Detection and enumeration of bacteriophages

BS ISO 10705-3-2003 pdf free.Water quality一 Detection and enumeration of bacteriophages一 Part 3: Validation of methods for concentration of bacteriophages from water.
For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and the following apply:
bacteriophages
bacterial viruses which are capable of infecting selected host strains
NOTE Bacteriophages produce visible plaques (clearance zones) in a confluent lawn of the host strain grown under appropriate culture conditions.
4 Principle
The sample is treated according to a method of choice, by which the bacteriophages are concentrated from a relatively large volume of sample (100 ml up to several litres) to a smaller volume (typically from a few to 20 ml). The concentrated sample is then analysed for bacteriophages according to an International Standard method or other suitable protocol.
The concentration method to be evaluated should be carefully described in a protocol, following ISO standard layout as much as possible. The description should include the target group(s) of bacteriophages and their detection method(s), the types of water and ranges of volumes to be analysed, as well as exceptions to the field of application, e.g. turbidity.
The method is validated according to principles laid down in this part of ISO 10705. The validation procedure consists of determining the recovery of bacteriophages from a series of samples, seeded with naturally polluted water (raw or treated sewage). The recovery is studied in a range of volumes, and particular attention is paid to its reproducibility.
5 Reagents
Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media. For information on storage see ISO 8199. except where indicated in this part of ISO 10705. Alternatively, use dehydrated complete media and follow strictly the manufacturer’s instructions.
Other grades of chemicals may be used provided they can be shown to lead to the same results.
5.1 Water, for the preparation of media, glass-distilled water or de ionized water free from substances that might inhibit bacterial growth under the conditions of the test, and at least Grade 3 as specified in ISO 3696.
5.2 Diluent, for making dilutions, peptone-saline solution or another suitable diluent in accordance with Iso 6887-1 or Iso 8199.
5.3 Culture media and reference cultures, as specified in the corresponding standard method of Iso 10705-1, IsO 10705-2 and Iso 10705-4 for the phage assay.
5.4 Glycerol (ρ= 870 g/), autoclaved at (121 3) °C for 15 min and stored in the dark at room temperature for a period no longer than 1 year.
6 Apparatus and glassware
SAFETY PRECAUTIONS — Field apparatus should be disinfected before use. Apply safety precautions appropriate to the disinfectant solution used. Some stages of the concentration process may involve the application of hydrostatic or pneumatic pressure. Observe relevant safety precautions.
Use usual microbiological laboratory equipment as specified in the method for the phage assay (Clause 8), and the protocol for the concentration method.
7 Sampling
Samples up to 10 I can conveniently be transported to the laboratory. Take the samples and deliver them to the laboratory as specified in ISO 8199 (see also ISO 19458121). For larger samples, it is advisable to perform the first step of the concentration procedure in the field. This process may take up to several hours. If parallel examination for indicator bacteria or other micro-organisms is carried out, take a time-proportional sample for these analyses, preferably by filling a sample bottle with a side flow from the concentration apparatus. Filters, precipitates or other products from the first concentration step may be further treated in the field, or may be transported to the laboratory. Include the transport and storage conditions of intermediate stages of the process in the validation procedure.
If the phages are randomly distributed within and between the vials, T1 and T2 follow approximately a
distribution with respectively 2 and 1 degrees of freedom. Accept the samples if 0,01 <TI <5,99 and T2 < 3,84.
NOTE Somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragiis naturally occurring in raw sewage partially purified as indicated above, do not suffer significant inactivation when frozen with a volume fraction of 5 % glycerol and they can be preserved frozen below (-20 ± 5) C and preferably at (-70 10) C without significant decrease in numbers for at least one year
9 Procedure
9.1 Preparation of spiked samples
9.1.1 Batch methods
Obtain samples from all types of water mentioned in the scope of the concentration procedure. Obtain the samples on different days. preferably representing different seasons and climatic conditions. Study a minimum of five samples for each sample water type. Let ‘max be the maximum volume of sample to be treated by the concentration method under evaluation. The volume of sample to be obtained for the validation procedure shall then at least be 3 x Vmax. Prepare containers with the following volumes of sample:
To each container, add 1 ml of spiking material (see Clause 8) pre-warmed at room temperature. Preserve the remainder of the spiking material on melting ice.
9.1.2 In-line concentration methods
Perform field studies for all types of water mentioned in the scope of the concentration procedure. Carry out the studies on different days, preferably representing different seasons and climatic conditions. Study a minimum of five samples for each water type. Let Vmax be the maximum volume of sample to be treated by the concentration method. Perform field studies with the following volumes of sample:
Treat each volume as described in the protocol of the concentration procedure. Add 1 ml of spiking material (see Clause 8) pre-warmed to room temperature to approximately 10 ml of diluent (5.2). Allow the concentration apparatus to operate under stable conditions. Inject then the total volume of diluent plus spiking material in the inflow to the concentration apparatus (e.g. by piercing the needle of a syringe through a hose) in four similar portions, each after passage of approximately one-fifth of the water volume to be treated.
9.2 Evaluation of recovery
Treat the spiked samples as described in the protocol of the test concentration method, including all sample transport and conservation steps, imitating as much as possible the sample transport steps of natural samples. Assay the total volume of the final concentrate in 1 ml portions, or fractions if the final volume of concentrate is not a whole number of millilitre. Any additional bacteriophages remaining on the concentration surfaces should be assayed when possible, e.g. phages retained in the filters.
In parallel, assay two 0,5-mI aliquots of spiking material. The values obtained shall be used to calculate the concentration efficiency, which will allow the determination of the number of phages introduced in the different volumes to be concentrated and will also allow the calculation of T1 and T2 of each one of the bottles with regard to other bottles. If more than 20 % (1 in 5) of the spiking material samples do not comply with the acceptable values of ‘1 andlor T, discard the spiking material. If . 20 % of the spiking material samples do not comply with T1 andlor T2. discard the results of this assay and perform a new assay.
Anomalous or extreme results are characteristic of microbiological measurement. Occasionally it is acceptable to discard a result on the basis of simple observation of the data. However, it is preferable to apply an appropriate statistical test. Use the Dixon test to discard extreme values.
Perform a minimum of five experiments with results that have not been rejected before data analysis.
If the method is evaluated using a natural water suspected of containing phages detected by the same bacterial host as the test bacteriophages, then determine the background counts of phages. Plaque an aliquot or concentrate and count the concentrate. If the sample contains a number of phages > 20 % of the phages spiked into the sample, heat the water and keep it at 80 C for 30 mm and allow it to cool prior to use. If the number of naturally occurring bacteriophages is <20 % of the added phages, then enumerate them and take them into account in the data analysis (Clause 10).BS ISO 10705-3 pdf free download.Detection and enumeration of bacteriophages