ISO/TC 147/SC 4 - Microbiological methods

This document specifies a method for the detection, semi-quantitative and quantitative (MPN) enumeration of thermotolerant Campylobacter species. The method can be applied to all kinds of waters including: drinking water, ground water and well water, fresh, brackish and saline surface water, swimming pools, spa and hydrotherapy pools, recreational waters, agricultural waters and runoff, untreated and treated wastewater and also sand and other sediments. This method can be used for the detection of Campylobacter species in a specified sample volume. Clean water samples with low turbidity can be membrane filtered for either a qualitative method, semi-quantitative or quantitative (MPN) method. Water samples with higher turbidity, such as primary and secondary wastewater effluents and sediments, are analysed using the same qualitative, semi-quantitative or quantitative MPN method by direct inoculation of material into bottles or tubes. Sediments can be suspended in a suitable diluent or inoculated directly into enrichment broths. Users wishing to employ this method are expected to verify its performance for the particular matrix under their own laboratory conditions.

This document specifies a method for the detection and quantification of Legionella spp. and L. pneumophila using a quantitative polymerase chain reaction (qPCR). It specifies general methodological requirements, performance evaluation requirements, and quality control requirements. Technical details specified in this document are given for information only. Any other technical solutions complying with the performance requirements are suitable. NOTE 1 For performance requirements, see Clause 9. This document is intended to be applied in the bacteriological investigation of all types of water (hot or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or accompanying flora interfere with the determination. This interference can result in an adverse effect on both the detection limit and the quantification limit. NOTE 2 For validation requirements, see 9.7. The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per litre of sample. The method described in this document is applicable to all types of water. However, some additives, such as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method. The qPCR methods do not give any information about the physiological state of the Legionella.

This document specifies requirements and gives guidance for performing the manipulations common to each culture technique for the microbiological examination of water, particularly the preparation of samples, culture media, and general apparatus and glassware, unless otherwise required in the specific standard. It also describes the various techniques available for detection and enumeration by culture and the criteria for determining which technique is appropriate. This document is mainly intended for examinations for bacteria, yeasts and moulds, but some aspects are also applicable to bacteriophages, viruses and parasites. It excludes techniques not based on culturing microorganisms, such as polymerase chain reaction (PCR) methods.

This document specifies a method for the enumeration of Pseudomonas aeruginosa in water. The method is based on the growth of target organisms in a liquid medium and calculation of the most probable number (MPN) of organisms by reference to MPN tables. This document is applicable to a range of types of water. For example, hospital waters, drinking water and non‑carbonated bottled waters intended for human consumption, groundwater, swimming pool and spa pool waters including those containing high background counts of heterotrophic bacteria. This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower waters or marine waters, for which the method has not been validated. These waters are, therefore, outside the scope of this document. Laboratories can employ the method presented in this document for these matrices by undertaking appropriate validation of performance of this method prior to use. The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa through the hydrolysis of a 7‑amino‑4‑methylcoumarin aminopeptidase substrate present in a special reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the 7‑amido‑coumarin aminopeptidase substrate releasing a product which fluoresces under ultraviolet (UV) light. The test described in this document provides a confirmed result within 24 h with no requirement for further confirmation of positive wells.

ISO 13843:2017 deals with characterization of microbiological methods. In terms of ISO 13843:2017, characterization means the study of parameters that can be measured to describe how the method is likely to perform in a given set of conditions, which can be described as performance characteristics. ISO 13843:2017 describes procedures for the determination of performance characteristics which can be used for subsequent validation or verification of methods. The emphasis is on selective quantitative methods and ISO 13843:2017 applies to all types of water. For methods that are not based upon direct microscopic count, colony count or most probable number, the applicability of the procedures described in ISO 13843:2017 should be considered carefully.

ISO 11731:2017 specifies culture methods for the isolation of Legionella and estimation of their numbers in water samples. These methods are applicable to all kinds of water samples including potable, industrial, waste and natural waters. These methods can be used for water related matrices, e.g. biofilms, sediments, etc. Not all Legionella species are culturable; therefore, the methods described in this document do not recover all species of Legionella.

ISO 9308-1:2014 specifies a method for the enumeration of Escherichia coli (E. coli) and coliform bacteria. The method is based on membrane filtration, subsequent culture on a chromogenic coliform agar medium, and calculation of the number of target organisms in the sample. Due to the low selectivity of the differential agar medium, background growth can interfere with the reliable enumeration of E. coli and coliform bacteria, for example, in surface waters or shallow well waters. This method is not suitable for these types of water. ISO 9308-1:2014 is especially suitable for waters with low bacterial numbers that will cause less than 100 total colonies on chromogenic coliform agar (CCA). These may be drinking water, disinfected pool water, or finished water from drinking water treatment plants. Some strains of E. coli which are β-D-glucuronidase negative, such as Escherichia coli O157, will not be detected as E. coli. As they are β-D-galactosidase positive, they will appear as coliform bacteria on this chromogenic agar.

