ISO 4407
(Main)Hydraulic fluid power — Fluid contamination — Determination of particulate contamination by the counting method using an optical microscope
Hydraulic fluid power — Fluid contamination — Determination of particulate contamination by the counting method using an optical microscope
This International Standard specifies methods for determining the level of particulate contamination in liquids used in hydraulic systems by counting the number of particles deposited on the surface of a membrane filter using an optical microscope. It includes particle counting by two manual methods and image analysis, using either transmitted or incident lighting systems. Particle sizes greater than or equal to 2 micrometres can be sized and counted by this method, but the resolution and accuracy of the results will depend upon the optical system used and the capabilities of the operator. All hydraulic fluids with a wide range of contamination levels can be analysed according to this International Standard. However, the counting uncertainty at the larger particle sizes increases if the volume filtered is reduced to allow smaller sized particles to be counted, where a fine precipitate or a high particle concentration is present.
Transmissions hydrauliques — Pollution des fluides — Détermination de la pollution particulaire par comptage au microscope optique
La présente Norme internationale définit des méthodes permettant de déterminer le niveau de pollution particulaire des liquides utilisés dans les systèmes hydrauliques, par comptage au microscope optique du nombre de particules déposées à la surface d'une membrane filtrante. Elle traite du comptage des particules par deux méthodes manuelle et par analyse d'image, en utilisant soit une lumière transmise, soit une lumière incidente. Ces méthodes permettent de classer et de dénombrer des particules de taille supérieure ou égale à 2 micromètres, mais la résolution et l'exactitude des résultats dépend du système optique utilisé et des aptitudes de l'opérateur. Tous les liquides hydrauliques qui présentent un large éventail de niveaux de pollution sont analysables selon la présente Norme internationale. Cependant, l'incertitude de comptage aux plus grandes tailles de particules augmente si le volume filtré est réduit pour permettre le dénombrement des particules de plus petites tailles, lorsqu'on observe un léger précipité ou que la concentration des particules est élevée.
General Information
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Standards Content (Sample)
FINAL DRAFT
International
Standard
ISO/FDIS 4407
ISO/TC 131/SC 6
Hydraulic fluid power — Fluid
Secretariat: BSI
contamination — Determination of
Voting begins on:
particulate contamination by the
2025-03-28
counting method using an optical
Voting terminates on:
microscope
2025-05-23
Transmissions hydrauliques — Pollution des fluides —
Détermination de la pollution particulaire par comptage au
microscope optique
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
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INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/FDIS 4407:2025(en) © ISO 2025
FINAL DRAFT
ISO/FDIS 4407:2025(en)
International
Standard
ISO/FDIS 4407
ISO/TC 131/SC 6
Hydraulic fluid power — Fluid
Secretariat: BSI
contamination — Determination of
Voting begins on:
particulate contamination by the
counting method using an optical
Voting terminates on:
microscope
Transmissions hydrauliques — Pollution des fluides —
Détermination de la pollution particulaire par comptage au
microscope optique
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
ISO/FDIS 4407:2025(en) © ISO 2025
ii
ISO/FDIS 4407:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Apparatus . 5
5 Rinsing and cleaning fluids . 7
5.1 General .7
5.2 Glassware cleaning procedure .8
5.3 Environmental conditions .8
5.4 Solvent cleaning .8
6 Calibration . 8
6.1 Microscope calibration for automatic counting .8
6.2 Validation of system for automatic counting .9
6.3 Microscope calibration for manual counting .9
6.4 Determination of the EFA (effective filtration area) .9
7 Membrane filter preparation . 10
7.1 Blank analysis.10
7.2 Sample preparation .11
7.3 Capturing the particulate contamination on the filter membrane .11
7.4 Mounting of membrane filters for observation under transmitted light . 12
8 Particle sizing and counting procedure .13
8.1 Automated counting . 13
8.1.1 Evaluation of suitability for counting . 13
8.1.2 Image capturing . 13
8.1.3 Sequentiation . . .14
8.1.4 Particle counting .14
8.1.5 Validation of results.14
8.2 Procedure for manual counting with statistical counting and extrapolation .14
8.3 Calculation of total count .16
9 Verification of data . 16
10 Identification statement (reference to this document) .16
Annex A (normative) Report for automated counting .18
Annex B (informative) Report for manual counting .20
Annex C (informative) Binary pictures with different threshold settings .21
Annex D (informative) Membrane pictures where counting is impossible .22
Annex E (informative) Reference slides for system validation and adjustment.24
Bibliography .25
iii
ISO/FDIS 4407:2025(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 131, Fluid power systems and components,
Subcommittee SC 6, Contamination control.
This third edition cancels and replaces the second edition (ISO 4407:2002) which has been technically
revised.
