C
• Examples: Escherichia, Citrobacter, Klebsiella
and Enterobacter
• High numbers (2 x 109 per capita per day) in human AND animal feces
• < 1 coliform per 100 mL drinking water ensures the prevention of bacterial
waterborne disease outbreaks
Drawbacks
• Coliforms may grow in aquatic environments, particularly if organic
matter levels and temperatures are elevated
• Coliforms may form biofilms in drinking water distribution systems – this
is a problem because, for example, E. coli is 2400 times more resistant
to chlorine in a biofilm than when planktonic
• Coliforms may recover from disinfectant injury
• Growth of heterotrophic bacteria on media selective for coliforms can
mask coliform population in water (occurs when heterotrophic counts
exceed 500/mL)
• More vulnerable to disinfection and environmental trauma than enteric
viruses or parasites
• Do not necessarily indicate fecal contamination
Regrowth of coliforms and E. coli in sewage effluents after inactivation
with 5 mg/L chlorine
100
% Bact er ia r emaining
Coliforms
6
10
10
Die-off rate depends on
amount/type of organic
matter present and the
water temperature
can lead to
false positives
1
0.1
0.01
0.001
5
10
4
10
3
10
2
10
E. coli
1
0.0001
10
0
1
2
3
4
Time (days)
5
6
7
Colif or ms or E. coli / 100 ml
7
10
Fecal coliforms
• Subgroup of total coliforms
• Able to ferment lactose and produce both acid and gas at 44.5°C in
24 hours
• Include Escherichia and Klebsiella, which are exclusively fecal in
origin (perhaps. . .)
• Drawbacks
– same drawbacks as for total coliforms
– indicates fecal contamination for sure, but can’t distinguish
between animal and human feces
– can survive and grow for extended periods of time in tropical
waters
• may be natural inhabitants of these waters!
Fecal Streptococci
• do not multiply in water
• are more resistant to stress/disinfection
• last longer in the environment
• used as indicators of enteric viruses, and gastroenteritis for
swimmers
• Members of the lactic acid bacteria
• Gram positive, non-motile, non-spore-forming, aerotolerant
anaerobic bacteria that ferment sugars to lactic acid
• FC/FS ratio - ratio of fecal coliform counts to fecal strep counts
• FC/FS >4 : fecal contamination of human origin
• FC/FS < 0.7: fecal contamination of animal origin
•
This relationship is only valid for recent fecal contamination (within
the last 24 hours)
Anaerobic bacteria
Clostridium perfringens – one example
• Gram positive, anaerobic spore-forming rod-shaped bacterium
• Spores are heat resistant (can survive 75°C for 15 min), resist
disinfection, can remain viable in the environment for a long time
• May be used as indicator of resistant pathogens (viruses, parasites),
past fecal contamination, or tracing fecal contamination in a marine
environment
Drawbacks
• A common soil bacterium; may not necessarily indicate fecal
contamination
• Pathogenic (causes gas gangrene if it infects wounds, produces
enterotoxin in small intestine causing gastroenteritis)
• Anaerobic culture is difficult
Bacteriophage
Coliphage – one example
• bacteriophage that infect coliforms, particularly E. coli
• similar to enteric viruses in size, morphology, and performance in
environment
• found in higher numbers than enteric viruses in wastewater and
other waters
• rapid and easy detection methods available
• survive for 7 days in shellfish without increasing in numbers
• routinely used as indicator microorganisms to determine the
effectiveness of wastewater treatment processes
• resistant to disinfection
Detection Methods
Most Probable Number (MPN)
•
Used to detect coliforms
•
This test consists of two to three steps:
- gas production
1. Presumptive test
2. Confirming test
3. Completed test
+ gas production
•
Presumptive test: dilute water sample
•
Inoculate 3 or 5 tubes of lauryl sulfate-tryptose-lactose broth
containing upside-down Durham tubes with water dilutions
•
Incubate at 35°C for 48 hours
•
Determine number of tubes at each dilution that are positive for gas
production (contain bubble in Durham tube)
Sample MPN Table
2. Confirming test – select a positive tube and inoculate a Levines
EMB agar and Endo Agar plate
Levines EMB agar
+
Endo Agar
-
Coliforms produce
“nucleated” colonies
+
-
Coliforms and surrounding
medium turn red
3. Completed test – inoculate a colony back into MPN media and
confirm acid and gas production. (Not always performed)
What would you do to detect fecal coliforms instead of
coliforms ????
