ISO 10705-4:2001 pdf download

ISO 10705-4:2001 pdf download.Water quality一Detection and enumeration of bacteriophages一 Part 4:Enumeration of bacteriophages infecting Bacteroides fragilis.
NOTE 2 A general description of bacteriophages infecting B. fragilis is given in annex A.
4 Safety precautions
The host strain used in this part of ISO 10705 is non-pathogenic to man and animals and should be handled in accordance with the normal (national or international) safety procedures for bacteriological laboratories. Bacteriophages infecting Bacteroides fragilis are non-pathogenic for man and animals, but some types are very resistant to drying. Appropriate precautions shall be taken to prevent cross-contamination of test materials, particularly when examining or handling cultures of high titre or when inoculating cultures of the host strains. Such procedures shall be carried out in a biohazard cabinet or a separate area of the laboratory. Chloroform is a carcinogenic substance. Observe relevant safety precautions or use an alternative method of equal efficacy.
It is recommended that personnel using this method have or acquire some experience in handling anaerobic bacteria.
5 Principle
The sample is mixed with a small volume of semi-solid nutrient medium. A culture of host-strain is added and plated on a solid nutrient medium. After this, incubation and reading of plaques take place. The results are expressed as the number of plaque-forming particles (also named plaque-forming units, pfu) per unit of volume (pfp/ml, pfp/l, etc.).
6 Reagents
6.1 Basic materials.
Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagents and follow the instructions given in annex B. 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.
6.2 Water, for the preparation of media, glass-distilled or deionized, free from substances which might inhibit
bacterial growth under the conditions of the test, and complying with ISO 3696.
6.3 Diluent, for making sample dilution, such as peptone-saline solution or another diluent complying with
ISO 6887-1.
7 Apparatus
Apart from apparatus supplied sterile, sterilize any glassware and other equipment in accordance with ISO 8199.
Usual sterile, microbiological laboratory equipment and glassware or disposable plastics-ware in accordance with ISO 8199 and including the following:
7.1 Hot-air oven, for dry-heat sterilization, and an autoclave.
7.2 Incubator or water bath, thermostatically maintained at (36 + 2) °C.
7.3 Incubator or water bath, thermostatically maintained at (36 ± 2) °C with a shaking device.
7.4 Water bath or heating block, thermostatically maintained at (45 + 1) °C.
7.5 Water bath or equivalent device, for melting of agar media.
7.6 pH meter, and pH paper.
7.7 Counting apparatus, with indirect, oblique light.
7.8 Deep freezer, thermostatically maintained at (—20 + 5) °C.
7.9 Deep freezer, thermostatically maintained at (—70 ± 10) 00 or liquid nitrogen storage vessel.
7.10 Spectrophotometer, equipped with a filter for the range of 500 nm to 650 nm with a maximum bandwidth of
± 10 nm, capable of holding cuvettes (7.21) having an optical path length of 1 cm or Hungate glass tubes (7.20) with
butyl rubber stopper and screw cap or screw-capped glass culture tubes.
7.11 Anaerobic cabinet, or jars or bags, as well as anaerobiosis generators and anaerobiosis indicators.
7.12 Refrigerator, temperature set at (5 + 3) °C.
7.13 Petri dishes, having a diameter of 9 cm, and vented.
7.14 Graduated pipettes, having a capacity of 0,1 ml, 1 ml, 5 ml and 10 ml and Pasteur pipettes.
7.15 Glass bottles, of suitable volumes.
7.16 Screw-capped glass bottles, of suitable volumes.
7.17 Culture tubes, with caps or suitable alternatives.
7.18 Screw-capped glass culture tubes.
7.19 Measuring cylinders, of suitable capacity.
7.20 Hungate glass tubes, with butyl rubber stopper and screw cap or screw-capped glass culture tubes which can fit in the spectrophotometer (see Figure 1).
7.21 Cuvettes, having an optical path length of 1 cm.
7.22 Membrane filter units, for decontamination, having a pore size of 0,2 jim, preferably low protein-binding
membranes, as for example, those composed of polyvinylidene difluoride.
7.23 Plastics vials, lidded, having a capacity of 3 ml.
7.24 Glass vials, screw-capped, having a capacity of 3 ml.
7.25 Syringes and needles.
7.26 Cotton swabs.
8 Microbiological reference cultures
The recommended host strain is Bacteroides fragilis RYC2056 (ATCC 700786).Ehl
Use bacteriophage B56-3 (ATCC 700786-Bi) infecting Bacteroides fragilis RY02056 for the preparation of reference materials (11.4).