International Standard
INTERNATIONAL ORGANIZATION FOR STANDARDIZATlON.ME>K~YHAPOflHAR OPTAHM3AL&lR I-IO CTAH~APTM3AL(MM*ORGANlSATlON INTERNATIONALE DE NORMALISATION
Water quality - Evaluation of membrane filters used
for microbiological analyses
Qua/it6 de l eau - Evaha tion des membranes filtran tes u tifis&es pour des analyses microbiologiques
First edition - 1985-03-15
iTeh STANDARD PREVIEW
(standards.iteh.ai)
ISO 7704:1985
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
a6c27509f037/iso-7704-1985
UDC 628.163.067 : 579.68.08 Ref. No. IS0 7704-1985 (E)
Descriptors : water, quality, microbiological analysis, membranes, filters, tests.
Prrce based on 4 pages
Preisgr.C
Foreword
IS0 (the international Organization for Standardization) is a worldwide federation of
national standards bodies (IS0 member bodies). The work of preparing International
Standards is normally carried out through IS0 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, govern-
mental and non-governmental, in liaison with ISO, also take part in the work.
Draft International Standards adopted by the technical committees are circulated to
the member bodies for approval before their acceptance as International Standards by
the IS0 Council. They are approved in accordance with IS0 procedures requiring at
least 75 % approval by the member bodies voting.
International Standard
iTeh STANDARD PREVIEW
IS0 7704 was prepared by Technical Committee ISO/TC 147,
Water quality.
(standards.iteh.ai)
ISO 7704:1985
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
a6c27509f037/iso-7704-1985
0 International Organization for Standardization, 1985
Printed in Switzerland
INTERNATIONAL STANDARD IS0 77044985 (E)
Water quality - Evaluation of membrane filters used
for microbiological analyses
0 introduction 4 Principle
Many membrane filter comparison studies which have been 4.1 Filtration of aqueous or pure cultures in liquid suspension
reported in the literature indicate that there are significant dif- through test membrane filters, using conventional procedures.
ferences between various chemical compositions, brands and Five replicates are minimum sample requirements; a total of
batches of membranes in their ability to recover bacteria from 200 colonies is considered the minimum number for statistical
water samples. comparison.
Thus, it is very important that one of the basic tools of aquatic
microbiology, the membrane filter, be standardized as much as 4.2 Evaluation of the efficiency of each type of membrane
possible, not only to provide consistent results, but also to filter by
enable the development of standardized procedures for
enumerating specific micro-organisms. a) counts obtained on non-selective medium using spread
or pour plate technique versus membrane filtration tech-
nique counts on the same medium (experience indicates
1 Scope iTeh STANDARD PREVIEW that under these conditions the best membrane filter counts
are 80 to 90 % of those obtained by plate counts) ;
1.1 This International
evaluation and comparison
(standards.iteh.ai)
Standard specifies a method for the
of water-testing membrane filters b) results for specific organisms obtained on selective
intended for the enumeration of specific organisms and mixed membrane filter medium using spread or pour plate tech-
microbial populations. ISO 7704:1985 niques versus membrane filtration technique on the same
medium.
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
1.2 The method provides general guidelines for a6c27509f037/iso-7704-1985
comparative
NOTE - Pour plate control may provide fewer colonies than spread
testing of the recoveries of bacteria, yeasts and other fungi on
plate control.
membrane filters, as compared to recoveries by the spread
plate and pour plate techniques.
2 Field of application 5 Diluent, culture media and reagent
2.1 This method is applicable to the user’s evaluation of any
5.1 Basic material
microporous filter intended for use with aquatic samples. Its
range covers any pore size filter which may be useful in a
In order to improve the reproducibility of the results, it is
specific application.
recommended that, for the preparation of the diluents and cul-
ture media, dehydrated basic components or complete dehy-
2.2 For specific applications, it is expected that suitable drated media be used. The manufacturer’s instructions shall be
media, incubation temperature, incubation duration, incu- rigorously followed.
bation atmosphere and controls (spread or pour plate) will be
used. Results obtained from one species or group of micro- The chemical products used for the preparation of the culture
organisms may not be valid for other groups. media and the reagents shall be of recognized analytical quality.
The water used shall be distilled or deionized water, free from
3 Definition substances that might inhibit the growth of micro-organisms
under the test conditions.
For the purpose of this International Standard, the following
definition applies. Measurements of pH shall be made using a pH meter, measure-
ments being referred to room temperature.
membrane filter : A thin non-fibrous filtration medium for
liquids and gases, having a mean pore size larger than 0,Ol pm If the prepared culture media are not used immediately, they
in diameter, on which particles larger than the rated pore size shall, unless otherwise stated, be stored in the dark at
are retained at or near the delivery surface when suction or 4 -+ 2 OC, for no longer than 1 month, in conditions which do
pressure is applied. not produce any change in their composition.
1
IS0 77044985 (E)
5.2 Diluent 6.5 Colony counting apparatus, with suitable illumination
and magnification.
Any appropriate sterile diluent may be used. Use of peptone a
[O,l % bnlm)l water has been found to be suitable. This mini-
mizes the shock to the organisms of pure water.