ISO 17994:2014 specifies an evaluation procedure for comparing two methods with established performance characteristics according to ISO/TR 13843 and intended for the quantification of the same target group or species of microorganisms. It provides the mathematical basis for the evaluation of the average relative performance of two quantitative methods against chosen criteria for the comparison. It does not provide data for assessment of the precision of the methods being compared. It is appropriate that the precision of methods is assessed as part of their performance characterization. ISO 17994:2014 does not provide methods for the verification of method performance characterization in a single laboratory.

ISO 14189:2013 specifies a method for the enumeration of vegetative cells and spores of Clostridium perfringens by the membrane filtration method in samples of water intended for human consumption. However, the method can be applied to all types of water samples provided they do not contain particulate or colloidal matter that interferes with filtration.

ISO 9308-2:2012 specifies a method for the enumeration of E. coli and coliform bacteria in water. The method is based on the growth of target organisms in a liquid medium and calculation of the "Most Probable Number" (MPN) of organisms by reference to MPN tables. This method can be applied to all types of water, including those containing an appreciable amount of suspended matter and high background counts of heterotrophic bacteria. However it must not be used for the enumeration of coliform bacteria in marine water. When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically required, although the method has been shown to work well with some marine waters that have a lower than normal concentration of salts. In the absence of data to support the use of the method without dilution, a 1→10 dilution is used. This method relies upon the detection of E. coli based upon expression of the enzyme b‑D‑glucuronidase and consequently does not detect many of the enterohaemorhagic strains of E. coli, which do not typically express this enzyme. Additionally, there are a small number of other E. coli strains that do not express b‑D‑glucuronidase. The choice of tests used in the detection and confirmation of the coliform group of bacteria, including E. coli, can be regarded as part of a continuous sequence. The extent of confirmation with a particular sample depends partly on the nature of the water and partly on the reasons for the examination. The test described in ISO 9308-2:2012 provides a confirmed result with no requirement for further confirmation of positive wells.

This International Standard gives guidelines for the evaluation of uncertainty in quantitative microbiological analyses based on enumeration of microbial particles by culture. It covers all variants of colony count methods and most probable number estimates. Two approaches, the component (also known as bottom‑up or step‑by‑step) and a modified global (top‑down) approach are included. The aim is to specify how values of intralaboratory operational variability and combined uncertainty for final test results can be obtained. The procedures are not applicable to methods other than enumeration methods. NOTE 1 Most annexes are normative. However, only the annexes relevant to each case are to be applied. If the choice is the global approach, then all normative annexes that belong to the component approach can be skipped and vice versa. NOTE 2 Pre-analytical sampling variance at the source is outside the scope of this International Standard, but needs to be addressed in sampling designs and monitoring programmes. NOTE 3 The doubt or uncertainty of decisions based on the use of analytical results whose uncertainty has been estimated is outside the scope of this International Standard. NOTE 4 The extra‑analytical variations observed in proficiency tests and intercalibration schemes are also not detailed in this International Standard, but it is necessary to take them into consideration in analytical control. The use of intercalibration data in uncertainty estimation offers the possibility for the bias between laboratories to be included (Nordtest Report TR 537[12]).

ISO 19250:2010 specifies a method for the detection of Salmonella spp. (presumptive or confirmed) in water samples. It is possible that, for epidemiological purposes or during outbreak investigations, other media are also required.

ISO 15553:2006 specifies a method that is applicable for the detection and enumeration of Cryptosporidium oocysts and Giardia cysts in water. It is applicable for the examination of surface and ground waters, treated waters, mineral waters, swimming pool and recreational waters. This method does not allow identification to species level, the host species of origin or the determination of viability or infectivity of any Cryptosporidium oocyst or Giardia cyst which may be present. These procedures are for use by experienced analysts who have successfully completed competency tests prior to commencing analysis.

ISO 19458:2006 provides guidance on planning water sampling regimes, on sampling procedures for microbiological analysis and on transport, handling and storage of samples until analysis begins. ISO 19458:2006 focuses on sampling for microbiological investigations.

ISO 16266:2006 specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in samples of bottled water by a membrane filtration technique. This method can also be applied to other types of water with a low background flora, for example, pool waters and waters intended for human consumption.

