SIST ISO 10705-4:2007
(Main)Water quality -- Detection and enumeration of bacteriophages -- Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
Water quality -- Detection and enumeration of bacteriophages -- Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water, sediments and sludge extracts, where necessary after dilution. In the case of low phage numbers, a pre-concentration step may be necessary for which a separate International Standard has been developed. The method is also applicable to shellfish extracts.
Qualité de l'eau -- Détection et dénombrement des bactériophages -- Partie 4: Dénombrement des bactériophages infectant Bacteroides fragilis
La présente partie de l'ISO 10705 spécifie une méthode de détection et de dénombrement des bactériophages
infectant la bactérie Bacteroides fragilis par incubation de l'échantillon avec une souche-hôte appropriée. La
méthode est applicable ŕ tous les types d'eaux, aux extraits de sédiments et de boues si nécessaire aprčs dilution.
Dans le cas de faibles populations de phages, une étape de préconcentration peut s'avérer nécessaire pour laquelle
une Norme internationale distincte a été élaborée. La méthode est également applicable aux extraits de coquillages.
NOTE Il est souhaitable que les Normes internationales soient adoptées le plus largement possible. La présente partie de
l'ISO 10705 comporte des références ŕ des procédures alternatives qui évitent d'avoir ŕ utiliser du matériel ou des équipements
coűteux qui peuvent ne pas ętre aisément disponibles dans les pays en voie de développement. L'utilisation de ces procédures
alternatives n'affectera pas les performances de la présente méthode.
Kakovost vode - Ugotavljanje prisotnosti in števila bakteriofagov - 4. del – Štetje bakteriofagov, ki okužijo Bacteroides fragilis
General Information
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 10705-4
First edition
2001-12-15
Water quality — Detection and enumeration
of bacteriophages —
Part 4:
Enumeration of bacteriophages infecting
Bacteroides fragilis
Qualité de l'eau — Détection et dénombrement des bactériophages —
Partie 4: Dénombrement des bactériophages infectant Bacteroides fragilis
Reference number
ISO 10705-4:2001(E)
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ISO 10705-4:2001(E)
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ISO 10705-4:2001(E)
Contents Page
1 Scope . 1
2 Normative references . 1
3 Term and definition . 1
4 Safety precautions . 2
5 Principle . 2
6 Reagents . 2
7 Apparatus . 2
8 Microbiological reference cultures . 3
9 Sampling . 4
10 Preparation of test materials . 4
11 Procedure . 7
12 Expression of results . 9
13 Test report . 10
Annexes
A General description of bacteriophages infecting Bacteroides fragilis. 11
B Culture media and diluents . 12
C Alternative method for preparing inoculum cultures. 17
D Culturing of Bacteriophage B56-3. 18
Bibliography. 19
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ISO 10705-4:2001(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technical
committees. Each member body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, governmental and non-governmental, in
liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this part of ISO 10705 may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO10705-4 was prepared by Technical Committee ISO/TC147, Water quality,
Subcommittee SC 4, Microbiological methods.
ISO 10705 consists of the following parts, under the general title Water quality — Detection and enumeration of
bacteriophages:
— Part 1: Enumeration of F-specific RNA bacteriophages
— Part 2: Enumeration of somatic coliphages
— Part 3: Validation of methods for concentration of bacteriophages from water
— Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
Annexes A, B, C and D of this part of ISO 10705 are for information only.
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INTERNATIONAL STANDARD ISO 10705-4:2001(E)
Water quality — Detection and enumeration of bacteriophages —
Part 4:
Enumeration of bacteriophages infecting Bacteroides fragilis
1 Scope
This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides
fragilis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water,
sediments and sludge extracts, where necessary after dilution. In the case of low phage numbers, a pre-
concentration step may be necessary for which a separate International Standard has been developed. The method
is also applicable to shellfish extracts.
NOTE It is desirable for International Standards to be adopted as widely as possible. This part of ISO 10705 includes reference
to alternative procedures which obviate the need for expensive materials or equipment which may not be readily available in
developing countries. Use of these alternatives will not affect the performance of this method.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 10705. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 10705 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated references,
the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of
currently valid International Standards.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension
and decimal dilutions
ISO 8199, Water quality — General guide to the enumeration of micro-organisms by culture
3 Term and definition
For the purposes of this part of ISO 10705, the following term and definition applies.
3.1
bacteriophage infecting Bacteroides fragilis
bacterial virus which is capable of infecting selected Bacteroides fragilis host strains by attachment to the bacterial
cell wall as the first step of the infectious process
NOTE 1 Such bacteriophages produce visible plaques (clearance zones) in a confluent lawn of host bacteria grown under
appropriate culture conditions.
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ISO 10705-4:2001(E)
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.
5Principle
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.).
6Reagents
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 ISO10705. 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.
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ISO 10705-4:2001(E)
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) C 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 9cm, 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 1cm.
7.22 Membrane filter units, for decontamination, having a pore size of 0,2µm, preferably low protein-binding
membranes, as for example, those composed of polyvinylidene difluoride.
7.23 Plastics vials, lidded, having a capacity of 3ml.
7.24 Glass vials, screw-capped, having a capacity of 3ml.
7.25 Syringes and needles.
7.26 Cotton swabs.
8 Microbiological reference cultures
[1]
The recommended host strain is Bacteroides fragilis RYC2056 (ATCC 700786).
Use bacteriophage B56-3 (ATCC 700786-B1) infecting Bacteroides fragilis RYC2056 for the preparation of reference
materials (11.4).
NOTE The ATCC strains are available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas,
VA 20110-2209, USA. This information is given for the convenience of users of this part of ISO 10705 and does not constitue an
endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
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ISO 10705-4:2001(E)
Figure 1 — Hungate glass tube with rubber stopper and screw cap
9 Sampling
Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2 and
ISO 5667-3.
10 Preparation of test materials
10.1 Culturing and maintenance of host strains
10.1.1 General
The culturing and maintenance of host strains involves several stages which are summarized in Figure 2.
Bacteroides fragilis is an obligate anaerobe. However, it does not require handling under conditions of strict
anaerobiosis. Incubation of cultures in solid media should be carried out in an anaerobic cabinet, or anaerobic jars or
bags. When using liquid media it is sufficient to ensure that containers are completely filled and closed with a screw
cap.
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ISO 10705-4:2001(E)
Figure 2 — Scheme for the culturing and maintenance of host strains
10.1.2 Preparation of stock cultures
Rehydrate the content of a lyophilized ampoule of the reference culture of the host strain in 1ml of Bacteroides
phage recovery medium broth (BPRMB) (B.1) using a Pasteur pipette (7.14). Inoculate the suspensions in 10 ml of
◦
BPRMB (B.1) in a 10 ml screw-capped glass tube (see 10.1.1) and incubate at (36± 2) C for (21± 3) h.
Aseptically soak a sterile cotton swab with the culture and streak it onto a plate of Bacteroides phage recovery
◦
medium agar (BPRMA) (B.2). Incubate the culture in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h.
Alternatively, if a culture in slant is available, streak it with a sterile cotton swab directly onto a plate of BPRMA (B.2).
◦
Incubate the culture in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h.
Inoculate cells (mass inoculation with a sterile cotton swab) from the plate into 10 ml of BPRMB (B.1) in a 10 ml
screw-capped glass tube (7.18). Be sure that the tube is completely filled (see 10.1.1). If dense growth occurs,
inoculate 1/81 of the growth onto the BPRMA plate; if poor growth occurs, use /2 of the growth onto the plate.
◦
Incubate the culture at (36± 2) C for (21± 3) h.
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ISO 10705-4:2001(E)
Mix culture and cryoprotector (B.6) in a ratio 1:1 (volume). Mix well avoiding bubble formation. Distribute into screw-
◦
capped, preferably glass, vials (7.24) in aliquots of approximately 1,0 ml and store at (−70± 10) C or in liquid
nitrogen for up to five years.
