microbiological analysis

aquatic organisms provides an overall description of the bacterial realm and methods used to identify the main aquatic micro-organisms with an emphasis on controlling the sanitary control of waters.

principle for the sanitary control

Water intended for human consumption should not jeopardise consumer health and as such it should be free of any pathogenic micro-organism.

Looking for all pathogenic micro-organisms (bacteria, viruses, parasites) is an impossible task that would be far too costly. There are many species (Salmonella, Shigella, Campylobacter, Vibrio cholerae, Leptospir, Giardia, Cryptosporidium, Rotavirus, poliomyelitis virus, hepatitis A virus…). Searching for these species is often complex and onerous with analytical procedures that can only be carried out in highly specialised laboratories. Additionally, their presence is irregular and random.

Most of the pathogen micro-organisms carried in water are from the fecal group. Therefore, the suggested control procedure consists in “searching for certain species or groups of bacteria that can act as faecal pollution or contamination indicator controls”. These indicator germs are commensal bacteria that are naturally present in the intestine of man and warm blood animals. They and consistently and abundantly excreted within fecal matter.

The potability standards applicable to water are based on the hypothesis that the principles governing the inactivation of pathogenic and indicator micro-organisms are comparable Consequently, a significant reduction in indicator mirco-organisms should result in an advanced inactivation of pathogenic germs. In essence, the presence of indicators micro-organisms is considered as an alarm suggesting the presence of pathogens. Similarly, their absence indicates an absence of pathogens.

Regulations have used enterococcal and heat tolerant coliform bacteria as indicator of fecal contamination. These have now been replaced by the Escherichia coli.

To complement these indicator micro-organisms the following alternative exist:

• indicators of treatment effectiveness indicators: spores of sulphur reducing anaerobic bacteria (French decree) or spores of the Clostridiumperfringens (European directive);

• counts of aerobic germs that regrow at temperatures of 22°C and 36°C. Referenced as Common Germs, they are used to monitor bacterial levels in water distribution networks. Their counts provide secondary information. Actually, it is the fluctuation of those counts and specifically sudden increase that may indicate changes in the microbial quality of the water being distributed.

bacteriological analyses

Laboratories have standardised methods that are applied to comply with the requirements of bacterial controls.

Microbial counting is carried out via introduction of a small amount of the sample into a solid nutrient medium.

For indicator pathogens, the most widespread method involves filtration through a membrane and then placing the filter on a specific agar culture medium that is appropriate to the target bacteria. The medium is incubated under optimum growth conditions in terms of temperature and time.

After incubation, each bacteria present in the sample will produce a colony that is visible to the naked eye. Confirmation tests, frequently based on biochemical properties, are then carried out on the colonies regarded as characteristic of the target species.

• Results are obtained within a few days. They are usually expressed as CFU (colony forming units).
• Sampling conditions (sterility, transport) are crucial as they govern the validity of the result (see sampling).

For a more comprehensive analysis, the laboratory may be required to look for bacteria that are pathogenic to humans such as Salmonella, Shigella, Legionella, Staphylococcus aureus, Pseudomonas aeru­ginosa…

virological analyses

The most widely used analytical technique consists in counting enteroviruses.

A sufficient concentration is essential. The most widespread techniques are filtration or ultrafiltration through membranes, adsorption-elution methods using various mediums (cellulose nitrate, micro fibre glass cartridge, powdered glass), organic flocculation methods.

The grand majority of the viruses that are searched in the hydric medium can be isolated using different types of cell cultures. The most widely used are continuous BGM (Buffalo Green Monkey) kidney cell lines, or human neoplastic cells (Hela cells). The probability of isolating a virus increases with the number of cell cultures used. Consequently, the use of at least two different types of cell cultures is recommended.

