Page 18: Guidelines for Canadian Recreational Water Quality – Third Edition

Appendix B: Microbiological sampling and analysis

B.1 Sampling procedures for water

Samples for microbiological examination should be collected in clean, sterilized, environmentally sensitive bottles with screw cap closures. Borosilicate glass or autoclavable plastic bottles capable of withstanding repeated sterilization at 121°C or 170°C are recommended. Bottles capable of holding volumes of 200-500 mL should be adequate for most analyses; however, certain circumstances may require the collection of greater volumes of water (e.g., 1 L, 10 L).

When sampling by hand, the sterilized bottle should be opened with the base firmly held in one hand and with the opening facing downward. Exceptional care should be taken at all times during sampling to avoid accidental contact with the mouth of the bottle or the bottle cap. The bottle mouth is plunged downward into the water 15-30 cm below the surface (for both deep and shallow waters). The bottle is then turned towards the current (if there is one), tilted slightly upwards to displace the air and then gently pushed forward away from the hand, body, boat or other sampling platform. Samples collected from a boat or other platforms should be collected from the upstream side of these objects.

When collecting is done with a sampling pole, the bottle should be fit into the holder in the recommended manner. With the cover removed, the sample should be collected upstream and away from the collector by simulating the scooping method used during the collection of a hand-collected sample.

The volume collected should be sufficient to enable all of the required testing to be performed. Before recapping the bottle, a small amount of sample should be discarded, leaving an airspace to allow for proper mixing prior to analysis. Once capped, the bottle should be properly labelled and placed in an insulated cooler that contains pre-frozen cooling packs or ice. Additional data, such as the time of sample collection, temperature of the water and similar observations, should be recorded at this time as part of the sample record.

Responsible authorities may also wish to include within their monitoring program a requirement to collect supplemental data on various water quality and meteorological parameters at the areas being monitored. Several researchers have reported on the usefulness of such information in developing mathematical models for predicting recreational water quality (Nevers and Whitman, 2005; Olyphant and Whitman, 2005). Potentially useful measurements may include the following:

  • amount of rainfall;
  • degree of sunlight and cloud cover;
  • temperature (air, water);
  • tidal stage and water level;
  • wave height;
  • number of swimmers;
  • wind direction and speed;
  • bird populations (gulls, ducks, geese); and
  • turbidity.

An example of a sample collection and reporting form is provided in Appendix E.

B.2 Sampling procedures for sand and sediments

When epidemiological or other evidence indicates that swimming beaches could be the source of waterborne diseases among swimmers, sand and sediment sampling and analysis for suspected pathogens may be warranted. Many investigations have demonstrated that faecal indicator bacteria and faecal pathogens can persist for extended periods in sand and sediments.

At present, there is no single preferred procedure for the collection and analysis of sand and sediment samples. A variety of methods have been proposed for the collection of sand samples, including the use of sterilized scoops, spatulas, corers, probes and other sterilized collection vessels. Authorities are advised to consult the scientific literature to determine which methods may be the most suitable for their needs. Sediment samples may be collected with sterile, 250- to 500-mL wide-mouth jars, and the same precautions that were used with water sampling should be followed to ensure aseptic collection. In shallow waters, the jars are pushed along the bottom, collecting the material at the sediment-water interface. When it has been half filled, the container is retrieved, the excess water is poured off and the container is sealed. In deeper waters, sediment samplers used for collecting benthic invertebrates, such as the Ponar, Petersen or Ekman grabs, can also be used (APHA et al., 2005). Regardless of the method used, the use of sterilized equipment and aseptic technique is of utmost importance in minimizing the risk of accidental contamination of the sample. As with water samples, once collected, sand and sediment samples should be properly labelled and placed in an insulated cooler containing pre-frozen cooler packs or ice.

B.3 Sample transport, preservation and storage

After collection, samples should be held at temperatures below 10°C and in the absence of light until the time of analysis. Insulated coolers containing pre-frozen cooling packs or ice may be used during sample transport to the laboratory. To prevent the possibility of contamination, samples should be packed in such a manner so as to prevent contact between the bottle lids and any free water (from thawing/melting of the cooling material or otherwise) that may be retained within the cooler. As well, the samples should never be frozen. Thus, they should be protected from direct contact with the ice or cooling packs as necessary to prevent freezing.