This first passage of the host strain should be stored as a reference in the laboratory. Purity of the culture should be
checked before storage by Gram staining, by testing absence of growth under aerobic conditions and by testing
sensitivity to a reference bacteriophage (i.e. B56-3).
10.1.3 Preparation of working cultures
Remove a vial of stock culture (10.1.2) from frozen storage, allow the vial to equilibrate to room temperature (i.e.
◦ ◦
C30 C
15 to ) and streak the culture with a sterile cotton swab onto a plate of BPRMA (B.2). Incubate the culture in
◦
an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h. Inoculate cell material (mass inoculation with a sterile
cotton swab) from the plate into of prewarmed BPRMB (B.1) kept in a screw-capped glass tube (see
10 ml 10 ml
10.1.1). If dense growth occurs, inoculate 1/8 of the growth onto the BPRMA plate; if poor growth occurs, use 1/2 of
◦
(36± 2) C (21± 3) h
the growth onto the plate. Incubate the culture at for .
Add BPRMB (B.1) to a tube for anaerobic cultures (7.18) and warm to at least room temperature [faster growth will
◦
occur if the broth is prewarmed to (36± 2) C]. Transfer an aliquot of the above-mentioned culture, without shaking
the tube and taking the aliquot from the middle part of the tube, to the tube containing prewarmed BPRMB in a ratio
of culture-to-BPRM of 1,5:10 (volume). Be sure that the inoculated tube is completely filled (see 10.1.1). Incubate the
◦ 9
culture at (36± 2)C2 to reach approximately × 10 cfu/ml.
Mix working culture and cryoprotector (B.6) in a ratio of 1:1 (volume) avoiding bubble formation. Distribute into screw-
◦
capped, preferably glass, vials (7.24) in aliquots of approximately 1,5 ml and store at (70± 10) C for a maximum
of 12 months.
Ensure that the culture does not reach the stationary phase before mixing it with the cryoprotector. Absorbance
stabilization will indicate the end of the log phase, which may last 5h to 8h.
10.2 Calibration of absorbance measurements for counts of viable host bacteria
Remove a vial of working culture (10.1.3) from the deep freeze (7.9) and allow the vial to equilibrate to room
◦ ◦
temperature (i.e. 15C3 to 0 C). Add BPRMB (B.1) to a tube for anaerobic cultures (7.18) and warm to at least
◦
room temperature [faster growth will occur if the broth is prewarmed to (36± 2) C]. Before inoculation, adjust the
spectrophotometer to zero. Transfer the working culture into BPRMB (B.1) in a ratio of respectively 1:10 (volume),
completely filling the tube. Tubes for anaerobic cultures may be inoculated/sampled by puncture with sterile syringes
◦
and needles (7.25). Incubate the culture at (36± 2)C3. Every 0min, measure the absorbance (using 7.10) and
withdraw, by puncture, a 0,3 ml sample for viable cell counts. Ensure that the tube is out of the incubator for as short
a time as possible.
Melt 50 ml of semi-solid BPRMA (ssBPRMA) (B.3) (basal agar) by putting bottles in a boiling water bath. Then place
◦
the bottles in a water bath at (45± 1) C. Aseptically add haemin solution, Na CO and antibiotics and adjust pH to
2 3
6,8± 0,5 (B.1) according to Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps, placed in a water bath
◦
at .(45± 1) C
−8 −6 −7 −8
Dilute the aliquots sampled from the culture to 10 and add 1ml volumes of the 10 , 10 and 10 dilutions to
each tube of 2,5 ml of melted ssBPRMA, in duplicate. Pour onto a layer of BPRMA in a 90 mm Petri dish (B.2)
prewarmed at room temperature. Distribute evenly, allow to solidify on an horizontal, cool surface and incubate the
◦
plates upside down in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h. Ensure that the process is
performed in a period of time as short as possible and that the diluents had been autoclaved immediately just before
use (to have them free of oxygen). Be sure that the diluents have cooled to room temperature before use. Count the
total number of colonies in each plate yielding between 30 colonies and 300 colonies and calculate the number of
cfu/ml (consult ISO 8199 if necessary).
This procedure should be carried out several times (approximately 4 to 5 times) to establish the relationship between
absorbance measurements and colony counts. If sufficient data have been obtained, further work can then be based
only on absorbance measurements.
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ISO 10705-4:2001(E)
11 Procedure
11.1 Preparation of inoculum cultures
Remove a vial of working culture (10.1.3) from the deep freeze (7.9) and allow the vial to equilibrate to room
◦ ◦
temperature, (i.e. 15C3 to 0 C). Add BPRMB (B.1) to a screw-capped tube and warm to at least room temperature
◦
[faster growth will occur if the broth is prewarmed to (36± 2) C]. Before inoculation, adjust the spectrophotometer
to zero for the tube (10.2). Transfer the working culture into the tube filled with BPRMB (B.1) in a ratio of 1:10
◦
(volume). Be sure that the tube is completely filled (see 10.1.1). Incubate the culture at (36± 2)C2. After h,
measure the absorbance of the culture every 30 min. At an absorbance corresponding to a cell density of
8
approximately 2× 10 cfu/ml (based on data obtained in 10.2) take the inoculum culture from the incubator and
either use it immediately or quickly cool the culture by placing it in melting ice. Use the culture within 6h. Cell
8 8
densities ranging from 1× 10 cfu/ml to 4× 10 cfu/ml give similar plaque counts, however cell densities of
8 8
1× 10 cfu/ml or 2× 10 cfu/ml give larger plaques.
An alternative method for preparing inoculum cultures is given in annex C.
8
NOTE If the cell density of approximately 2× 10 cfu/ml is not reached within 3 h, it is possible to increase the amount of
working culture transferred into the BPRMB to a ratio of respectively 1,5:10 (volume).
11.2 Standard procedure
◦ ◦
Prepare an inoculum culture as described in 11.1. Allow sample to equilibrate to room temperature (15C3 to 0 C).
Melt 50 ml of ssBPRMA (B.3) (basal agar) by putting the bottles in a boiling water bath (7.5) then place them in a
◦
water bath at (45± 1) C. Aseptically add haemin, Na CO and antibiotics and adjust the pH to 6,8± 0,5 (B.1)
2 3
according to Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps (7.17), which are placed in a water bath
◦
at .(45± 1) C
To each culture tube (7.17), add 1ml of sample (or diluted or concentrated sample) prewarmed to room temperature.
Examine each aliquot at least in duplicate.
Add 1ml of inoculum culture to each culture tube containing the aliquots of sample and ssBPRMA, then mix carefully
avoiding the formation of air bubbles and pour the contents on a layer of complete BPRMA (B.2) in a 90 mm Petri dish
prewarmed to room temperature. Distribute the contents evenly allowing the agar to solidify on a horizontal, cool
surface. B. fragilis is an anaerobic bacterium. Therefore, once dried, distribute inoculated tubes as fast as possible
and place the plates into the anaerobic jars as soon as possible. Incubate the plates upside down in an anaerobic
◦
cabinet, jar or bag at (36± 2) C for (21± 3) h.
After incubation, count the number of plaques on each plate. If it is not possible to count the plates after finishing
◦
incubation, keep the plates at (5± 3) C until reading.
For samples containing high background flora, it is recommended to decontaminate the samples by filtration through
low protein-binding membranes, as for example, those composed of polyvinylidene difluoride 0,2µm pore size
(7.22), or to increase the amount of ssBPRMA kanamycine monosulfate to obtain a final concentration in the final
−1 −1
medium of 300µgml instead of 100µgml (the concentration recommended for growing B. fragilis).
NOTE 1 Freshly prepared triphenyltetrazoliumchloride solution (B.10) can be added to enhance contrast for counting plaques. If
this procedure is used, plates should remain under aerobic conditions for 1h to 2h before counting plaques.
NOTE 2 The addition of ice-cold inoculum culture (11.1) to the ssBPRMA may lead to a sharp drop in temperature and
solidification of the medium. To avoid this, prewarm the ice-cold inoculum culture to room temperature before adding it to the tubes
containing ssBPRMA and the aliquot of the sample.