6.6 Hand tally counter.
5.3 Agar media 6.7 Autoclave, or other sterilizing equipment.
53.1 Non-selective medium. 68. Turntable (optional) and glass spreading rod.
Tryptone soya agar o ra similar medium may be used as the
non-selective medium this test. 6.9 Sterile vented Petri dishes, appropriate sizes.
Prepare the medium and dispense a measured amount of
6.10 Sterile calibrated pipettes, of capacities 0,l; 1,O; and
medium into Petri dishes (in the case of membrane filter counts
10 ml.
and spread plate counts) or into suitable tubes (in the case of
pour plate counts). The depth of agar in the Petri dishes should
be at least 3 mm. All media used for each test should be
prepared from the same batch and prepared at the same time. 7 Preparation of cultures for testing
5.3.2 Selective medium. 7.1 Whether natural water, effluent samples or pure cultures
are being used to evaluate the membranes, they should be
The media used shou d be appropriate for the organisms being analysed prior to the test in order to obtain the dilution to be
used, either in mixed culture or pure culture. used (9.2.1). At all stages mix the samples or cultures
thoroughly (6.2) to obtain homogeneous distribution.
Prepare the medium and dispense a measured amount of
medium into Petri dishes (in the case of membrane filter counts
iTeh STANDARD
and spread plate counts) or into suitable tubes (in the case of
pour plate counts). The depth of agar in the Petri dishes should
7.2
PREVIEW If pure cultures in stressed or unstressed state are used,
establish the concentration of the test organism on an ap-
be at least 3 mm. All media used for each test should be
(standards.iteh.ai)
prepared from the same batch and prepared at the same time.
propriate medium to ensure that the correct dilution factor is
used to obtain the proper counting range (9.2.1).
ISO 7704:1985
6 Apparatus and glassware 7.3 The samples used to establish proper dilutions and
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
counting ranges may be used in the formal test if refrigerated
Clean all glassware and filtration equipment thoroughly, a6c27509f037/iso-7704-1985
using a immediately after testing and not held for an excessively long
suitable detergent in hot water, rinse with hot water, and then period (maximum 48 h). Mix samples thoroughly to obtain
rinse with distilled water. maximum homogeneity.
Follow standard microbiological laboratory practices for prepar-
ing glassware and filtration equipment prior to sterilization in 8 Membrane filters
the autoclave. Autoclave at 121 OC for 20 min for wet steriliz-
ation or for dry sterilization heat at 170 OC for at least 60 min The membrane filters shall be sterile.
(6.7).
Usual microbiological and laboratory equipment and
9 Procedure
6.1 Filtration units, for membrane filters, with vacuum flask
tubing, moisture trap flask, and connectable to a vacuum 91. Inoculation and incubation
source.
Prior to conducting the test, aseptically dry the Petri dishes
(6.91 containing the nutrient non-selective agar (5.3.1) or the
6.2 Vortex mixer, or similar mixer to mix cul tures for testing agar specific for the test organism (5.3.21, in accordance with
(optional). one of the following methods.
6.3 Forceps, with flat, non-serrated tips.
a) With covers on, store the Petri dishes inverted in the
dark if the media are light sensitive at 25 to 30 OC for 15 to
6.4 Incubator, water-bath or heat sink capable of being 17 h.
maintained at a variety of temperatures
b) With covers on, store the Petri dishes inverted in the
The appropriate incubator should be frequently checked to en- dark at 45 to 50 OC for 2 to 3 h.
sure that it is capable of maintaining the required temperature
for at least 24 h. A thermometer that has been checked for ac- c) With covers removed, place the Petri dishes in a filtered
curacy should be placed in the incubator on the shelf where the air laminar flow hood at room temperature for 1 h. If this
plates will be incubated. procedure is chosen, sterility controls shall be used.
2
IS0 7704-1985 (E)
Cultures for testing (see clause 7) should be readily available for
conducting the tests without delay. The same cultures shall be
used to inoculate the different media. Equal volumes, of be-
tween 0,l to 0,5 ml of an appropriate dilution of the culture,
should be used to inoculate control plates or for filtration 9.1.3 Pour plate controls
through membranes. The volume 0,5 ml shall not be exceeded
because of spread plate requirements. 9.1.3.1 Aseptically deliver (6.10) the same volume from the
same well-mixed sample dilution that was used in the mem-
To avoid test bias, spread plate controls should be carried out brane filtration procedure (9.1.2.1) into the bottom of a sterile
alternatively with the membrane filter test, using the same Petri dish (6.9). To this add one standard portion of melted agar
nutrient or selective media as the membrane filter test.
(45 “C), either non-selective nutrient agar (5.3.1) or membrane
filter selective agar (5.3.2) and thoroughly mix the two sol-
If more than one brand of membrane filter is being compared, utions by back and forth and circular motions of the dish.
the membranes should be alternated with control spread or
pour plates until the required number of replicates has been
made with the specific culture. 9.1.3.2 Allow the agar to solidify thoroughly, then invert the
dish and incubate (6.4) for the required period at the required
The media with and without membrane filters are incubated temperature.
after seeding under temperature and time conditions ap-
propriate to the organisms being studied. 9.1.3.3 For sterility control of the agar, prepare plates from
several tubes of melted agar.
