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Neil Leat
Rand Water
Date 30/09/2014
Addressing challenges associated with the detection
of faecal coliform organisms in water matrices
Definitions of coliforms are based on biochemical characteristics rather
than phylogenetic affiliations.
What are coliforms?
Definitions of coliforms are based on biochemical characteristics rather
than phylogenetic affiliations.
Typical biochemical characteristics that define coliforms.
• Aerobic and facultative anaerobic.
• Gram negative.
• Non-spore-forming.
• Rod-shaped bacteria.
• Oxidase negative.
• Grow in the presence of bile salts or other surface-active agents.
• Ferment lactose with gas and acid formation within 48 h at 35-37°C.
• Produce aldehyde during lactose fermentation.
• Express β-D-galactosidase.
What are coliforms?
Definitions of coliforms are based on biochemical characteristics rather
than phylogenetic affiliations.
Typical biochemical characteristics that define coliforms.
• Aerobic and facultative anaerobic.
• Gram negative.
• Non-spore-forming.
• Rod-shaped bacteria.
• Oxidase negative.
• Grow in the presence of bile salts or other surface-active agents.
• Ferment lactose with gas and acid formation within 48 h at 35-37°C.
• Produce aldehyde during lactose fermentation.
• Express β-D-galactosidase.
The coliform group typically includes genera from the Enterobacteriaceae
family (eg Citrobacter, Klebsiella, Escherichia, Enterobacter).
What are coliforms?
Thermotolerant (faecal) coliforms are bacteria which exhibit
typical coliforms biochemical characteristics when incubated at
elevated temperatures (43.5 to 45°C).
What are thermotolerant (faecal) coliforms?
Example of a definition
Schematic representation of the relationship between
coliform groups.
Total coliforms
Schematic representation of the relationship between
coliform groups.
Total coliforms
Faecal coliforms
Schematic representation of the relationship between
coliform groups.
Total coliforms
Faecal coliforms
E.coli K.pneumoniae
Standard Methods for the Examination of Water and Wastewater
(APHA et al., 1998)
How are thermotolerant (faecal) coliforms detected
9221E
Multiple Tube
Fermentation.
9222D
Membrane filter
procedure
9223B
Enzyme
Substrate Test
Biochemical characteristic tested: Fermentation of lactose in a liquid medium
containing bile salts at 44.5 ± 0.2°C with the
production of acid and gas.
9221E Multiple Tube Fermentation
Biochemical characteristic tested: Fermentation of lactose in a liquid medium
containing bile salts at 44.5 ± 0.2°C with the
production of acid and gas.
Stage 1: Conduct presumptive coliform test as follows (9221B) • Inoculate water samples into lauryl tryptose broth.
• Incubate at 35 ± 0.5°C for 24 hours.
• Examine for growth, gas and acid (if not observed incubate for a total of 48 hours).
• Production of an acidic reaction or gas within 48 hours constitutes a positive
presumptive reaction for total coliforms.
9221E Multiple Tube Fermentation
Biochemical characteristic tested: Fermentation of lactose in a liquid medium
containing bile salts at 44.5 ± 0.2°C with the
production of acid and gas.
Stage 1: Conduct presumptive coliform test as follows (9221B) • Inoculate water samples into lauryl tryptose broth.
• Incubate at 35 ± 0.5°C for 24 hours.
• Examine for growth, gas and acid (if not observed incubate for a total of 48 hours).
• Production of an acidic reaction or gas within 48 hours constitutes a positive
presumptive reaction for total coliforms.
Stage 2: Faecal coliform procedure (9221E) • Transfer growth from presumptive tubes to tubes containing EC medium.
• Incubate in a water bath at 44.5 ± 0.2°C for 24 ± 2hours.
• Examine for growth and gas production.
• Growth in EC broth with gas production within 24 ± 2 hours is considered a positive
faecal coliform reaction.
9221E Multiple Tube Fermentation
Biochemical characteristic tested: Production of acid on a lactose containing
medium at 44.5 ± 0.2°C in the presence
of bile salts.
9222D Membrane Filter Procedure
Biochemical characteristic tested: Production of acid on a lactose containing
medium at 44.5 ± 0.2°C in the presence
of bile salts.
The method uses an enriched lactose medium containing bile salts and aniline
blue. Fermentation of lactose and production of acid is indicated by a colour
change of the aniline blue dye.
9222D Membrane Filter Procedure
Biochemical characteristic tested: Production of acid on a lactose containing
medium at 44.5 ± 0.2°C in the presence
of bile salts.