Drawback to MPN test: HPC can outcompete
coliforms and fecal coliforms for nutrients in the
environment and mask their detection by this method.
Membrane Filter Test
• Used to detect coliforms
• Filter 100 mL water through a 0.45 m filter
• Incubate filter on pad soaked with a differential medium (Endo
medium; contains lactose and Basic Fuchsin dye) at 35°C for 18-24
hours
• Count colonies that grow on filter
coliforms will be dark red with metallic gold sheen
• To enumerate Fecal Streptococci, grow on Streptococcus agar at
37°C for 24 hours. Fecal streptococci reduce 2,4,5triphenyltetrazolium chloride to formazan, which makes colonies
appear red
• Much quicker and easier than MPN method
Presence-Absence Tests, e.g., Colilert Test
• Qualitative NOT quantitative
• Used to detect total coliforms and E. coli
• Add packet of salts and nutrients to water sample and incubate 24
hours
• Total coliforms can convert o-nitrophenyl--D-galactopyranoside
(ONPG) to yellow nitrophenol with -galactosidase
• E. coli can metabolize 4-methylumbelliferone glucuronide (MUG)
to a molecule that fluoresces under UV light with glucuronidase
• May not detect up to 1/3 of E. coli strains (including pathogenic
ones!)
• Broth and agar plate techniques involving ONPG and MUG also
exist
MUG
ONPG
Heterotrophic Plate Counts (HPC)
• Enumeration of all aerobic and facultative anaerobic
chemoheterotrophs in water
– includes Pseudomonas, Aeromonas, Klebsiella,
Flavobacterium, Enterobacter, Citrobacter, Serratia,
Acinetobacter, Proteus, Alcaligenes, and Moraxella
• Varies from 1 to 104 CFU/mL, and depends on temperature,
residual chlorine concentration, and availability of organic
nutrients
• Indicates general quality of water (particularly levels of
organic matter in water)
• HPC > 500 CFU/mL indicates poor water quality
Plaque Assay
• Used to detect bacteriophage
• Filter phage from water with charged membrane filter
• Elute with beef extract, pH 9.0
• Flocculate solids (including phage) with HCl.
• Centrifuge. Remove supernatant and resuspend pellet in beef extract.
Neutralize solution.
• Inoculate 4 mL loose (0.7%) agar with host bacterial culture and 100
L phage concentrate.
• Pour loose agar onto a solid agar plate. Incubate for 8-18 hours
• Host bacteria will form lawn on plate. Bacteriophage will lyse small
holes in the lawn (plaques)
• Count plaques and compare to the volume of filtered water to
determine bacteriophage population in the water sample
Water Quality Standards and Guidelines
• Regulated at both the Federal and State levels
• Water quality standards are legally enforceable!!
Authority
Standards
.
U.S. E.P.A.
Safe Drinking Water Act
Clean Water Act
Wastewater discharges
Sewage sludge
0 coliforms/100ml
200 fecal coliforms/100 ml
<1000 fecal coliforms/4 g
<3 Salmonella/4 g
<1 enteric virus/4 g
<1 helmintha ova/4 g
California
Wastewater reclamation
for irrigation
<2.2 MPN coliforms
Arizona
Wastewater reclamation
for irrigation of golf
courses
25 fecal coliforms/100ml
125 enteric virus/40 L
No detectable Giardia/40 L
Water Quality Criteria and Guidelines
• Comprise recommendations for acceptable levels of indicator
microorganisms
• NOT legally enforceable!!!
Guidelines for Recreational Water Quality Standards
Country
U.S.EPA
Regime
(samples/time)
Criteria or standard
5/30 days
200 fecal coliforms/100ml
<10% to exceed 400/ml
Fresh water
33 enterocci/100 ml
126 fecal coliforms/100 ml
Marine water
35 enterococci/100 ml