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ISO 10705-4:2001(E)
11.3 Presence/absence test
This test can be used to examine the presence of bacteriophages in different volumes of sample. For instance, to
determine the presence/absence in 100 ml, proceed as follows:
Add 100 ml of double-strength BPRMB (B.9) to a 250 ml screw-capped sterile glass bottle and prewarm at least to
◦
room temperature [faster growth will occur if the broth is prewarmed to (36± 2) C]. Add 100 ml of the sample
prewarmed to room temperature. Add 30 ml of an inoculum culture, in its exponential (log) growth phase, containing
8 8
approximately 2× 10 cells/ml to 5× 10 cells/ml (11.1). To ensure anaerobic conditions it is necessary to fill the
bottle completely with medium and to tighten the cap if the bottle is incubated under aerobic conditions. Incubate the
◦
culture at (36± 2) C for (21± 3) h. Gentle magnetic stirring during incubation is recommended.
To avoid the toxic effect of oxygen dissolved in the sample on the host cells, mainly for the presence/absence test, it
is recommended to treat the sample to remove oxygen. This can be achieved by either bubbling nitrogen for 5min at
5 l/min 0,04 %
a rate of or by the addition of a reducing solution, such as Na S (final mass concentration ). By adding
2
a resazurine solution (0,5 ml/100 ml of a solution having a concentration of 0,025 g/100 ml), anaerobiosis is
indicated by a change of colour from blue to straw.
Melt 50 ml of ssBPRMA (basal medium) (B.3) by putting the bottles in a boiling water bath (7.5). Then place them in
◦
a water bath (7.4) at (45± 1) C. Aseptically add haemin, Na CO and antibiotics and adjust pH to 6,8± 0,5 (B.1)
2 3
as indicated in Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps, which are placed in a water bath at
◦
(45± 1)C1. To each tube add ml of inoculum culture, in its exponential (log) growth phase, containing
8
approximately 2× 10 bacteria/ml (11.1). Mix carefully to avoid the formation of air bubbles and pour the contents
onto a layer of BPRMA (B.2) in a 90 mm Petri dish prewarmed to room temperature. Distribute the contents evenly,
allowing the agar to solidify on a horizontal, cool surface.
Transfer 1 ml of the enrichment culture to a centrifuge tube, add 0,4 ml of chloroform, mix well and centrifuge at
3 000g for 5min. Place one drop of the supernatant from each one of the chloroform-treated cultures onto the
inoculated plates using a fine capillary or a pipette. Do not damage the top agar layer. Leave the spot to dry and
◦
incubate the plates upside down in an anaerobic cabinet, jar or bag at (36± 2) C for (21± 3) h.
Examine the plate for a clear zone in the spotted area, which is indicative of the presence of B. fragilis phages in the
original sample.
This procedure will result in high-titre phage suspensions. Take appropriate precautions, for example working in a
biohazard cabinet or in a separate area of the laboratory, always disinfecting the work area by wiping over with 70 %
alcohol or a hypochlorite solution at the end of each work session.
NOTE This procedure can also be used in an MPN format (ISO 8199). More than one spot can be placed on the surface of an
inoculated plate.
11.4 Quality assurance
11.4.1 Plaque count procedure
For each series of samples examine, a procedural-blank using sterile diluent as the sample and a reference standard
preparation of bacteriophage B56-3, prepared as follows:
From a high titre phage culture (e.g. as described in annex D), prepare a decimal dilution series and plate out
according to 11.2. Store the dilution series in a refrigerator overnight. Count the number of plaques. From the dilution
series, prepare 100 ml to 1 000 ml of a suspension with an expected concentration of plaque-forming particles of
approximately 100/ml. Add 5 % (volume) of glycerol (B.7). Distribute into plastic vials in 2,4 ml aliquots and store at
◦
(−70± 10) C. Thaw vials of the reference control of B56-3 before use and plate out according to the procedure
used (11.2
...
SLOVENSKI STANDARD
SIST ISO 10705-4:2007
01-februar-2007
Kakovost vode - Ugotavljanje prisotnosti in števila bakteriofagov - 4. del – Štetje
bakteriofagov, ki okužijo Bacteroides fragilis
Water quality -- Detection and enumeration of bacteriophages -- Part 4: Enumeration of
bacteriophages infecting Bacteroides fragilis
Qualité de l'eau -- Détection et dénombrement des bactériophages -- Partie 4:
Dénombrement des bactériophages infectant Bacteroides fragilis
Ta slovenski standard je istoveten z: ISO 10705-4:2001
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST ISO 10705-4:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 10705-4:2007
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SIST ISO 10705-4:2007
INTERNATIONAL ISO
STANDARD 10705-4
First edition
2001-12-15
Water quality — Detection and enumeration
of bacteriophages —
Part 4:
Enumeration of bacteriophages infecting
Bacteroides fragilis
Qualité de l'eau — Détection et dénombrement des bactériophages —
Partie 4: Dénombrement des bactériophages infectant Bacteroides fragilis
Reference number
ISO 10705-4:2001(E)
©
ISO 2001
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
Contents Page
1 Scope . 1
2 Normative references . 1
3 Term and definition . 1
4 Safety precautions . 2
5 Principle . 2
6 Reagents . 2
7 Apparatus . 2
8 Microbiological reference cultures . 3
9 Sampling . 4
10 Preparation of test materials . 4
11 Procedure . 7
12 Expression of results . 9
13 Test report . 10
Annexes
A General description of bacteriophages infecting Bacteroides fragilis. 11
B Culture media and diluents . 12
C Alternative method for preparing inoculum cultures. 17
D Culturing of Bacteriophage B56-3. 18
Bibliography. 19
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technical
committees. Each member body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, governmental and non-governmental, in
liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this part of ISO 10705 may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO10705-4 was prepared by Technical Committee ISO/TC147, Water quality,
Subcommittee SC 4, Microbiological methods.
ISO 10705 consists of the following parts, under the general title Water quality — Detection and enumeration of
bacteriophages:
— Part 1: Enumeration of F-specific RNA bacteriophages
— Part 2: Enumeration of somatic coliphages
— Part 3: Validation of methods for concentration of bacteriophages from water
— Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
Annexes A, B, C and D of this part of ISO 10705 are for information only.
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SIST ISO 10705-4:2007
INTERNATIONAL STANDARD ISO 10705-4:2001(E)
Water quality — Detection and enumeration of bacteriophages —
Part 4:
Enumeration of bacteriophages infecting Bacteroides fragilis
1 Scope
This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides
fragilis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water,
sediments and sludge extracts, where necessary after dilution. In the case of low phage numbers, a pre-
concentration step may be necessary for which a separate International Standard has been developed. The method
is also applicable to shellfish extracts.
NOTE It is desirable for International Standards to be adopted as widely as possible. This part of ISO 10705 includes reference
to alternative procedures which obviate the need for expensive materials or equipment which may not be readily available in
developing countries. Use of these alternatives will not affect the performance of this method.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 10705. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 10705 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated references,
the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of
currently valid International Standards.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension
and decimal dilutions
ISO 8199, Water quality — General guide to the enumeration of micro-organisms by culture
3 Term and definition
For the purposes of this part of ISO 10705, the following term and definition applies.
3.1
bacteriophage infecting Bacteroides fragilis
bacterial virus which is capable of infecting selected Bacteroides fragilis host strains by attachment to the bacterial
cell wall as the first step of the infectious process
NOTE 1 Such bacteriophages produce visible plaques (clearance zones) in a confluent lawn of host bacteria grown under
appropriate culture conditions.
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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.
5Principle
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.).
6Reagents
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 ISO10705. 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.
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ISO 10705-4:2001(E)
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) C 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 9cm, 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 1cm.
7.22 Membrane filter units, for decontamination, having a pore size of 0,2µm, preferably low protein-binding
membranes, as for example, those composed of polyvinylidene difluoride.
