An Introduction to Water

Microbiology
What we shall talk about in this section:
Introduction - Presence of microbes in water
 Microbiological contamination of water sources
 The effect of storage on the microbial load in the bottled water
Introduction – indicator/index microorganisms
 What are indicator organisms, why do we use them?
 How are they chosen?
 What do they tell us about water quality?
Review groups of indicator organisms / Analytical methods
 Heterotrophic Plate Count
 Total Coliforms
 Escherichia coli and thermotolerant Coliforms
 Fecal streptococci
 Pseudomonas aeruginosa
 Sulfite reducing anaerobes
Standards & TCCC testing requirements

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Many factors influence the number of microorganisms in water

Type of water
Age of water
Autochthon microbiota
Allochthon microbiota
Aquifer location
Sources of contamination
Surrounding bedrock
Soil constituents
Substrate/nutrient content
Oxygen content
Temperature
Mineral content
Water flow

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Microbiological contamination caused by
different groups of microbes…
Bacteria
 Indigenous bacteria (Pseudomonaceae, Acinetobacter spp)
 Indigenous saprophytic species (Enterobacter spp.)
 Human pathogenic contaminants (Salmonella spp.)
 Fecal contaminants (Escherichia spp.)

Yeasts and Molds

Parasites
Algae
Viruses

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Here are some examples of types of water in our system

• Well water

• City (municipal) water

• Treated water

• Packaged treated water (e.g., ozonated, or thermally

processed)

• Packaged untreated water (no processing permitted)

All of these must be free of pathogenic microorganisms

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Different types of water support
different organisms

Bacteria are mostly found in untreated water (for example,

natural mineral waters in Europe)

Yeasts and Molds are generally associated with treated

waters such ozonated bottled water

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Microorganisms will grow in packaged water
The characteristics of bottled water support growth of bugs:
pH:

Growth is possible !
 Still water: ± 7

Up to 104-107CFU/mL
 Carbonated water

Nutrients:
 Minerals
 Dead autolyzed indigenous microorganisms
 Metabolites of indigenous autotrophs
 Bottle components
Oxygen:
 Oxygenation during bottling process
 Diffusion

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What is the microbiological risk associated with drinking water?

World Health Organization Guidelines for Drinking Water Quality:

“The greatest risk from microbes in water is associated with

consumption of drinking water that is contaminated with human or
animal excreta [feces]”

We must ensure that all the water we use is free of harmful

pathogenic bacteria

Let’s look at how we do that….

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Which microorganisms are we concerned about?

In water, some of the main harmful or pathogenic bacteria

include:

• Vibrio cholerae

• Salmonella spp.

• Shigella spp.

• Yersinia spp.

How do we check our water for these organisms?

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Which methods do we use to test for these microorganisms?

In general, testing for pathogens is expensive, time consuming

and technically difficult….

….. so instead of testing for pathogens we test for

organisms that will provide the following
2 pieces of information….
1. Signal and suggest the presence of fecal contamination and
therefore the presence of the pathogens
2. Provide a measure of the effectiveness of control measures
against pathogens (e.g. disinfection)

The organisms that we test for are called index and indicator organisms

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Index vs. Indicator organism: what’s the difference?

Index organisms:
 Point to the presence of
pathogenic organisms. For
example they are used as an
index of fecal contamination

Indicator organisms:
 Used to measure the
effectiveness of a process. For
example, a process indicator or
disinfection indicator

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Key Microbiological Parameters for Water Quality…
We test for these organisms as indicator or index organisms to determine
that the water we are using is safe

Heterotrophic plate count / Total Count

Total Coliforms
Escherichia coli and thermotolerant
coliforms
Fecal Streptococci
Pseudomonas aeruginosa
Sulfite reducing anaerobes

Let’s look at these in more detail

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Heterotrophic Plate Count (Total Viable Count)

Indicator of the efficiency of water treatment processes, disinfection,

growth (or re-growth) of biofilms

Monitoring of trends is important v.s single results

No direct correlation to possible health risk

A typical method includes

 Using a nutritive agar such as TGE
 Incubation time 72 hrs
 Incubation temperature at 35oC

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Total Coliforms…
Includes both fecal and environmental bacteria belonging to 4 main genera:
 Citrobacter spp.
 Klebsiella spp.
 Enterobacter spp.
 Escherichia spp.
Biochemical attributes
 Facultative anaerobic, Gram-negative, non-spore forming rods.
 Members of the family Enterobacteriaceae capable of growth in the presence
of bile salts.
 Ferment lactose, produce gas within 48 h at 35-37 °C.
 The enzyme ß-galactosidase is present in 94-96 %.
 Detection by incubation of selective media at 35-37 °C.
 Occur in the bowel of humans and warm-blooded animals, soil/environment,
fresh surface water

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Total Coliform count is used as an indicator…

Generally signifies a safe supply and is used to indicate:

Poor sanitation practices

Post processing contamination

Fluctuation of the source water quality

Effectiveness of disinfection

BUT: It is not used as an index of fecal pathogens because

some Coliform organisms are not associated with fecal matter

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Escherichia coli & thermotolerant Coliforms are index organisms