ISO 10705-3:2003 specifies the general principles for assessing the performance of methods for the concentration of bacteriophages from water. Concentration is recommended for those water samples expected to contain ISO 10705-3:2003 does not give specific details of concentration methods, but outlines the fundamental principles for evaluating the suitability of a particular method for a given type and volume of water. Annex A gives examples of methods that have been found satisfactory and their fields of application.

This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water, sediments and sludge extracts, where necessary after dilution. In the case of low phage numbers, a preconcentration step may be necessary for which a separate International Standard has been developed. The method is also applicable to shellfish extracts. NOTE It is desirable for International Standards to be adopted as widely as possible. This part of ISO 10705 includes reference to alternative procedures which obviate the need for expensive materials or equipment which may not be readily available in developing countries. Use of these alternatives will not affect the performance of this method.

Specifies a method for the detection and enumeration of F-specific ribonucleic acid bacteriophages. The sample is incubated with an appropriate host strain. Applicable to all kinds of water, sediments and sludges, even for shellfish extracts. The presence of F-specific RNA bacteriophages in a water sample indicates pollution by wastewater contaminated by human or animal faeces.

The procedure is applicable to all types of water, including turbid water. The principle covers several steps from selection by applying heat to destroy vegetative bacteria to the indication by inoculating volumes of the sample into media followed by incubation at 27 ± °C in anaerobic conditions.

The procedure is applicable to all types of water, except when a large amount of particulate material is liable to be retained by the membrane. The principle covers several steps from selection by applying heat to destroy vegetative bacteria to filtration of the water sample through a membrane filter having a suitable pore size (0,2 µm) to retain the spores in or on it. The filter is placed on a selective culture medium, followed by incubation and counting of black colonies.

Gives a procedure for the evaluation and comparison of water-testing filters intended for the enumeration of specific organisms and mixed microbial populations. The procedure provides general guidelines for comparative testing of the recoveries of bacteria, yeasts and other fungi on membrane filters, as compared to recoveries by the spread plate and pour plate techniques.

ISO/TS 12869:2012 specifies a method for the detection and quantification of Legionella spp. and L. pneumophila using a quantitative polymerase chain reaction (qPCR). It specifies general methodological requirements, performance evaluation requirements, and quality control requirements. Technical details specified in ISO/TS 12869:2012 are given for information only. Any other technical solutions complying with the performance requirements are suitable. ISO/TS 12869:2012 is intended to be applied in the bacteriological investigation of all types of water (both hot and cold), unless the nature and/or content of suspended matter and/or accompanying flora interfere with the determination. This interference can result in an adverse effect on both the detection limit and the quantification limit. The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per litre of sample. The method described in ISO/TS 12869:2012 is applicable to all types of water. However, some additives, e.g. chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method. The qPCR methods do not give any information about live or dead cells.

ISO 17995:2005 specifies a method for the detection and semiquantitative enumeration of thermotolerant Campylobacter species. The method can be applied to all kinds of filterable waters. The method can also be used as a presence/absence test for Campylobacter species in a specified sample volume.

ISO 8199:2005 presents guidance for carrying out manipulations which are common to each technique for the microbiological examination of water, particularly the preparation of samples, culture media and apparatus. It also describes the various enumeration techniques available and the criteria for the choice of a particular technique. It is mainly intended for bacteria, yeasts and moulds. Some aspects are also applicable to viruses and parasites.

ISO 17994:2004 defines an evaluation procedure for comparing two methods intended for the detection or quantification of the same target group or species of microorganisms. ISO 17994:2004 provides the mathematical basis for the evaluation of the average relative performance of two methods against chosen criteria of equivalence. Any two enumeration methods based on counts (of colonies or positive tubes) or any two detection methods (presence/absence methods) intended for the same purpose can be compared. ISO 17994:2004 provides no solution to directly compare a quantitative method (colony count or MPN) with a detection method (P/A).

ISO 11731-2:2004 describes a monitoring method for the isolation and enumeration of Legionella organisms in water intended for human use (e.g. hot and cold water, water used for washing), for human consumption and for treated bathing waters (e.g. swimming pools). It is especially suitable for waters with prospected low numbers of Legionella. As the growth of Legionella may be inhibited by overgrowth of other bacterial colonies on the membrane the method is only suitable for waters containing low bacterial.

Specifies a method for the detection of Salmonella in water samples for monitoring purposes. Applicable to all kinds of water, except raw sewage.

Describes a method used for the comparison and evaluation of the same medium prepared from different lots of materials, and deals with the comparison and evaluation of different media which are used for the same purpose. Annexes A and B form an integral part of this Standard.

Presents guidance for carrying out manipulations which are common to each technique for the microbiological examination of water, particularly the preparation of samples, culture media and apparatur. It also describes the various enumeration methods available and the criteria for the choice of a particular technique.