7.23 Plastics vials, lidded, having a capacity of 3ml.
7.24 Glass vials, screw-capped, having a capacity of 3ml.
7.25 Syringes and needles.
7.26 Cotton swabs.
8 Microbiological reference cultures
[1]
The recommended host strain is Bacteroides fragilis RYC2056 (ATCC 700786).
Use bacteriophage B56-3 (ATCC 700786-B1) infecting Bacteroides fragilis RYC2056 for the preparation of reference
materials (11.4).
NOTE The ATCC strains are available from American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas,
VA 20110-2209, USA. This information is given for the convenience of users of this part of ISO 10705 and does not constitue an
endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
Figure 1 — Hungate glass tube with rubber stopper and screw cap
9 Sampling
Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2 and
ISO 5667-3.
10 Preparation of test materials
10.1 Culturing and maintenance of host strains
10.1.1 General
The culturing and maintenance of host strains involves several stages which are summarized in Figure 2.
Bacteroides fragilis is an obligate anaerobe. However, it does not require handling under conditions of strict
anaerobiosis. Incubation of cultures in solid media should be carried out in an anaerobic cabinet, or anaerobic jars or
bags. When using liquid media it is sufficient to ensure that containers are completely filled and closed with a screw
cap.
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
Figure 2 — Scheme for the culturing and maintenance of host strains
10.1.2 Preparation of stock cultures
Rehydrate the content of a lyophilized ampoule of the reference culture of the host strain in 1ml of Bacteroides
phage recovery medium broth (BPRMB) (B.1) using a Pasteur pipette (7.14). Inoculate the suspensions in 10 ml of
◦
BPRMB (B.1) in a 10 ml screw-capped glass tube (see 10.1.1) and incubate at (36± 2) C for (21± 3) h.
Aseptically soak a sterile cotton swab with the culture and streak it onto a plate of Bacteroides phage recovery
◦
medium agar (BPRMA) (B.2). Incubate the culture in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h.
Alternatively, if a culture in slant is available, streak it with a sterile cotton swab directly onto a plate of BPRMA (B.2).
◦
Incubate the culture in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h.
Inoculate cells (mass inoculation with a sterile cotton swab) from the plate into 10 ml of BPRMB (B.1) in a 10 ml
screw-capped glass tube (7.18). Be sure that the tube is completely filled (see 10.1.1). If dense growth occurs,
inoculate 1/81 of the growth onto the BPRMA plate; if poor growth occurs, use /2 of the growth onto the plate.
◦
Incubate the culture at (36± 2) C for (21± 3) h.
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
Mix culture and cryoprotector (B.6) in a ratio 1:1 (volume). Mix well avoiding bubble formation. Distribute into screw-
◦
capped, preferably glass, vials (7.24) in aliquots of approximately 1,0 ml and store at (−70± 10) C or in liquid
nitrogen for up to five years.
This first passage of the host strain should be stored as a reference in the laboratory. Purity of the culture should be
checked before storage by Gram staining, by testing absence of growth under aerobic conditions and by testing
sensitivity to a reference bacteriophage (i.e. B56-3).
10.1.3 Preparation of working cultures
Remove a vial of stock culture (10.1.2) from frozen storage, allow the vial to equilibrate to room temperature (i.e.
◦ ◦
C30 C
15 to ) and streak the culture with a sterile cotton swab onto a plate of BPRMA (B.2). Incubate the culture in
◦
an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h. Inoculate cell material (mass inoculation with a sterile
cotton swab) from the plate into of prewarmed BPRMB (B.1) kept in a screw-capped glass tube (see
10 ml 10 ml
10.1.1). If dense growth occurs, inoculate 1/8 of the growth onto the BPRMA plate; if poor growth occurs, use 1/2 of
◦
(36± 2) C (21± 3) h
the growth onto the plate. Incubate the culture at for .
Add BPRMB (B.1) to a tube for anaerobic cultures (7.18) and warm to at least room temperature [faster growth will
◦
occur if the broth is prewarmed to (36± 2) C]. Transfer an aliquot of the above-mentioned culture, without shaking
the tube and taking the aliquot from the middle part of the tube, to the tube containing prewarmed BPRMB in a ratio
of culture-to-BPRM of 1,5:10 (volume). Be sure that the inoculated tube is completely filled (see 10.1.1). Incubate the
◦ 9
culture at (36± 2)C2 to reach approximately × 10 cfu/ml.
Mix working culture and cryoprotector (B.6) in a ratio of 1:1 (volume) avoiding bubble formation. Distribute into screw-
◦
capped, preferably glass, vials (7.24) in aliquots of approximately 1,5 ml and store at (70± 10) C for a maximum
of 12 months.
Ensure that the culture does not reach the stationary phase before mixing it with the cryoprotector. Absorbance
stabilization will indicate the end of the log phase, which may last 5h to 8h.
10.2 Calibration of absorbance measurements for counts of viable host bacteria
Remove a vial of working culture (10.1.3) from the deep freeze (7.9) and allow the vial to equilibrate to room
◦ ◦
temperature (i.e. 15C3 to 0 C). Add BPRMB (B.1) to a tube for anaerobic cultures (7.18) and warm to at least
◦
room temperature [faster growth will occur if the broth is prewarmed to (36± 2) C]. Before inoculation, adjust the
spectrophotometer to zero. Transfer the working culture into BPRMB (B.1) in a ratio of respectively 1:10 (volume),
completely filling the tube. Tubes for anaerobic cultures may be inoculated/sampled by puncture with sterile syringes
◦
and needles (7.25). Incubate the culture at (36± 2)C3. Every 0min, measure the absorbance (using 7.10) and
withdraw, by puncture, a 0,3 ml sample for viable cell counts. Ensure that the tube is out of the incubator for as short
a time as possible.
Melt 50 ml of semi-solid BPRMA (ssBPRMA) (B.3) (basal agar) by putting bottles in a boiling water bath. Then place
◦
the bottles in a water bath at (45± 1) C. Aseptically add haemin solution, Na CO and antibiotics and adjust pH to
2 3
6,8± 0,5 (B.1) according to Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps, placed in a water bath
◦
at .(45± 1) C
−8 −6 −7 −8
Dilute the aliquots sampled from the culture to 10 and add 1ml volumes of the 10 , 10 and 10 dilutions to
each tube of 2,5 ml of melted ssBPRMA, in duplicate. Pour onto a layer of BPRMA in a 90 mm Petri dish (B.2)
prewarmed at room temperature. Distribute evenly, allow to solidify on an horizontal, cool surface and incubate the
◦
plates upside down in an anaerobic cabinet, jar or bag at (36± 2) C for (44± 4) h. Ensure that the process is
performed in a period of time as short as possible and that the diluents had been autoclaved immediately just before
use (to have them free of oxygen). Be sure that the diluents have cooled to room temperature before use. Count the
total number of colonies in each plate yielding between 30 colonies and 300 colonies and calculate the number of
cfu/ml (consult ISO 8199 if necessary).
This procedure should be carried out several times (approximately 4 to 5 times) to establish the relationship between
absorbance measurements and colony counts. If sufficient data have been obtained, further work can then be based
only on absorbance measurements.
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SIST ISO 10705-4:2007
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11 Procedure
11.1 Preparation of inoculum cultures
Remove a vial of working culture (10.1.3) from the deep freeze (7.9) and allow the vial to equilibrate to room
◦ ◦
temperature, (i.e. 15C3 to 0 C). Add BPRMB (B.1) to a screw-capped tube and warm to at least room temperature
◦
[faster growth will occur if the broth is prewarmed to (36± 2) C]. Before inoculation, adjust the spectrophotometer
to zero for the tube (10.2). Transfer the working culture into the tube filled with BPRMB (B.1) in a ratio of 1:10
◦
(volume). Be sure that the tube is completely filled (see 10.1.1). Incubate the culture at (36± 2)C2. After h,
measure the absorbance of the culture every 30 min. At an absorbance corresponding to a cell density of
8
approximately 2× 10 cfu/ml (based on data obtained in 10.2) take the inoculum culture from the incubator and
either use it immediately or quickly cool the culture by placing it in melting ice. Use the culture within 6h. Cell
8 8
densities ranging from 1× 10 cfu/ml to 4× 10 cfu/ml give similar plaque counts, however cell densities of
8 8
1× 10 cfu/ml or 2× 10 cfu/ml give larger plaques.