Storing samples under refrigerated temperatures will have only a limited effect in terms of preserving the distribution of microbiological populations in a recreational water sample. As a result, microbiological analysis of water samples should be initiated as soon as possible to avoid unpredictable changes in the microbial population (APHA et al., 2005). Samples should be analysed within 24 hours from the time of collection (Bartram and Rees, 2000); however, analysis within 8 hours is recognized as the preferred time interval (Bartram and Rees, 2000; APHA et al., 2005). If the time required for transport is expected to exceed 6 hours, it is recommended that field analysis be considered (APHA et al., 2005). Similarly, if the results are to be used in legal action, it is recommended that special means (e.g., rapid transport, courier service) be used to deliver the samples within the specified time limits and to maintain the chain of custody (APHA et al., 2005).

Data such as the temperature of the samples when received at the laboratory and the times of sample collection, reception and analysis should be recorded as part of the sample record. Such information may prove valuable during the interpretation of results (Bartram and Rees, 2000).

B.4 Methods for microbiological analysis

B.4.1 Recommended indicators of faecal contamination

Consideration should be given to the type of water being analysed when selecting the most appropriate method for analysis. Currently, two main types of methods are used for the routine detection and enumeration of E. coli and enterococci in recreational waters: the multiple-tube fermentation (MTF) method and the membrane filtration (MF) method.

Multiple-tube fermentation (MTF) procedure

In this method, serial dilutions of a 100-mL sample are prepared in replicate sets of tubes or wells containing differential media. These are then incubated and examined for the number of positive test reactions. The number of positive tubes per dilution is then matched to a most probable number (MPN) table, which provides a statistical estimate of the number of target organisms in the original 100-mL sample.

An advantage of the MTF method is that it can be useful for samples where conditions have made MF unusable--for example, with turbid, coloured or grossly contaminated water (Health Canada, 2006a). As well, the use of liquid media may more easily permit the recovery of stressed organisms. Limitations are that the method can be time-consuming, can require large amounts of media and glassware and can require a long turnaround time to obtain results, particularly if confirmation steps are required. As well, the method provides only a statistical estimate of the presence of the target organism and not a true count of the total number of bacteria present.

Membrane filtration (MF) procedure

In this procedure, the water sample (usually 100 mL) is passed through a filter that retains the bacteria. The filter is then placed on an appropriate differential or selective medium and incubated. Following incubation, the appropriate colonies are counted, and the result is recorded as the number of target organisms per 100 mL.

Advantages of the MF method are the ability to test greater volumes of water, reduced requirements for labour and materials and the greater reliability and reproducibility associated with a direct count. For these reasons, the MF procedure is largely favoured over the MTF procedure for the routine examination of recreational waters. There are some limitations associated with the MF method, however. Samples that are high in turbidity can interfere with filtration, and highly contaminated samples may overwhelm the filter, preventing its ability to produce an accurate count. As well, the direct transfer onto solid, selective media may hinder the recovery of some organisms, and thus a resuscitation step may be required.

Defined substrate technologies

With both procedures, more recent methods have been based on the ability to detect specific enzymes considered to be characteristic for the target organisms. E. coli methods are based on the detection of the enzyme β-glucuronidase, which is thought to be restricted to this organism and a few strains of Salmonella and Shigella. Enterococci methods are based on the detection of the enzyme β-glucosidase, which is characteristic for this group. Specifically tailored chromogenic or fluorogenic substrates are incorporated into the growth media, which, when metabolized by the target organism, confer a unique property to the developing colony or surrounding media that can be used for diagnostic purposes. Chromogenic substrates produce a distinct colour change when hydrolysed, whereas fluorogenic substrates produce a fluorescent product that can be detected upon viewing under long-wavelength ultraviolet (UV) light.

Many currently available commercialized methods have used these principles in what has been termed "defined-substrate technology." With this technology, the indicator substrates are specifically designed to function as both the principal source of carbon and energy for the target bacteria. Other competitive bacteria cannot utilize the substrate and are therefore unable to interfere with the recovery of the target.

