Addressing challenges associated with the detection of faecal coliform organisms in water matrices. Neil Leat Rand Water Date 30/09/2014

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1 Addressing challenges associated with the detection of faecal coliform organisms in water matrices Neil Leat Rand Water Date 30/09/2014

2 What are coliforms? Definitions of coliforms are based on biochemical characteristics rather than phylogenetic affiliations.

3 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 C. Produce aldehyde during lactose fermentation. Express β-d-galactosidase.

4 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 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).

5 What are thermotolerant (faecal) coliforms? Thermotolerant (faecal) coliforms are bacteria which exhibit typical coliforms biochemical characteristics when incubated at elevated temperatures (43.5 to 45 C).

6 Example of a definition

7 Schematic representation of the relationship between coliform groups. Total coliforms

8 Schematic representation of the relationship between coliform groups. Total coliforms Faecal coliforms

9 Schematic representation of the relationship between coliform groups. Total coliforms Faecal coliforms K.pneumoniae E.coli

10 How are thermotolerant (faecal) coliforms detected Standard Methods for the Examination of Water and Wastewater (APHA et al., 1998) 9221E Multiple Tube Fermentation. 9222D Membrane filter procedure 9223B Enzyme Substrate Test

11 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.

12 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.

13 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.

14 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.

15 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.

16 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.

17 9223B Enzyme Substrate Test Biochemical characteristic tested: Activity of the enzyme β-d-galactosidase cleaves a defined chromogenic substrate, resulting in the release of a chromogen. β-d-galactosidase enzyme Example: IDEXX Colilert medium contains ortho-nitrophenyl-β-d-galactopyranoside (ONPG)

18 Day 2 Day 1 Overview of the use of Colilert-18 medium with Quanti-Trays 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

19 What could lead to differences in results between laboratories. Differences in faecal coliform definitions. Differences in fundamental methodology. Differences in media and consumables. Differences in incubation conditions.

20 A lack of consistency with respect to prescribed incubation conditions Recommended incubation conditions 44.3 to 44.7 C Reference Standard Methods for the Examination of Water and Wastewater (APHA et al., 1998) 44 to 45 C to C or to C Effectively to to 44.5 C World Health Organization (2011) Guidelines for drinking-water quality 4 th edition (page 296) ISO :1990 Water quality-detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli Part 2: Multiple tube (most probable number) method. AFNOR (Association Francaise de Normalisation), Eauxmethodes d essais. Recueil de Normes Francaises, 4th edn. La Defense, Paris, 735 pp.

21 Challenges associated with thermometer calibration

22 Challenges associated with thermometer calibration

23 Maintaining a uniform thermal incubation environment Standard Methods for the Examination of Water and Wastewater (Part c; APHA et al., 1998) The specificity of the faecal coilform test is related directly to the incubation temperature.

24 Maintaining a uniform thermal incubation environment Standard Methods for the Examination of Water and Wastewater (Part c; 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.

25 Maintaining a uniform thermal incubation environment Standard Methods for the Examination of Water and Wastewater (Part c; 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.

26 Maintaining a uniform thermal incubation environment Standard Methods for the Examination of Water and Wastewater (Part c; 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.

27 Discussion points for laboratories participating in the NLA water microbiology scheme Differences in faecal coliform definitions. Differences in fundamental methodology. Differences in media and consumables. Differences in incubation conditions.