An alternative method for preparing inoculum cultures is given in annex C.
8
NOTE If the cell density of approximately 2× 10 cfu/ml is not reached within 3 h, it is possible to increase the amount of
working culture transferred into the BPRMB to a ratio of respectively 1,5:10 (volume).
11.2 Standard procedure
◦ ◦
Prepare an inoculum culture as described in 11.1. Allow sample to equilibrate to room temperature (15C3 to 0 C).
Melt 50 ml of ssBPRMA (B.3) (basal agar) by putting the bottles in a boiling water bath (7.5) then place them in a
◦
water bath at (45± 1) C. Aseptically add haemin, Na CO and antibiotics and adjust the pH to 6,8± 0,5 (B.1)
2 3
according to Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps (7.17), which are placed in a water bath
◦
at .(45± 1) C
To each culture tube (7.17), add 1ml of sample (or diluted or concentrated sample) prewarmed to room temperature.
Examine each aliquot at least in duplicate.
Add 1ml of inoculum culture to each culture tube containing the aliquots of sample and ssBPRMA, then mix carefully
avoiding the formation of air bubbles and pour the contents on a layer of complete BPRMA (B.2) in a 90 mm Petri dish
prewarmed to room temperature. Distribute the contents evenly allowing the agar to solidify on a horizontal, cool
surface. B. fragilis is an anaerobic bacterium. Therefore, once dried, distribute inoculated tubes as fast as possible
and place the plates into the anaerobic jars as soon as possible. Incubate the plates upside down in an anaerobic
◦
cabinet, jar or bag at (36± 2) C for (21± 3) h.
After incubation, count the number of plaques on each plate. If it is not possible to count the plates after finishing
◦
incubation, keep the plates at (5± 3) C until reading.
For samples containing high background flora, it is recommended to decontaminate the samples by filtration through
low protein-binding membranes, as for example, those composed of polyvinylidene difluoride 0,2µm pore size
(7.22), or to increase the amount of ssBPRMA kanamycine monosulfate to obtain a final concentration in the final
−1 −1
medium of 300µgml instead of 100µgml (the concentration recommended for growing B. fragilis).
NOTE 1 Freshly prepared triphenyltetrazoliumchloride solution (B.10) can be added to enhance contrast for counting plaques. If
this procedure is used, plates should remain under aerobic conditions for 1h to 2h before counting plaques.
NOTE 2 The addition of ice-cold inoculum culture (11.1) to the ssBPRMA may lead to a sharp drop in temperature and
solidification of the medium. To avoid this, prewarm the ice-cold inoculum culture to room temperature before adding it to the tubes
containing ssBPRMA and the aliquot of the sample.
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SIST ISO 10705-4:2007
ISO 10705-4:2001(E)
11.3 Presence/absence test
This test can be used to examine the presence of bacteriophages in different volumes of sample. For instance, to
determine the presence/absence in 100 ml, proceed as follows:
Add 100 ml of double-strength BPRMB (B.9) to a 250 ml screw-capped sterile glass bottle and prewarm at least to
◦
room temperature [faster growth will occur if the broth is prewarmed to (36± 2) C]. Add 100 ml of the sample
prewarmed to room temperature. Add 30 ml of an inoculum culture, in its exponential (log) growth phase, containing
8 8
approximately 2× 10 cells/ml to 5× 10 cells/ml (11.1). To ensure anaerobic conditions it is necessary to fill the
bottle completely with medium and to tighten the cap if the bottle is incubated under aerobic conditions. Incubate the
◦
culture at (36± 2) C for (21± 3) h. Gentle magnetic stirring during incubation is recommended.
To avoid the toxic effect of oxygen dissolved in the sample on the host cells, mainly for the presence/absence test, it
is recommended to treat the sample to remove oxygen. This can be achieved by either bubbling nitrogen for 5min at
5 l/min 0,04 %
a rate of or by the addition of a reducing solution, such as Na S (final mass concentration ). By adding
2
a resazurine solution (0,5 ml/100 ml of a solution having a concentration of 0,025 g/100 ml), anaerobiosis is
indicated by a change of colour from blue to straw.
Melt 50 ml of ssBPRMA (basal medium) (B.3) by putting the bottles in a boiling water bath (7.5). Then place them in
◦
a water bath (7.4) at (45± 1) C. Aseptically add haemin, Na CO and antibiotics and adjust pH to 6,8± 0,5 (B.1)
2 3
as indicated in Table B.1. Distribute 2,5 ml aliquots into culture tubes with caps, which are placed in a water bath at
◦
(45± 1)C1. To each tube add ml of inoculum culture, in its exponential (log) growth phase, containing
8
approximately 2× 10 bacteria/ml (11.1). Mix carefully to avoid the formation of air bubbles and pour the contents
onto a layer of BPRMA (B.2) in a 90 mm Petri dish prewarmed to room temperature. Distribute the contents evenly,
allowing the agar to solidify on a horizontal, cool surface.
Transfer 1 ml of the enrichment culture to a centrifuge tube, add 0,4 ml of chloroform, mix well and centrifuge at
3 000g for 5min. Place one drop of the supernatant from each one of the chloroform-treated cultures onto the
inoculated plates using a fine capillary or a pipette. Do not damage the top agar layer. Leave the spot to dry and
◦
incubate the plates upside down in an anaerobic cabinet, jar or bag at (36± 2) C for (21± 3) h.
Examine the plate for a clear zone in the spotted area, which is indicative of the presence of B. fragilis phages in the
original sample.
This procedure will result in high-titre phage suspensions. Take appropriate precautions, for example working in
...
NORME ISO
INTERNATIONALE 10705-4
Première édition
2001-12-15
Qualité de l'eau — Détection et
dénombrement des bactériophages —
Partie 4:
Dénombrement des bactériophages
infectant Bacteroides fragilis
Water quality — Detection and enumeration of bacteriophages —
Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
Numéro de référence
ISO 10705-4:2001(F)
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ISO 2001
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ISO 10705-4:2001(F)
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ISO 10705-4:2001(F)
Sommaire Page
1 Domaine d'application . 1
2 Références normatives . 1
3 Terme et définition . 2
4 Précautions relatives à la sécurité . 2
5 Principe . 2
6 Réactifs . 2
7 Appareillage . 3
8 Cultures microbiologiques de référence . 5
9 Échantillonnage . 5
10 Préparation des matériaux d'essai . 5
11 Mode opératoire . 7
12 Expression des résultats . 10
13 Rapport d'essai . 11
Annexes
A Description générale des bactériophages infectant Bacteroides fragilis. 12
B Milieux de culture et diluants. 13
C Méthode alternative pour la préparation des précultures. 18
D Mise en culture du bactériophage B56-3 . 19
Bibliographie. 20
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ISO 10705-4:2001(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée aux
comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du comité
technique créé à cet effet. Les organisations internationales, gouvernementales et non gouvernementales, en liaison
avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec la Commission électrotechnique
internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI, Partie 3.
Les projets de Normes internationales adoptés par les comités techniques sont soumis aux comités membres pour
vote. Leur publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments de la présente partie de l'ISO 10705 peuvent faire l'objet
de droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne pas
avoir identifié de tels droits de propriété et averti de leur existence.
La Norme internationale ISO 10705-4 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-
comité SC 4, Méthodes microbiologiques.
L'ISO 10705 comprend les parties suivantes, présentées sous le titre général Qualité de l'eau — Détection et
dénombrement des bactériophages:
— Partie 1: Dénombrement des bactériophages ARN F spécifiques
— Partie 2: Dénombrement des coliphages somatiques
— Partie 3: Validation des méthodes de concentration des bactériophages dans l'eau
— Partie 4: Dénombrement des bactériophages infectant Bacteroides fragilis
Les annexes A, B, C et D de la présente partie de l'ISO 10705 sont données uniquement à titre d'information.