Escherichia coli

Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) recommends the mTEC method--an MF method developed by the U.S. Environmental Protection Agency (EPA)--for the detection and enumeration of E. coli at natural swimming beaches. This method involves placement of the filter on selective (mTEC) media, a resuscitation step at 35°C for 2 hours to rejuvenate stressed organisms, incubation at 45°C for 22 hours to detect all thermotolerant coliforms and then transfer to a urea substrate medium to differentiate urease-negative E. coli from other thermotolerant coliforms, which are mostly urease-positive.

The U.S. EPA has published a list of approved methods for enumerating E. coli in recreational waters (U.S. EPA, 2006c). Method 1103.1 is the original mTEC method. Method 1603 is a modified (single-step) mTEC method that uses a single medium containing a chromogenic substrate (5-bromo-6-chloro-3-indolyl-β-D-glucuronide, or BCIG). Hydrolysis of the BCIG substrate by E. coli imparts a red or magenta colour to the resulting colonies, differentiating them from other thermotolerant coliforms. Method 1604 is another MF method that uses MI medium, which contains both a fluorogen (4-methylumbelliferyl-β-D-galactopyranoside, or MUGal) and a chromogen (indoxyl-β-D-glucuronide, or IBDG) for the simultaneous detection of both total coliforms and E. coli, respectively.

Other methods using similar principles have been approved for the enumeration of E. coli in water supplies (ISO, 1998; APHA et al., 2005; U.S. EPA, 2006b). Similarly, various commercial methods - prepackaged miniaturized or simplified versions of traditional MF/MTF tests - have also received approval (U.S. EPA, 2006b). Laboratories in Canada may wish to evaluate the applicability of specific methods to recreational waters in their regions.


Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) contains two official methods for the examination of enterococci at swimming beaches--an MTF procedure and an MF procedure. The MTF procedure involves inoculation of a series of tubes of azide dextrose broth, incubation at 35°C for 48 hours and confirmation of tubes showing growth on a bile-esculin-type agar by the appearance of brownish-black colonies with brown halos. The MF procedure represents the mE method, originally described by the U.S. EPA in 1985. Filters are placed on mE agar, incubated at 41°C for 48 hours and then transferred to esculin-iron agar (EIA) medium to confirm esculin hydrolysis. Pink to red colonies with black or reddish-brown precipitates are considered enterococci.

The U.S. EPA has similarly published a list of approved methods for the enumeration of enterococci from recreational waters (U.S. EPA, 2006c). Method 1106.1 is the original mE method. Method 1600 is a modified (single-step) method (mEI), which reduces the time of analysis from 48 hours to 24 hours. The method uses a single medium that contains a chromogenic substrate (indoxyl-ß-D-glucoside). Hydrolysis of the substrate by enterococci confers a blue halo to the resulting colonies, distinguishing them from other non-enterococci.

As with E. coli, other methods using similar principles have been approved for the enumeration of enterococci in water supplies (ISO, 2000; APHA et al., 2005; U.S. EPA, 2006b). Various commercial methods - prepackaged miniaturized or simplified versions of traditional MF/MTF tests - have also received approval (U.S. EPA, 2006b). Laboratories in Canada may wish to evaluate the applicability of specific methods to recreational waters in their regions.

B.4.2 Pathogenic microorganisms

The routine testing of recreational waters for the presence of pathogenic microorganisms (bacteria, viruses, protozoa) is not recommended. However, certain circumstances may warrant testing for the presence of specific organisms, such as during investigations of potential waterborne disease outbreaks.

Bacterial pathogens

Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) describes methods for the isolation, detection and identification of the bacterial pathogens of concern for recreational waters: Campylobacter, E. coli O157:H7, Salmonella, Shigella, Aeromonas, Legionella, Mycobacterium, Pseudomonas, Leptospira and Staphylococcus aureus.

In general, methods for the isolation and detection of bacterial pathogens in recreational waters follow the same basic design: concentration of the target organism (through MF, centrifugation or growth in enrichment media), differentiation from non-target organisms (e.g., via growth in selective media or using antibody-based methods of detection) and confirmation (through a combination of morphological assessment, response to biochemical tests and serological identification).