Coliforms that are able to breakdown lactose at 44 oC ± 0.5 °C

within 48 hours
Predominant organisms are Escherichia coli and thermotolerant
Klebsiella spp. strains
Source and occurrence of E.coli
 Present in very high numbers in human and animal feces
 Found in sewage and waters subject to recent fecal
contamination
 Water temperature and nutrient conditions in a water distribution
system unlikely to support growth of E.coli
Indicator value
 E. coli considered the most suitable index of fecal contamination
 May also be used as an indicator of disinfection efficiency

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What methods do we use for detecting Coliforms?
Membrane filtration and incubation onto selective agar (m-Endo
agar, TTC-Tergitol agar). Routinely used in The Coca-Cola
System
Sample size for testing total Coliforms and themotolerant
Coliforms varies by regulatory body (e.g. US 100ml, EU 250ml)

 Total Coliforms incubate at 35-37oC for 24-48 hours, read

plates at 24- 48 hours depending on medium
 E.coli and thermotolerant Coliforms incubate at 44-45oC for
24-48 hours, read plates at 24- 48 hours depending on
medium

Further confirmatory microbiological testing is required to

determine whether the growth is a coliform and to determine if
the coliform is E.coli or another species

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 Here are some examples of Coliforms on plates

Tergitol Medium
Coliforms form yellow color in the
medium under the membrane

Endo agar
A typical Coliform colony has pink to dark red
color with a metallic sheen
BUT: some non Coliforms may produce colonies
with sheen
AND some Coliforms may not produce a sheen

We will look at these methods

during the lab exercises

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 Other methods for Coliform detection rely upon biochemical reactions and provide
simultaneous detection of Coliforms and E.coli. Examples include Colilert and
ReadyCult. Wwe will look at these in more detail during the lab exercises
Most Coliforms

As Coliforms grow in medium, they use ß-galactosidase to metabolize ONPG and change it from colorless to yellow.

E. Coli only

Simultaneously E. coli use ß-glucuronidase to metabolize MUG and create fluorescence.

.

Taken from Idexx colilert website

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Another group of index organisms are called Fecal Streptococci
(Intestinal Enterococci)
Includes two genera of bacteria: Enterococcus and Streptococcus.
Enterococcus spp more specific for human feces
Particular species include
 Enterococcus faecalis
 Enterococcus faecium
 Enterococcus durans
 Enterococcus hirae
 Streptococcus bovis
 Streptococcus equinus
Source and occurrence
 Excreted in human feces and other warm blooded animals .
 Present in large numbers in sewage and waters polluted by human or animal
waste
 Survive longer in water than Coliforms and more resistant to drying and
chlorination
Indicator value
 Used as an index of fecal pathogens

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How do we detect Fecal Streptococci?
Membrane filtration and incubation onto AZID containing
selective agar (m-E agar for Enterococcus)
Sample size for testing fecal Streptococci may vary by
regulatory body (EU 250 mL)
Further confirmatory microbiological testing is required to
determine the presence of Fecal Streptococci
Biochemical tests such as Enterolert can also be used

41oC for
24 hours

Taken from Idexx colilert website

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What about Pseudomonas aeruginosa?

Tested on: 250 mL – Cetrimide agar / 48 hrs / 37 °C

Highly motile (irregular colony shapes)
Produce PIGMENTS (esp. pyocyanin)
Characteristic FRUITY ODOR
Used as an indicator of sanitation

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We also use Sulfite Reducing Anaerobes as an index organism
Clostridium perfringens is the species of Clostridium that is most
often associated with feces of warm blooded animals
Clostridium perfringens produces spores that are very resistant to
 UV irradiation
 Temperature
 Disinfection (especially chlorination)
Source and occurrence
 Always present in sewage
 Does not grow in water environments
Index value
 Due to resistance to disinfection, C. perfringens may be a index
of enteric viruses and protozoa in drinking water
 Index of fecal pollution that took place previously and therefore
an intermittent contamination source

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Here is a method for detecting Sulphite Reducing Clostridia…

Sample size for testing C. perfringens may vary by regulatory

body (EU 50 mL)
Membrane filtration and incubation onto tryptose sulfite agar
selective agar / anaerobic jar
Because C. perfingens is an anaerobic organism the plates
must incubated anaerobically (i.e. without oxygen)
Methods are not as simple and inexpensive as tests for other
index/indicator organisms
No enzyme/substrate test

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What are TCCC specifications for packaged water?
TCCC uses the following guidelines for packaged water microbiological
testing (local regulations may be stricter)

Treated packaged water: International Bottled Water Model Code

Coliforms Absent in 100 ml

Untreated packaged water (natural mineral waters) : Codex standard

Coliforms Absent in 250 ml

E. coli or thermotolerant Coliforms Absent in 250 ml
Fecal streptococci Absent in 250 ml
Pseudomonas aeruginosa Absent in 250 ml
Sulphite-reducing anaerobes Absent in 50 ml

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Questions?

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