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Célia Martinez
In Drinking water

Why, when and how check for somatic and RNA F-specific coliphages?

Viral indicators, parameter now included in regulations

The European regulation recently added the monitoring of coliphages to control the quality of drinking water and reuse water.

For the monitoring of drinking water, the EU Directive 2020/2184 requires the detection of somatic coliphages only. It is the first time that a viral parameter has been introduced in the field of tap water. They must be checked for in raw water. If the result is above 50 PFU per 100 ml, then, the water after treatment must be analysed.

Regarding water reuse system, total coliphages must be enumerated, i.e. somatic coliphages and RNA F-specific coliphages (Regulation (EU) 2020/741). This parameter is now required for the reuse of reclaimed water for irrigation of all food crops consumed raw where the edible part is in direct contact with reclaimed water and root crops consumed raw.

The enumeration of somatic and RNA F-specific coliphages is used to qualify the efficacy of drinking water and wastewater treatment plants.

See this article form more information on somatic coliphages.

 Indicators in the fight against covid-19

In the fight against SARS-CoV-2, ANSES was charged to evaluate two viruses, somatic and RNA F-specific coliphages, to monitor the reduction of SARS-CoV-2 in wastewater and sludges¹.

Reference values:
Regulation Application field Virus Reference value
Before treatment		 Reference value
After treatment
Directive EU 2020/2184 Water intended for human consumption Somatic coliphages < 50 PFU/ 100 ml 0 / 100 ml
Regulation EU 2020/741 Reused water for agricultural irrigation Total coliphages* – 6 LOG10 reduction**

The performance targets established by the regulations ask that a sample concentration method is used in analytical laboratories.

*If analysis of total coliphages is not feasible, at least one of them (F-specific or somatic coliphages) shall be analysed.
** If a biological indicator is not present in sufficient quantity in raw waste water to achieve the log10 reduction, the absence of such biological indicator in reclaimed water shall mean that the validation requirements are complied with.

What standards should you use for the analysis? 

Standards ISO 10705-1 and 10705-2 describe the detection method of RNA F-specific coliphages and somatic coliphages respectively. The enumeration is done using a double agar layer plaque assay. With this method, up to 5 ml of sample can be analysed on the same petri dish. Thus, if 20 plates are inoculated in parallel, this procedure will allow detection of one virus in 100 ml. However, this method is extremely time and effort consuming, as well as costly. Therefore, it cannot be used as a routine method.

Standard 10705-3 recommends several concentration methods, detailed below. Each laboratory must implement and validate its procedure based on the performance criteria described in the standard.

Concentration methods of ISO 10705-3

Méthode Principle Benefits Flaws
Adsorption/ elution 

Adsorption of virus on a filter by electrostatic interactions.

Elution in 10-15 ml or 500-1000 ml then reconcentration by ultrafiltration.

Recommended for large samples (10 to 100 litres).
  • Easy
  • High yields
  • High investment costs
  • Expensive consumables
  • Clogging
  • Low reproducibility
Flocculation

Flocculation of viruses with Mg(OH)2.

Elution in 30 ml.

Recommended for samples ranging from 100 ml to 1 l with turbidity > 2 NTU.
  • Cheap
  • Efficient for turbid samples
  • Labour intensive
  • Use of chemical components
  • Low reproducibility
Membrane filtration

Concentration of viruses on a filter.

Elution in a small volume <5ml.

Recommended for samples ranging from 100 ml to 1 l with turbidity < 2 NTU.
  • Low labour costs
  • Easy
  • Reproducible
  • Yields impacted by the filtration flowrate
  • Few references available

Membrane filtration is perfectly adapted for treated water (drinking and reclaimed water). Indeed, concentration of 100 ml to 1 litre of sample is enough to meet the performances required. Besides, the treatment generally guarantees a high quality water with low total suspended solids and low turbidity, which enable the filtration of one litre of water without clogging issues.

Therefore, membrane filtration is the best compromise possible, offering simplicity, rapidity and performances.

Membrane filtration: VIRAPREP®

This method is a simple membrane concentration of samples ranging from 100 ml to 1 litre on a specific filter that has an affinity for the concerned viruses. They are then eluted in a solution that guarantees their integrity and infectivity. The elution volume (5 ml) and the membrane filter are inoculated on a double layer agar (10705-1 and -2) in order to analyse the whole sample.

The VIRAPREP® kit, that GL BIOCONTROL produces, is a turnkey solution to concentrate somatic and F-specific RNA coliphages. This kit, fully compliant with the ISO 10705-3 requirements, limits the analysis to the inoculation of two to three petri dishes.

Several analysis laboratories were accredited by the French Accreditation Committee (COFRAC) using VIRAPREP® as their concentration method.

Célia Martinez
In ATP test, Drinking water, Microbiologique testing

ATP testing: A predictive indicator of bacteriological non-compliance in water

HPC (Heterophoric Plate Count) culture mediums such as YEA, PCA or R2A, frequently used for environmental bacteria counting, detect less than 1% of the total flora (WHO, 2003). Indeed, a large proportion of bacteria cannot multiply on those mediums. For example, there are:

  • Anaerobic organisms (tolerant or obligate): oxygen presence slows down or inhibits their growth.
  • Bacteria requiring a specific temperature to grow such as psychrophile (low temperature) or thermophile (high temperature).
  • Germs requiring a specific environment such as acidophiles (high acidic conditions) or halophiles (high salinity).
  • Germs requiring specific elements such as rare amino acids, complex sugars, vitamins, cations…
  • Non-culturable bacteria whose culture is impossible using traditional methods.
Total bacterial flora

Diagram of total bacterial flora

  • VBNC bacteria (viable but non-culturable) which have momentarily lost their growing capacities as a response to stress. The use of biocides, physical treatments (ex: UV) or the environmental parameter modifications (temperature, pH…) can cause that change of state.

Furthermore, the bacteria needs to form a colony to be detected by the technicians’ eye. Which means growing from one to several billions within the allocated time period. That implies a short lag phase and a fast growth. Those parameters partly depend on the incubating temperature and the type of medium used.

 

In the end, we only detect aerobic mesophilic flora able to grow between 20°C (68°F) and 45°C (113°F) in the given time and for which the nutrients are adequate.
Why you should not speak of “total flora” with culture method?

Each culture medium will only detect a part of the bacteria depending on chosen conditions.
Thus, to be thorough, we should speak of “culturable flora” indicating the medium, temperature and incubation time chosen.

Diagram ATP vs cultural method

Example of bacterial growth monitored by culture and ATP-metry

As for ATP testing, it detects the whole living bacterial flora, even non culturable bacteria. For all these reasons, it is often observed an increase of total flora by ATP testing long before the appearance of the first colonies on the culture medium.

ATP-metry is an early indicator of microbiological contamination.
ATP tests for industrial waterATP tests for industrial water
ATP tests for drinking waterATP tests for drinking water
Célia Martinez
In Drinking water, News, Project

Research project: development of a detection kit for somatic coliphages

By Estelle Alaume

In Europe, water intended for human consumption is highly regulated by the Drinking Water Directive. Water intended for human consumption includes distribution systems, drinking water in bottles or containers and water used in the food-processing industry. Recently, the revision of the Drinking Water Directive (n°98/83/CE) adds new parameters to the list of criteria determining water safety, such as control of somatic coliphages. These viruses are used as indicator organisms for faecal contamination. Its control represents an important step towards a high quality of drinking water.

“Somatic coliphages, new faecal contamination indicator” Continue Reading
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