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NORME INTERNATIONALE ISO 10705-4:2001(F)
Qualité de l'eau — Détection et dénombrement des
bactériophages —
Partie 4:
Dénombrement des bactériophages infectant Bacteroides fragilis
1 Domaine d'application
La présente partie de l'ISO 10705 spécifie une méthode de détection et de dénombrement des bactériophages
infectant la bactérie Bacteroides fragilis par incubation de l'échantillon avec une souche-hôte appropriée. La
méthode est applicable à tous les types d'eaux, aux extraits de sédiments et de boues si nécessaire après dilution.
Dans le cas de faibles populations de phages, une étape de préconcentration peut s'avérer nécessaire pour laquelle
une Norme internationale distincte a été élaborée. La méthode est également applicable aux extraits de coquillages.
NOTE Il est souhaitable que les Normes internationales soient adoptées le plus largement possible. La présente partie de
l'ISO 10705 comporte des références à des procédures alternatives qui évitent d'avoir à utiliser du matériel ou des équipements
coûteux qui peuvent ne pas être aisément disponibles dans les pays en voie de développement. L'utilisation de ces procédures
alternatives n'affectera pas les performances de la présente méthode.
2Références normatives
Les documents normatifs suivants contiennent des dispositions qui, par suite de la référence qui y est faite,
constituent des dispositions valables pour la présente partie de l'ISO 10705. Pour les références datées, les
amendements ultérieurs ou les révisions de ces publications ne s'appliquent pas. Toutefois, les parties prenantes
aux accords fondés sur la présente partie de l'ISO 10705 sont invitées à rechercher la possibilité d'appliquer les
éditions les plus récentes des documents normatifs indiqués ci-après. Pour les références non datées, la dernière
édition du document normatif en référence s'applique. Les membres de l'ISO et de la CEI possèdent le registre des
Normes internationales en vigueur.
ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d'essai
ISO 5667-1, Qualité de l'eau — Échantillonnage — Partie 1: Guide général pour l'établissement des programmes
d'échantillonnage
ISO 5667-2, Qualité de l'eau — Échantillonnage — Partie 2: Guide général sur les techniques d'échantillonnage
ISO 5667-3, Qualité de l'eau — Échantillonnage — Partie 3: Guide général pour la conservation et la manipulation
des échantillons
ISO 6887-1, Microbiologie des aliments — Préparation des échantillons, de la suspension mère et des dilutions
décimales en vue de l'examen microbiologique — Partie 1: Règles générales pour la préparation de la suspension
mère et des dilutions décimales
ISO 8199, Qualité de l'eau — Guide général pour le dénombrement des micro-organismes sur milieu de culture
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ISO 10705-4:2001(F)
3 Terme et définition
Pour les besoins de la présente partie de l'ISO 10705, le terme et la définition suivants s'appliquent.
3.1
bactériophage infectant Bacteroides fragilis
virus des bactéries capable d'infecter certaines souches-hôtes sélectionnées de Bacteroides fragilis en se fixant sur
la paroi cellulaire au début du processus d'infection
NOTE 1 De tels bactériophages provoquent l'apparition de plages visibles (zones de lyse) sur un tapis confluent de bactéries-
hôtes cultivées dans des conditions appropriées.
NOTE 2 Une description générale des bactériophages infectant B. fragilis est donnée dans l’annexe A.
4Précautions relatives à la sécurité
La souche-hôte utilisée pour les besoins de la présente partie de l'ISO 10705 est une souche non pathogène pour
l'homme et l'animal, et qu'il convient de manipuler conformément aux procédures normales (nationales ou
internationales) des laboratoires d'analyses bactériologiques. Les bactériophages infectant Bacteroides fragilis ne
sont pathogènes ni pour l'homme ni pour l'animal, mais certains types sont très résistants à la dessiccation. Des
précautions appropriées doivent être prises pour éviter tout risque de contamination croisée des échantillons, en
particulier lors de l'examen ou de la manipulation de cultures de concentration élevée ou lors de l'ensemencement
de cultures de souches-hôtes. Ces opérations doivent être réalisées dans une enceinte pour risque biologique ou
dans une zone séparée du laboratoire. Le chloroforme est une substance cancérigène. Prendre les précautions de
sécurité applicables ou utiliser une autre méthode d'efficacité égale.
Il est recommandé que le personnel en charge d'appliquer la présente méthode ait acquis ou acquière un certain
niveau d'expérience dans la manipulation des bactéries anaérobies.
5Principe
L'échantillon est mélangé à un faible volume de milieu de culture nutritif semi-solide. Une culture de la souche-hôte
y est ajoutée et le mélange est coulé dans une boîte contenant du milieu de culture nutritif solide. Après cela, les
boîtes sont incubées et examinées pour y repérer l'apparition de plages. Les résultats son exprimés en nombre de
particules formant plages (également identifiées par unités formant plages, ufp) par unité de volume (ufp/ml, ufp/l,
etc.).
6Réactifs
6.1 Matériaux de base.
Pour la préparation des milieux de culture et des réactifs, utiliser des produits de qualité constante ainsi que des
réactifs de qualité analytique, et suivre les instructions données dans l'annexe B. Pour toute information relative à la
conservation, se reporter à l’ISO 8199, sauf indication spécifique donnée dans la présente partie de l'ISO 10705.
Des milieux de culture complets déshydratés peuvent également être utilisés. Dans ce cas, suivre scrupuleusement
les instructions du fabricant.
Des produits chimiques d'autres qualités peuvent être utilisés, pourvu qu’il puisse être démontré qu’ils conduisent
aux mêmes résultats.
6.2 Eau, pour la préparation des milieux de culture, distillée dans des récipients en verre ou déminéralisée,
exempte de substances susceptibles d'inhiber la croissance des bactéries dans les conditions de l'essai, et conforme
à l’ISO 3696.
6.3 Diluant, pour faire des dilutions d'échantillon, tel qu’une solution peptonée saline ou tout autre diluant
conforme à l’ISO 6887-1.
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ISO 10705-4:2001(F)
7 Appareillage
En dehors des appareils fournis sous forme stérile, stériliser la verrerie et les autres matériels conformément à
l'ISO 8199.
Matériel et verrerie de laboratoire stériles d'usage courant en microbiologie, ou matériel à usage unique en plastique
conformes à l'ISO 8199, et comprenant notament les éléments suivants:
7.1 Étuve à air chaud, pour stérilisation en chaleur sèche, et autoclave.
◦
7.2 Incubateur ou bain d'eau, maintenu thermostatiquement à .(36± 2) C
◦
7.3 Incubateur ou bain d'eau, maintenu thermostatiquement à (36± 2) Cet équipé d'un agitateur.
◦
7.4 Bain d'eau ou bloc chauffant, maintenu thermostatiquement à .(45± 1) C
7.5 Bain d'eau ou dispositif équivalent, pour faire fondre la gélose.
7.6 pH-mètre et papier indicateur de pH.
7.7 Appareil de comptage, équipé d'une source de lumière indirecte, oblique.
◦
7.8 Congélateur, maintenu thermostatiquement à .(−20± 5) C
◦
7.9 Congélateur, maintenu thermostatiquement à (−70± 10) Cou réservoir de stockage à azote liquide.
7.10 Spectrophotomètre, équipé d'un filtre pour la gamme de longueurs d'onde allant de 500 nm à 650 nmavec
± 10 nm 1 cm
une largeur de bande maximale de , capable de contenir des cuves de de trajet optique (7.21) ou des
tubes en verre de Hungate (7.20) avec un bouchon en caoutchouc butyle et une capsule à vis, ou encore des tubes
de culture en verre à capsule à vis.
7.11 Enceinte anaérobie, ou jarres ou sachets anaérobies, ainsi que générateurs d'anaérobiose et
indicateurs d'anaérobiose.