In vitro cultures are inoculated with concentrated samples and the appearance of a cytopathogenic effect revealed by microscope examination is indicative of viral proliferation and of the presence of the virus in the inoculum. The number of infectious viral plaques is then expressed as MPC (Most Probable Count [NPP in French]) for cells cultured in a liquid medium or as PFU (Plate Forming Units) for cells cultured in a solid medium. Recovery rates are typically low and final counts are obtained within 2 to 3 weeks.

parasitological analysis

Wastewaters carry many types of parasites.

The two protozoa Giardialambia and Cryptosporidium parvum are by far the most researched parasites in waters, as they have been found responsible for many waterborne epidemics in recent decades. Seeking out these parasites is therefore useful and often necessary, especially in groundwaters that are impacted by surface waters.

The same standard analytical procedure is used to detect both Giardia cysts and Cryptospori­dium. It comprises several phases as follows:

• concentration of a large volume of water by filtration through sulphone polyether cartridges followed by elution resulting from mechanical stirring in the presence of a detergent solution;

• reconcentration through centrifugation and the cysts are then extracted by magnetic immuno-separation (MIS [IMS in French]);

• immunofluorescence marking (monoclonal antibodies marked using a fluorochromium; fluorescein isothiocyanate (FITC), capable of fixing specifically on the antigens present at the surface of the cysts) and nucleic acid coloring using DAPI (4’,6-diamino-2-phenyl-indole);

• examination under a microscope at a 490 nm wavelength reveals fluorescent apple green colored cysts.

The result can be obtained within one day.

This analytical procedure only targets the encysted form of the parasite and not the infectious vegetative form. In order to evaluate cyst viability or infectivity, existing tests (specific coloration, encystation techniques, infectivity in young mice) are not particularly appropriate to water samples as the sensitivity threshold of these procedures is often too high.

molecular biology techniques

When applied to the sanitary control of drinking waters, molecular detection techniques are a revolution in the field of analysis providing for a more comprehensive and proactive monitoring of the microbiological quality of a water. To date, there are only a few applications but numerous research and development projects are ongoing.

Unlike traditional identification methods that examine the morphological and physiological nature of micro-organisms, molecular identification relies on the recognition of one or more nucleic sequences found in the DNA (or RNA ) are present in all living micro-organisms (bacteria, parasites, viruses ...). The main advantages of these analytical techniques are their rapidity, their high levels of selectivity, their sensitivity, and their capability for automated operation. These techniques are applicable to the analysis of all micro-organisms, even those that cannot be identified using traditional methods.

Of the different molecular techniques, the PCR (Polymerase Chain Reaction) technique is one of the most promising. This gene amplification stage consists of multiplying the proper DNA sequence of a target micro-organism by approximately one million times. Consequently, there is no longer a need to cultivate bacteria in Petri dishes or to isolate viruses using cell cultures. With the goal to highlight micro-organism viability and to ensure that these micro-organisms are not “dead”, researchers use the RT- PCR (reverse transcriptase-polymerase chain reaction) technique that targets RNA (messenger RNA, ribosomal RNA ) whose presence is indicative of cell metabolic activity.

The main drawback of these molecular biology techniques lies in the fact that the target pathogenic micro-organisms or fecal contamination indicators are only present in small amounts (when they are present!) within a frequently abundant “indigenous” flora. Consequently, operating conditions must be optimised in order to overcome potential interference and to eliminate any danger of false negatives.

algology

Algae count can be carried out using optical or electronic microscope. Species are distinguished by the nature of the pigments they synthesise, their morphology or their method of reproduction (see micro-organisms for which freshwater is their natural habitat).

Samples are usually stabilised by addition of formol (3 to 5 mL of a 40 % formal­dehyde solution per 100 mL of sample). When the water is low in algae, it requires concentration by centrifugation (5 000 rpm^–1), by sedimentation (1 week in a 1 L test tube) or by filtration through a membrane or through fine sand. An optical count is then carried out. These results are expressed as the number of organisms or cells, sometimes as standard planimetric units (1 spu = 400 mm²) per mL or per L of water.

It is also possible to assess a water overall algal content by measuring chlorophyll pigments, especially chlorophyll a and b, after concentration through a membrane and extraction with acetone or methanol.