1. 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

2. 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

3. 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 warm-blooded 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

4. 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 warm-blooded animals

5. Types of Indicators Total coliforms Fecal coliforms Fecal Streptococci
Anaerobic bacteria
Bacteriophage

6. Estimated levels of indicator organisms in raw sewage
Organism CFU/100ml .
Coliforms
Fecal coliforms
Fecal streptococci
Entercocci
Clostridium perfringens
Staphylococcus
Pseudomonas aeruginosa
Acid-fast bacteria
Coliphages
Bacteroides

7. 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

8. 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

9. 100 10 l m Coliforms 10 10 g 1 n / i n i i l a o 1 10 m c e . r E a
Regrowth of coliforms and E. coli in sewage effluents after inactivation with 5 mg/L chlorine
100 10 7 l m
Coliforms 10 10 6 g 1 n / i n
Die-off rate depends on
amount/type of organic
matter present and the
water temperature
can lead to
false positives i i l a o 1 10 5 m c e . r E a
0.1 10 4 r i o r e s t c m
0.01 10 3 a r B o f i % l
0.001 o 10 2
E. coli C
0.0001 10 1
Time (days)

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!

11. 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)

12. 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

13. 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

14. Most Probable Number (MPN)
Detection Methods
gas production
+ gas production
Most Probable Number (MPN)
Used to detect coliforms
This test consists of two to three steps:
Presumptive test
Confirming test
Completed test
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)

15. Sample MPN Table

16. Confirming test – select a positive tube and inoculate a Levines EMB agar and Endo Agar plate
Completed test – inoculate a colony back into MPN media and confirm acid and gas production. (Not always performed)
Levines EMB agar
Endo Agar + - + -
Coliforms produce
“nucleated” colonies
Coliforms and surrounding
medium turn red

17. 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.

18. 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 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 2,4,5-triphenyltetrazolium chloride to formazan, which makes colonies appear red
Much quicker and easier than MPN method

20. 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

21. ONPG
MUG

22. 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

23. 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

24. Water Quality Standards and Guidelines
Regulated at both the Federal and State levels
Water quality standards are legally enforceable!!

25. Authority Standards . U.S. E.P.A. California
Safe Drinking Water Act coliforms/100ml
Clean Water Act
Wastewater discharges fecal coliforms/100 ml
Sewage sludge <1000 fecal coliforms/4 g
<3 Salmonella/4 g
<1 enteric virus/4 g
<1 helmintha ova/4 g
California
Wastewater reclamation <2.2 MPN coliforms
for irrigation
Arizona
Wastewater reclamation fecal coliforms/100ml
for irrigation of golf enteric virus/40 L
courses No detectable Giardia/40 L

26. Guidelines for Recreational Water Quality Standards
Water Quality Criteria and Guidelines
Comprise recommendations for acceptable levels of indicator microorganisms
NOT legally enforceable!!!
Guidelines for Recreational Water Quality Standards
Regime
Country (samples/time) Criteria or standard
U.S.EPA /30 days 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