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 35C
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

10

% Bact er ia r emaining

100
Coliforms

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

10

10

10

10

E. coli

0.0001

10
0

3
4
Time (days)

Colif or ms or E. coli / 100 ml

Fecal coliforms
Subgroup of total coliforms
Able to ferment lactose and produce both acid and gas at 44.5C 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 cant 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 75C 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 35C 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 35C 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

37C 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

Regime
(samples/time)

U.S.EPA

5/30 days

Criteria or standard

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