9.1.1 Membrane filter cultures
9.1.1.1 With sterile forceps (6.31, aseptically remove the 9.2 Interpretation
membrane filter (see clause 8) to be tested and centre the
membrane grid side up or face-up on the filter holder base. 9.2.1 Membrane filter cultures
Place a filter funnel on to the assembly and secure as required
trap to a vacuum source (6.1).
iTeh STANDARD PREVIEW
by the specific holder. Connect the filtration flask and vacuum Use a colony counting apparatus (6.5) to count all typical col-
onies (mixed or pure cultures) on the surface of the membrane.
(standards.iteh.ai) If more than one dilution of a culture was employed for the test,
9.1.1.2 With vacuum off, add 20 to 30 ml of sterile dilution the dilution selected for counting should have a target colony
water (5.2). Aseptically transfer (6.10) the same volumeISO from count between 25 and 100 and a sufficient number of replicates
7704:1985
the same well-mixed sample dilution that was used in the to give at least 200 colonies per treatment.
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
spread plate procedure (9.1.1 .l), into the dilution water in the
a6c27509f037/iso-7704-1985
funnel. Apply the vacuum and filter the entire contents. Rinse 9.2.2 Spread plate and pour plate
the funnel with 20 to 30 ml of sterile dilution water twice, apply-
ing the vacuum continuously. Turn off the vacuum immediately Use the same culture dilution, selected for the membrane count
after the last rinse has passed through the filter. Remove the for the spread plate and pour plate counts for recovery com-
filter funnel and with sterile forceps remove the membrane filter parisons. Use a colony counting apparatus to help count (6.6)
from the base. all surface and subsurface colonies. Follow routine counting
procedures as established in the tester’s laboratory.
9.1.1.3 Place the test membrane on either the non-selective
medium (5.3.1) or the selective medium (5.3.21, depending on
the rotation order. Roll the membrane filter on to the agar sur-
face in the Petri dish, making sure that air is not entrapped be-
10 Expression of results
tween the membrane and the agar surface. If an air bubble is
observed, the membrane should be raised and again rolled on 10.1 Calculation
to the agar to eliminate the air.
For each culture used for testing, calculate the arithmetic mean
of the counts of the target organism from the five (or more)
9.1.1.4 All plates should be incubated (6.4) under tempera-
replicates of each treatment.
ture and time conditions appropriate to the organisms being
studied.
The recovery, R, expressed as a pe rcenta ge, for each culture, is
given by the equation
9.1.2 Spread plate controls
mm
9.1.2.1 Aseptically deliver (see 6.10) and spread [using a glass R=-x100
spreader (6.8) that has been dipped in ethanol and flamed] 0,l mc
to 0,5 ml of the appropriate dilution of the culture on to the
predried surface of either the nutrient non-selective agar or the where
membrane filter selective agar. To aid even distribution in the
inoculum, plates can be put on a turntable (6.8). Replace Petri mm is the mean of the membrane filter counts;
dish covers and allow the aliquot of culture to be completely
absorbed before inverting. mC is the mean of the pour plate or spread plate counts.
. 3
IS0 77044985 (E)
10.1.1 Establish the percentage recovery from the data ob- 10.2.2 When comparing membrane filter counts versus con-
tained with membrane filter count and plate count on non- trol plating counts (noting that spread plate procedures may
selective agar. produce the higher plating counts, thus the spread plate pro-
cedure is recommended), membranes producing counts 80 %
10.1.2 Establish the percentage recovery from the data ob- or more of the control plating counts will be considered accep-
tained with membrane filter count and plate count on selective table. If a membrane is to be used solely for the enumeration of
agar. a specific organism, then selective media data are used.
10.2 Interpretation of results
10.2.3 Consider a particular brand or type of membrane
preferable only if it yields mean colony counts, mm, which are
10.2.1 When testing batch to batch variation in membrane
20 % or more in excess of those on other membranes.
filters produced by one manufacturer, to be considered
equivalent, replicate counts should be within the 95 % con-
fidence interval of the batch with which they are being com-
pared. 11 Test report
The 95 % confidence is obtained by applying the The test report shall show the method used and the results ob-
following formulae : tained, indicating clearly the method of expression used. It shall
also mention any operating details not specified in this lnter-
a) for upper limit counts, m + 2 (JmTT + 1); national Standard, or regarded as optional, together with
details of any incidents likely to have influenced the results.
b) for lower limit counts, m - 2 (,/%%i - 1);
The test report shall include all the information necessary for
where m is the arithmetic mean of the bacterial counts on the the complete identification of the membrane filters, cultures,
batch of control membrane filters. media and diluents used.
iTeh STANDARD PREVIEW
(standards.iteh.ai)
ISO 7704:1985
https://standards.iteh.ai/catalog/standards/sist/7e484983-3be9-4164-8512-
a6c27509f037/iso-7704-1985