Methods for more rapid detection of bacterial pathogens in recreational waters using advanced biochemical, immunological or gene sequence-based technologies are currently being explored. Notably, developments in polymerase chain reaction (PCR)-based methods have generated interest in the application of this technology for such purposes. Currently, PCR methods have been described for the waterborne detection of all of the bacterial pathogens considered to be of concern. Similarly, quantitative or real-time PCR methods have been described for a number of important enteric bacterial pathogens, including Salmonella, Campylobacter and E. coli O157:H7. At present, more work is needed in this area to develop standardized methods that can be accurately, reliably and affordably used. Several authors have published reviews describing the current state of the knowledge with respect to emerging technologies for the enumeration and detection of recreational water pathogens (Ashbolt, et al., 2001; Noble and Weisberg, 2006; Savichtcheva and Okabe, 2006). Laboratories in Canada are advised to consult the literature for further information as to the potential applicability of specific methods to recreational waters in their areas.

Viral pathogens

The detection of pathogenic viruses is beyond the scope of most water microbiology laboratories. When necessary, testing should be performed only by trained virologists having adequate facilities for the proper handling of these organisms.

The methodology for detection of waterborne viruses has been standardized to a certain extent; however, even the most accepted methods continue to be researched, modified and improved. Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) contains procedures for the concentration of viruses and provides guidance on methods for virus detection and identification. As well, the U.S. EPA has published a Manual of Methods for Virology (U.S. EPA, 2001a), which provides detailed, step-by-step procedures for the recovery, detection, enumeration and identification of viruses from water, sewage and other related effluents.

In general, the recovery and detection of pathogenic viruses from surface waters samples are difficult processes. Viruses are present in small numbers in faecally contaminated waters; thus, large volumes of water (up to thousands of litres) must be concentrated in order to detect their presence. Adsorption-elution methods or ultrafiltration represent the main methods used to collect and concentrate viral particles from water samples. Further concentration of the filtered sample can be accomplished through the application of flocculation or precipitation techniques.

Traditionally, cell culture methods have been the most widely used for the detection of enteric viruses in water. These methods provide valuable information on virus infectivity and concentrations, yet they can be laborious and time-consuming, and not all viruses can be grown in cell culture. More recently, conventional PCR-based methods have been applied to the detection of viruses in environmental samples. Currently, no suitable alternative to cell culture exists to enable the assessment of virus viability. Specific applications of PCR technology (reverse transcriptase PCR, quantitative PCR, integrated cell culture PCR) are being explored for their ability to overcome some of the limitations associated with conventional PCR methods.

Numerous variations of the PCR method have been described that have been successfully used for the detection of viruses in recreational waters. Although much progress has been made in this area, standardized procedures have not yet been developed. Individuals are thus advised to consult the literature for further information regarding specific methods. Review articles summarizing the existing methods as well as potentially emerging technologies have been published (Bosch, 1998; Griffin, et al., 2003; Fong and Lipp, 2005).

Protozoan pathogens

Testing for pathogenic protozoa is outside the scope of analytical services provided by most water testing laboratories. Analysis requires the use of trained analysts and highly specialized equipment.

Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) does provide an overview of the methods that can be used for the recovery and detection of Giardia and Cryptosporidium in environmental samples; however, specific procedures are not described.

The U.S. EPA has approved two methods for the detection of Giardia and Cryptosporidium in water samples: Method 1622, which is a stand-alone method for Cryptosporidium, and Method 1623, which can be used for the simultaneous detection of both organisms (U.S. EPA, 2006c). These methods are considered the most widely used for the detection of Giardia and Cryptosporidium in water. The detection of Giardia cysts and Cryptosporidium oocysts is a complex and difficult procedure. Even the most widely used and accepted methods suffer from limitations associated with the recovery of these organisms. Work is ongoing to continue to refine and improve the accuracy and sensitivity of these methods.

The recovery and detection of Giardia cysts and Cryptosporidium oocysts in water samples comprise three basic steps: concentration (via flocculation, centrifugation or filtration), separation from interfering debris (through the use of density-gradient centrifugation, immunomagnetic separation or fluorescence activated cell sorting) and detection (via immunofluorescent staining or PCR-based methods).

Of the methods used for detection, immunofluorescent staining currently represents the most widely used technique. This procedure involves the application of fluorescent antibodies directed against cyst and oocyst antigens, followed by identification of the labelled cysts and oocysts under an immunofluorescence microscope.