◦
7.12 Réfrigérateur, réglé à une température de (5± 3) C.
7.13 Boîtes de Petri, ventilées, de 9cm de diamètre.
7.14 Pipettes graduées, de 0,1 ml, 1 ml, 5 ml et 10 ml de capacité et pipettes Pasteur.
7.15 Flacons en verre, de volume approprié.
7.16 Flacons en verre à capsule à vis, de volume approprié.
7.17 Tubes de culture, avec capsules ou dispositifs équivalents.
7.18 Tubes de culture en verre à capsule à vis.
7.19 Éprouvettes graduées, de capacité appropriée.
7.20 Tubes en verre de Hungate, avec bouchon en caoutchouc butyle et capsule à vis, ou tubes en verre à
capsule à vis pouvant s'adapter sur le spectrophotomètre (voir la Figure 1).
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ISO 10705-4:2001(F)
Figure 1 — Tube en verre de Hungate avec bouchon en caoutchouc et capsule à vis
7.21 Cuves optiques, de 10 mm de trajet optique.
7.22 Unités de filtration sur membrane, pour la décontamination, de 0,2µm de porosité, de préférence des
membranes à faible pouvoir de fixation des protéines, par exemple celles composées de difluorure de polyvinylidène.
7.23 Tubes en plastique, bouchés, de 3ml de capacité.
7.24 Tubes en verre, à capsule à vis, de 3ml de capacité.
7.25 Seringues et aiguilles.
7.26 Tampons d’ouate.
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ISO 10705-4:2001(F)
8 Cultures microbiologiques de référence
[1]
La souche recommandée est Bacteroides fragilis RYC2056 (ATCC 7007786) .
Utiliser le bactériophage B56-3 (ATCC 700786-B1) infectant la bactérie Bacteroides fragilis RYC2056 pour la
préparation des matériaux de référence (11.4).
NOTE Les souches ATCC sont disponibles auprès de American Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, VA 20110-2209, USA. Cette information est donnée à l’intention des utilisateurs de la présente partie de l'ISO 10705
et ne signifie nullement que l’ISO approuve ou recommande l’emploi exclusif du produit ainsi désigné. Des produits équivalents
peuvent être utilisés s’il est démontré qu’ils conduisent aux mêmes résultats.
9 Échantillonnage
Prélever des échantillons et les transporter au laboratoire conformément à l'ISO 8199, l'ISO 5667-1, l'ISO 5667-2 et
l'ISO 5667-3.
10 Préparation des matériaux d'essai
10.1 Mise en culture et entretien des souches-hôtes
10.1.1 Généralités
La mise en culture et l'entretien des souches-hôtes impliquent différentes étapes qui sont résumées sur la Figure 2.
La bactérie Bacteroides fragilis est une anaérobie stricte. Elle ne nécessite toutefois pas une manipulation dans des
conditions d'anaérobiose stricte. Il convient de procéder à l'incubation des cultures en milieux solides dans une
enceinte anaérobie, ou dans des jarres ou des sachets anaérobies. En cas d'utilisation de milieux liquides, il suffit de
s'assurer que les récipients sont entièrement remplis et bouchés avec une capsule à vis.
10.1.2 Préparation des cultures mères
Réhydrater le contenu d'une ampoule lyophilisée de la culture de référence de la souche-hôte dans 1ml de bouillon
de culture pour l’obtention des phages infectant Bacteroides (BPRMB) (B.1) à l'aide d'une pipette Pasteur (7.14).
Inoculer la suspension dans un tube en verre à capsule à vis de 10 ml (voir 10.1.1) contenant 10 ml de milieu
◦
BPRMB (B.1), puis incuber à (36± 2) C pendant (21± 3) h. En conditions d'asepsie, tremper un tampon d’ouate
stérile dans la culture et ensemencer dans une boîte contenant de la gélose de culture pour l’obtention des phages
◦
infectant Bacteroides (BPRMA) (B.2). Incuber dans une enceinte, une jarre ou un sachet anaérobie à (36± 2) C
pendant .(44± 4) h
Alternativement, si une culture inclinée est disponible, ensemencer avec un tampon d’ouate stérile directement dans
une boîte contenant du milieu BPRMA (B.2). Incuber dans une enceinte, une jarre ou un sachet anaérobie à
◦
(36± 2) C pendant (44± 4) h.
Inoculer les cellules (ensemencement de masse avec un tampon d’ouate stérile) de la boîte dans un tube en verre à
capsule à vis de 10 ml (7.18) contenant 10 ml de milieu BPRMB (B.1). S'assurer que le tube est entièrement rempli
(voir 10.1.1). En cas de croissance dense, inoculer 1/8 de la culture dans la boîte de BPRMA; en cas de croissance
◦
peu dense, utiliser la moitié de la culture dans la boîte. Incuber à (36± 2) Cpendant (21± 3) h.
Mélanger la culture et le cryoprotecteur (B.6) dans une proportion de 1:1 (en volume). Bien mélanger en évitant la
formation de bulles. Répartir dans des tubes à capsule à vis, de préférence en verre (7.24), par portions aliquotes de
◦
1,0 ml environ, et conserver à (−70± 10) C ou dans de l'azote liquide pendant cinq ans au maximum.
Il convient de conserver cette première culture de souche-hôte comme référence dans le laboratoire. Il convient de
vérifier la pureté de la culture avant stockage par la méthode de coloration de Gram, en contrôlant l'absence de
croissance en conditions aérobies et en testant la sensibilité à un bactériophage de référence (soit B56-3).
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ISO 10705-4:2001(F)
Figure 2 — Schéma pour la mise en culture et l'entretien de la souche-hôte
10.1.3 Préparation des cultures d'essai
◦ ◦
Retirer un tube de culture mère (10.1.2) du congélateur, laisser s'équilibrer à température ambiante (15 C3 à )0 C
et ensemencer avec un tampon d’ouate stérile dans une boîte contenant du milieu BPRMA (B.2). Incuber dans une
◦
enceinte, une jarre ou un sachet anaérobie à (36± 2) C pendant (44± 4) h. Inoculer le matériau cellulaire
(ensemencement de masse avec un tampon d’ouate stérile) de la boîte dans un tube en verre à capsule à vis de
10 ml (voir 10.1.1) contenant 10 ml de milieu BPRMB (B.1) préchauffé. En cas de croissance dense, inoculer 1/8 de
la culture dans la boîte de BPRMA; en cas de croissance peu dense, utiliser la moitié de la culture dans la boîte.
◦
Incuber à (36± 2) Cpendant (21± 3) h.
Ajouter du milieu BPRMB (B.1) dans un tube pour cultures anaérobies (7.18) et réchauffer au moins à température
◦
ambiante [la croissance sera plus rapide si le bouillon de culture est préchauffé à ]. (36± 2) CTransférer une
portion aliquote de la culture susmentionnée, sans agiter le tube et en prélevant l'aliquote dans la partie centrale du
tube, dans le tube contenant le milieu BPRMB préchauffé dans une proportion culture: BPRMB de 1,5:10 (en
◦
volume). S'assurer que le tube ensemencé est entièrement rempli (voir 10.1.1). Incuber à (36± 2) Cpour
9
atteindre environ 2× 10 ufc/ml.
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ISO 10705-4:2001(F)
Mélanger la culture d'essai et le cryoprotecteur (B.6) dans un proportion de 1:1 (en volume). Bien mélanger en
évitant la formation de bulles. Répartir dans des tubes à capsule à vis, de préférence en verre (7.24), par portions
◦
aliquotes de 1,5 ml environ, et conserver à (−70± 10) C pendant douze mois au maximum.
S'assurer que la culture n'atteint pas la phase stationnaire avant de la mélanger avec le cryoprotecteur. La
stabilisation de la densité optique indiquera la fin de la phase exponentielle qui peut durer de 5h à .8h
10.2 Étalonnage des mesurages d'absorbance pour le dénombrement des bactéries-hôtes
cultivables
◦
Retirer un tube de culture d'essai (10.1.3) du congélateur (7.9) et laisser s'équilibrer à température ambiante (15 C
◦
à 30 C). Ajouter du milieu BPRMB (B.1) dans un tube pour cultures anaérobies (7.18) et réchauffer à température
◦
ambiante [la croissance sera plus rapide si le bouillon de culture est préchauffé à une température de (36± 2) C].
