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

Indicator Microorganisms – Chapter 23

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 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

• 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

Criteria for an Ideal Indicator Organism

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

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

Total coliforms

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

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Coliforms

E. coli

Regrowth of coliforms and E. coli in sewage effluents after inactivation with 5 mg/L chlorine

Die-off rate depends on amount/type of organicmatter present and the water temperature can lead to false positives

• 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 coliforms

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)

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

Anaerobic bacteria

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:

1. Presumptive test

2. Confirming test

3. 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)

- gas production

+ gas production

Sample MPN Table

2. Confirming test – select a positive tube and inoculate a Levines EMB agar and Endo Agar plate

3. Completed test – inoculate a colony back into MPN media and confirm acid and gas production. (Not always performed)

Coliforms produce “nucleated” colonies

+ -Coliforms and surroundingmedium turn red

+ -

Levines EMB agar Endo Agar

Drawback to MPN test: HPC can outcompete

coliforms and fecal coliforms for nutrients in the

environment and mask their detection by this method.

What would you do to detect fecal coliforms instead of

coliforms ????

• 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 2,4,5-

triphenyltetrazolium chloride to formazan, which makes colonies

appear red

• Much quicker and easier than MPN method

Membrane Filter Test

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

MUGONPG

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

• Regulated at both the Federal and State levels

• Water quality standards are legally enforceable!!

Water Quality Standards and Guidelines

Authority Standards .

U.S. E.P.A. Safe Drinking Water Act 0 coliforms/100ml Clean Water Act Wastewater discharges 200 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 25 fecal coliforms/100ml for irrigation of golf 125 enteric virus/40 L courses 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

RegimeCountry (samples/time) Criteria or standard

U.S.EPA 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