Various conventional PCR-based methods have been described for the detection of Giardia and Cryptosporidium in recreational waters. Other analytical techniques, such as restriction fragment length polymorphism (RFLP) analysis, have been described for the further characterization of Cryptosporidium species and genotypes. Genotype information can be used to help identify the potential host sources of Cryptosporidium responsible for an outbreak.

One limitation of the current detection methods is that they do not provide information on the viability or infectivity of the cysts or oocysts. Separate assays have been described for these purposes, which involve observing the degree of excystation or the inclusion/exclusion of specific fluorescent dyes. Other methods require the use of cell culture or animal subjects. Specific variants of the PCR method (reverse transcriptase PCR, quantitative PCR) have also been designed to facilitate quantification of the organisms and to provide estimates of cyst or oocyst viability. In general, these tests can be expensive and difficult to perform and are typically reserved for specific research purposes.

Readers are advised to consult the literature for further information regarding specific methods. Published reviews of molecular techniques used for the detection and identification of Giardia and Cryptosporidium are similarly available (Caccio, 2003).

Cyanobacteria and their toxins

Several methods are available for the detection of cyanobacteria and microcystins in water and bloom samples. These methods vary markedly in their level of complexity and the level of information that they provide. Selection of an appropriate method will depend on the type and degree of information required as well as the availability of laboratory facilities and experienced personnel.

Published texts are available that discuss the methods that may be used for the detection of cyanobacteria and microcystins in recreational waters (Chorus and Bartram, 1999; Falconer, 2005). Review articles summarizing the existing and potentially emerging technologies have similarly been published (e.g., McElhiney and Lawton, 2005). Individuals are advised to consult the literature for information on specific methods.

Cell counting

Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005) contains procedures for the enumeration of cyanobacteria (as phytoplankton) in water. Concentrations are determined by direct microscopic count with a counting chamber of known dimensions and then back-calculating to the volume of the original sample. The majority of cyanobacteria are present as colonies or filaments, which can be difficult to distinguish; thus, the use of a trained microscopist with experience in identifying cyanobacteria is recommended.

Microcystin testing

Total toxin analysis requires the determination of both the free and cell-bound microcystins. Thus, initial processing steps are often required to extract and concentrate the toxins in the sample. These may include concentration of the cyanobacterial cells, cell lysis and toxin extraction and purification. A field (commercialized) test kit could be used as a screening tool to determine the presence or absence of toxin in a water supply. If the presence of toxin is detected in a sample using the field test kit, a sample can then be submitted to a recognized laboratory for confirmatory analysis.

The mouse bioassay had historically been used as the primary method for providing evidence of a bloom's toxicity. In recent years, it has been replaced by more sensitive and reliable laboratory methods, such as the protein phosphatase inhibition assay and enzyme-linked immunosorbent assay (ELISA) tests. One drawback of these methods is that they do not provide direct evidence of the sample's toxicity. A number of invertebrate and cell culture assays have been investigated as possible alternatives for the mouse bioassay.

The protein phosphatase inhibition assay is a biochemical assay used for the detection of microcystins based on their ability to inhibit the activity of protein phosphatase enzymes. It is a rapid, sensitive method for the detection of microcystins. The test provides a quantitative estimate of the total microcystins present, but does not provide information on the composition of microcystin variants within the sample. A commercialized kit for the detection of microcystins in water samples is currently available.

The ELISA technique is regarded as a very promising method for the detection of microcystins in bloom and water samples. ELISA methods are rapid and sensitive, but are presently not specific enough to distinguish between individual microcystin variants. A number of commercialized kits for the detection of microcystins in water samples are currently available.

Liquid chromatography (LC) coupled with mass spectroscopy (MS) is the most commonly used laboratory method for the identification and quantification of microcystins and represents the reference standard against which other methods are judged. Analysis using this method is time consuming, technically demanding and expensive as it requires specialized equipment. However, standardized procedures have been described, and many analytical laboratories possess the necessary instrumentation. The lack of available standards has limited this method's usefulness in identifying individual microcystin variants.

Recently, PCR-based methods have been described for the detection of cyanobacteria based on primers directed against gene fragments belonging to Microcystis spp., as well as the genes responsible for microcystin production (mcy genes). These methods have demonstrated some measure of success in the identification and quantification of cyanobacteria and their toxins in bloom samples; however, the techniques are still under development, and further investigation is required.

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