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.