The method uses an enriched lactose medium containing bile salts and aniline
blue. Fermentation of lactose and production of acid is indicated by a colour
change of the aniline blue dye.
Procedure:
• Filter a suitable volume/dilution of sample onto filters with a 0.45µm pore size.
• Place filters on mFC medium and incubate for 24 ± 2 hours at 44.5 ± 0.2°C.
• Colonies produced by faecal coliform bacteria on m-FC medium are various shades
of blue. Non Faecal coliform colonies are grey to cream coloured.
9222D Membrane Filter Procedure
Biochemical characteristic tested: Activity of the enzyme β-D-galactosidase cleaves a
defined chromogenic substrate, resulting in the
release of a chromogen.
9223B Enzyme Substrate Test
β-D-galactosidase
enzyme
Example: IDEXX Colilert medium contains ortho-nitrophenyl-β-D-galactopyranoside
(ONPG)
Step 1. Add dehydrated Colilert medium to
sample.
Step 2. Pour into Quanti-Tray
Step 3. Seal and incubate at 44.5 ± 2°C for 18
to 22 hours.
Step 4. Read results:
Yellow wells = Faecal Coliforms
Day
1
Day
2
Overview of the use of Colilert-18 medium with Quanti-Trays
Differences in faecal coliform definitions.
What could lead to differences in results between
laboratories.
Differences in fundamental methodology.
Differences in media and consumables.
Differences in incubation conditions.
A lack of consistency with respect to prescribed
incubation conditions
Recommended incubation
conditions
Reference
44.3 to 44.7°C
Standard Methods for the Examination of Water and Wastewater
(APHA et al., 1998)
44 to 45°C
World Health Organization (2011) Guidelines for drinking-water
quality 4th edition (page 296)
43.75 to 44.25°C
or
44.25 to 44.75°C
Effectively 43.75 to 44.75
ISO 9308-2:1990 Water quality-Detection and enumeration of coliform
organisms, thermotolerant coliform organisms and presumptive
Escherichia coli Part 2: Multiple tube (most probable number) method.
43.5 to 44.5°C
AFNOR (Association Francaise de Normalisation), 1990.
Eauxmethodes d’essais. Recueil de Normes Francaises, 4th edn. La
Defense, Paris, 735 pp.
Challenges associated with thermometer calibration
Challenges associated with thermometer calibration
Standard Methods for the Examination of Water and Wastewater (Part 9222 1c; APHA
et al., 1998)
• The specificity of the faecal coilform test is related directly to the incubation
temperature.
Maintaining a uniform thermal incubation environment
Standard Methods for the Examination of Water and Wastewater (Part 9222 1c; APHA
et al., 1998)
• The specificity of the faecal coilform test is related directly to the incubation
temperature.
• Static air incubation may be a problem in some types of incubator because of
potential heat layering within the chamber, slower heat transfer from air to the
medium and the slow recovery of temperature each time the incubator is opened
during daily operations.
Maintaining a uniform thermal incubation environment
Standard Methods for the Examination of Water and Wastewater (Part 9222 1c; APHA
et al., 1998)
• The specificity of the faecal coilform test is related directly to the incubation
temperature.
• Static air incubation may be a problem in some types of incubator because of
potential heat layering within the chamber, slower heat transfer from air to the
medium and the slow recovery of temperature each time the incubator is opened
during daily operations.
• To meet the need for greater temperature control use a water bath, a heat-sink
incubator or properly designed and constructed incubator shown to give equivalent
results.
Maintaining a uniform thermal incubation environment
Standard Methods for the Examination of Water and Wastewater (Part 9222 1c; APHA
et al., 1998)
• The specificity of the faecal coilform test is related directly to the incubation
temperature.
• Static air incubation may be a problem in some types of incubator because of
potential heat layering within the chamber, slower heat transfer from air to the
medium and the slow recovery of temperature each time the incubator is opened
during daily operations.
• To meet the need for greater temperature control use a water bath, a heat-sink
incubator or properly designed and constructed incubator shown to give equivalent
results.
• A temperature tolerance of 44.5 ± 0.2°C can be obtained with most types of water
baths that are also are equipped with a gable top for the reduction of water and heat
losses.
Maintaining a uniform thermal incubation environment
Differences in faecal coliform definitions.
Discussion points for laboratories participating in
the NLA water microbiology scheme
Differences in fundamental methodology.
Differences in media and consumables.
Differences in incubation conditions.