Avant de procéder à l'ensemencement, régler le spectrophotomètre sur zéro. Transférer la culture d'essai dans le
milieu BPRMB (B.1) dans une proportion de 1:10 (en volume) en remplissant entièrement le tube. Les tubes pour
cultures anaérobies peuvent être ensemencés/échantillonnés par ponction avec des seringues et des aiguilles
◦
stériles (7.25). Incuber à (36± 2)C3. Mesurer l'absorbance toutes les 0min (à l'aide du spectrophotomètre 7.10)
et prélever par ponction un échantillon de 0,3 ml pour le dénombrement des bactéries cultivables, en ayant soin de
réduire au minimum la durée de sortie du tube de l'incubateur.
Faire fondre 50 ml de gélose semi-solide de culture pour l’obtention des phages infectant Bactéroides (ssBPRMA)
◦
(B.3) (gélose de base) en plaçant les flacons dans un bain d'eau bouillante, puis dans un bain d'eau à .(45± 1) C
En conditions d'asepsie, ajouter la solution d'hémine, le Na CO et les antibiotiques, puis ajuster la valeur du pH à
2 3
6,8± 0,5 (B.1), conformément aux indications du Tableau B.1. Répartir des portions aliquotes de 2,5 ml dans des
◦
tubes de culture à capsule, placés dans un bain d'eau à .(45± 1) C
−8 −6 −7 −8
Diluer les aliquotes prélevées de la culture à 10 et ajouter 1ml des dilutions à , 10 10 et 10 dans chaque
2,5 ml
tube de de ssBPRMA fondue, ceci en double. Verser sur une couche de BPRMA dans une boîte de Petri de
90 mm (B.2) préchauffée à température ambiante. Répartir uniformément, laisser se solidifier sur une surface froide
◦
(36± 2) C
horizontale et incuber les boîtes retournées dans une enceinte, une jarre ou un sachet anaérobie à
pendant (44± 4) h. S'assurer que la procédure est exécutée dans le laps de temps le plus court possible, et que
les diluants ont bien été passés à l'autoclave juste avant utilisation (pour en éliminer l'oxygène). S'assurer également
que les diluants ont été refroidis à température ambiante avant utilisation. Compter le nombre total de colonies dans
chaque boîte contenant entre 30 et 300 colonies et calculer le nombre d’unités formant colonie par millilitres (ufc/ml)
(se reporter à l'ISO 8199 si nécessaire).
Il convient de répéter cette opération plusieurs fois (environ 4 à 5 fois) afin d'établir la relation entre les mesures
d'absorbance et le nombre de colonies. Si suffisamment de données ont été obtenues, les essais ultérieurs pourront
être basés uniquement sur les mesures d'absorbance.
11 Mode opératoire
11.1 Préparation des précultures
◦
Retirer un tube de culture d'essai (10.1.3) du congélateur (7.9) et laisser s'équilibrer à température ambiante (15 C
◦
à 30 C). Ajouter du BPRMB (B.1) dans un tube à capsule à vis et réchauffer au moins à température ambiante [la
◦
croissance sera plus rapide si le bouillon de culture est préchauffé à une température de (36± 2) C]. Avant de
procéder à l'ensemencement, régler le spectrophotomètre sur zéro avec le tube (voir 10.2). Transférer la culture
d'essai dans le tube rempli de BPRMB (B.1) dans une proportion de 1:10 (en volume) en s'assurant que le tube est
◦
entièrement rempli (voir 10.1.1). Incuber à . (36± 2)C2Après h, mesurer l'absorbance toutes les 30min. Lorsque
8
l'absorbance correspond à une densité cellulaire d'environ 2× 10 ufc/ml (sur la base des données obtenues en
10.2), sortir la préculture de l'incubateur; l'utiliser immédiatement ou la refroidir rapidement en la plaçant sur de la
8
glace fondante. Utiliser la préculture dans un délai de 6h. Les densités cellulaires comprises entre 1× 10 ufc/ml et
8 8
4× 10 ufc/ml donnent des nombres de plages similaires. Cependant, des densités cellulaires de 1× 10 ufc/ml ou
8
2× 10 ufc/ml donnent des plages plus importantes.
Une méthode alternative pour la préparation des précultures est donnée à l’annexe C.
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ISO 10705-4:2001(F)
8
NOTE Si une densité cellulaire de l'ordre de 2× 10 ufc/ml n'est pas atteinte dans un délai de 3 h, il est possible d'augmenter la
quantité de culture d'essai transférée dans le milieu BPRMB à une proportion de 1,5:10 (en volume).
11.2 Mode opératoire standard
◦
15 C
Préparer une préculture comme décrit en 11.1. Laisser l'échantillon s'équilibrer à température ambiante ( à
◦
30C5). Faire fondre 0ml de milieu ssBPRMA (B.3) (gélose de base) en plaçant les flacons dans un bain d'eau
◦
bouillante (7.5), puis dans un bain d'eau à . En conditions d'asepsie, ajouter la solution d'hémine, le
(45± 1) C
Na CO et les antibiotiques, puis ajuster la valeur du pH à 6,8± 0,5 (B.1) conformément aux indications du
2 3
Tableau B.1. Répartir des portions aliquotes de 2,5 ml dans des tubes de culture à capsule (7.17), placés dans un
◦
bain d'eau à .(45± 1) C
Ajouter 1mld'échantillon (ou d'échantillon dilué ou concentré) préalablement chauffé à température ambiante dans
chaque tube de culture (7.17). Examiner chaque aliquote au moins en double.
Ajouter 1ml de préculture dans chaque tube de culture contenant les aliquotes d'échantillon et le milieu ssBPRMA,
mélanger soigneusement en évitant la formation de bulles d'air et verser le contenu sur une couche de milieu
BPRMA complet (B.2) dans une boîte de Petri de 90 mm préalablement chauffée à température ambiante. Répartir
de façon homogène, laisser se solidifier sur une surface froide horizontale. B. fragilis est une bactérie anaérobie.
C'est pourquoi il faut répartir les tubes ensemencés le plus rapidement possible et placer les boîtes dans des jarres
anaérobies dès que possible après séchage. Incuber les boîtes retournées dans une enceinte, une jarre ou un
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sachet anaérobie à (36± 2) Cpendant (21± 3) h.
Après incubation, compter le nombre de plages dans chaque boîte. S'il n'est pas possible de dénombrer les plages
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après avoir terminé l'incubation, conserver les boîtes à (5± 3) Cjusqu'à la lecture.
Pour les échantillons contenant une flore résiduelle importante, il est recommandé de les décontaminer par filtration
à travers des membranes à faible pouvoir de fixation des protéines, comme par exemple celles composées de
difluorure de polyvinylidène d'une porosité de 0,2µm (7.22), ou d'ajouter du monosulfate de kanamycine au milieu
ssBPRMA jusqu’à une concentration finale dans le milieu final de 300µg/ml au lieu de 100µg/ml, ce qui correspond
à la concentration recommandée pour les cultures de B. fragilis.
NOTE 1 Une solution de chlorure de triphényltétrazolium (B.10) préparée extemporanément peut être ajoutée afin d'augmenter
le contraste pour le comptage des plages. Si ce mode opératoire est appliqué, il convient que les boîtes restent en conditions
anaérobies pendant 1h à 2h avant de procéder au comptage des plages.
NOTE 2 L'ajout de la préculture refroidie dans la glace (11.1) au milieu ssBPRMA peut provoquer une chute rapide de la
température et la solidification du milieu. Pour éviter cela, préchauffer la préculture refroidie dans la glace à la température
ambiante avant de l'ajouter dans les tubes contenant le milieu ssBPRMA et l'aliquote d'échantillon.
11.3 Essai de présence/absence
Cet essai peut être utilisé pour examiner la présence de bactériophages dans différents volumes d'échantillon. Par
exemple, pour l'essa
...
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