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AWQC Demystifying Microbial Detection Methods

Coliforms are bacteria that can be found in plant matter, soil and water. Thermotolerant (Faecal) Coliforms, including Escherichia coli (E. coli), are a group of coliforms that commonly inhabit the intestinal tracts of warm-blooded animals. Their presence in water is an indication of faecal pollution and the possible presence of enteric pathogens. There is a direct relationship between the densities of thermotolerant coliforms (including E. coli) in recreational water and the risk of gastrointestinal illness associated with swimming in that water.

Available Methods to Detect Coliforms at AWQC

Membrane Filtration (MF): This method, based on AS/NZS 4276.5:2007 and AS/NZS 4276.7:2007 standards, was developed to isolate and enumerate total coliforms, thermotolerant coliforms and E. coli in water. The recommended sample types are: potable, recreational, environmental, industrial, trade wastes, sewage and marine waters. The reporting unit is Colony Forming Unit (CFU)/100ml, the limit of detection is 1 CFU/100ml, and the test will take 18-20 hours to complete. The coliform colonies are counted via both manual and automated processes (colony counter). In 2012 the AWQC introduced a new NATA accredited test, which uses the membrane filtration technique and chromogenic agar (MI Agar) for the isolation and enumeration of coliforms. This test is recognised by health departments as an accepted alternative to Defined Substrate Technology (DST) using Colilert®18 for evaluating levels of E. coli and coliforms whilst maintaining the same short turnaround times. MI Agar also delivers a cost saving over the traditional DST.

Membrane Filtration of Thermotolerant Coliforms on Differential Faecal Coliform Agar         Membrane Filtration of Thermotolerant Coliforms (white) and E. coli (blue) on MI Agar

Multiple Tube Dilution technique (most probable number – MPN): This method, based on the AS/NZS 4276.6:2007 standard, was developed to estimate the most probable number (MPN) of total coliforms, thermotolerant coliforms, and E. coli in water or soil by the multiple tube dilution technique. The recommended sample types are: potable, recreational, environmental, industrial, trade wastes and wastewater. The reporting unit is MPN/100ml, the limit of detection is 1 MPN/100ml, and the test will take 96 hours to complete. The MPN method makes use of a statistical technique for estimating the most probable number of a bacterial species per specified unit of material under test. The results from a series can then be referred to McCrady's Tables from which the MPN of bacteria per unit sample can be calculated with a known degree of certainty. This method is predominantly used for the detection of coliforms in soils and biosolids.

Defined Substrate Technology (DST): This MPN-based method, based on the AS/NZS 4276.21:2005 standard, was developed for the simultaneous detection and confirmation of total coliforms and E. coli in water by the DST technique using Colilert®18. The recommended sample types are: recreational, environmental, industrial, trade wastes and wastewater. Water other than fresh water (estuarine, saline or brackish water) may contain non-target microorganisms (e.g. Aeromonas and Vibrionaecea) that may produce false positive results, consequently use of this method with these matrices is not recommended. The reporting unit is MPN/100ml, the limit of detection is 1 MPN/100ml, and the test will take 18 hours to complete. The MPN method makes use of a statistical technique for estimating the most probable number of a bacterial species per specified unit of material under test. The results from a series can then be referred to McCrady's Tables from which the MPN of bacteria per unit sample can be calculated with a known degree of certainty.

Defined Substrate Technology – yellow squares indicate Thermotolerant Coliforms         Interpreting DST results

Understanding the Reporting Units

A result from the membrane filtration technique is reported as colony forming units per 100mL (CFU/100mL). A colony of bacteria refers to a mass of individual cell(s) of the same organism, growing together.

A result from the multiple tube dilution and defined substrate technology technique is reported as the most probable number per 100 mL (MPN/100mL).

It is important to note both techniques estimate the number of viable microorganisms in the product in which the microorganisms are randomly distributed and that 1 CFU is equivalent to 1 MPN  (NOTE: uncertainty is calculated for every analysis and reported upon request as per NATA guidelines). Automated colony counter2

Do regulators treat the units differently?

The Australian Drinking Water Guidelines highlight that for total coliforms, thermotolerant coliforms and E. coli both membrane filtration and multiple tube dilution techniques (AS/NZS 4276.5. 2007 and AS/NZS 4276.6.2007) can be used for detection of the organisms in water.

The Environmental Protection Authority requirements generally vary between each state and territory but essentially 1 CFU = 1 MPN. (NOTE: all AWQC analyses undergo validation submission for NATA approval for testing). For further clarification on the most appropriate reporting unit for your requirements, contact AWQC directly or your EPA.

Which Test Should I Use?

All methods detect culturable cells, however, the multiple tube dilution and defined substrate technology methods are more useful when enumerating microbes in matrices where particulates make membrane filtration impractical. While the membrane filtration methods using MI agar is the preferred method for waters other than fresh due to the potential risk of DST delivering false positives.

Matrix:

Recreational Water – Membrane Filtration is recommended if the water is saline and DST is recommended if the turbidity of your water is high.

Saline Water – Membrane filtration is the preferred method.

Potable Water – Membrane filtration and DST are both suitable methods though membrane filtration using MI agar is the most cost effective approach.

Wastewater/Industrial – Membrane Filtration is recommended if the water is saline and DST is recommended if the turbidity of your water is high.

Biosolids and Soils – the most suitable method is Multiple Tube Dilution.

NOTE: membrane filtration using MI agar is the most cost effective approach.