The main changes are as follows:
— more detailed procedure for automatic particle counting by image analysing software.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO/FDIS 4407:2025(en)
Introduction
Fluids are used for a multitude of reasons over an array of industries. Whether they are used for hydraulic
power, lubricating or operational fluids, the presence of particulate contamination adversely affects
the fluids properties. This reduces the fluids capabilities and performance that can lead to damage of
components, equipment and eventual system failure.
The level of contamination in a fluid has a direct impact upon its performance and reliability.
Quantitative determination of particulate contamination requires precision in obtaining a representative
sample of the fluid to accurately ascertain the level of contamination. The method of particle counting using
an optical microscope is an accepted means of determining the extent of contamination. The accuracy of
particle counting can be affected by the different techniques and methods used. The accuracy when using
the automatic method described in ISO 4407 is typically in a range of +/- one ISO code according to ISO 4406.
This document details procedures that are acceptable methods for each step of the process of removing
particulate contamination from a fluid for analysis to achieve a uniform method, both manual and
automatic, for particle counting. These steps include sample preparation, vacuum filtration, filter membrane
preparation and, both manual and automatic counting methods.
v
FINAL DRAFT International Standard ISO/FDIS 4407:2025(en)
Hydraulic fluid power — Fluid contamination —
Determination of particulate contamination by the counting
method using an optical microscope
WARNING — The use of this document may involve hazardous materials, operations, and equipment.
This document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulative limitations prior to use.
1 Scope
This document specifies acceptable methods for determining the level of particulate contamination in a
known volume of fluid used in power systems by counting the number of particles captured on the surface
of a filter membrane using an optical microscope. It includes accepted methods of particle counting utilising
image analysis software or manual counting, see Annex B.
The aim of this document is to regulate a uniform, non-subjective filter membrane method of particle
counting that will ensure that the classification standard result is achieved by analysing, where feasible, the
entire effective filtration area (EFA) of the filter membrane.
The resolution and accuracy of the results will be dependent upon the optical system used, whether image
capture analysis software is used, and when opting for the manual counting method, the capabilities of the
operator.
All fluids that can be successfully vacuumed through the appropriate pore sized filter membrane will be
able to use the following 2 methods of membrane filter analysis:
— Automatic counting: automatic detection, size classification and counting of particles on a membrane
filter by using an optical microscope and image analysing software.
— Manual counting: manual detection, size classification and counting of particles on a membrane filter
using an optical microscope.
A known volume of sample fluid is vacuum filtered through a membrane filter to capture the particulate
contamination onto the surface of the membrane. The captured particles are analysed utilising an optical
microscope and, where possible, image analysis software to size, count and tabulate the particles according
to the particles longest chord.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 3722, Hydraulic fluid power — Fluid sample containers — Qualifying and controlling cleaning methods
ISO 5598, Fluid power systems and components — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 5598 and the following apply.
ISO/FDIS 4407:2025(en)
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
blank analysis
counting resulting from contaminants introduced from other sources, such as reagents, cleaning of
glassware and preparation of the membrane filter when performing a blank analysis
Note 1 to entry: See 5.2 for the glassware cleaning procedure.
3.2
effective filtration area
EFA
circular area of the membrane filter open to flow during filtration of liquid
3.3
effective filtration diameter
EFD
longest chord length of the membrane filter open to flow during filtration of liquid
Note 1 to entry: Both the EFA and the effective filtration diameter (EFD) are determined in 8.2.
3.4
measured filtration area
MFA
when using the automated counting method, it is the area that the software has analysed
3.5
fibre
particle longer than 100 µm with a length-to-width ratio greater than or equal to 10:1
3.6
fixative fluid
fluid that, as a result of a heat curing process, causes a membrane filter to adhere to a glass base slide, can
generate an opaque residue
3.7
grid square
square with side of nominally 3,1 mm printed on membrane filters
Note 1 to entry: Gridded membrane filters cannot be used in combination with automatic image analysis techniques.
3.8
image analysis system
image analysis system with integrated software to automatically size, count and tabulate the particulate
contamination captured on membrane filter
Note 1 to entry: A video image of the particle is digitally recreated based upon the difference in the grey scale contrast
of the particle and background. The sizing is either automatically determined or manually on the video screen.
3.9
mountant fluid
fluid that when heated, causes a membrane filter, previously treated with fixative liquid, to become
transparent and to adhere to the cover slip
3.10
particle size
size of particle as defined by the particle’s maximum ferret diameter
ISO/FDIS 4407:2025(en)
Figure 1 — Particle indicating the maximum ferret diameter
3.11
solvent
fluid that is physically and chemically compatible with and miscible in the sample fluid
Note 1 to entry: A solvent is used for diluting the sample fluid and can be used for cleaning and rinsing the apparatus.
The solvent should be chemically compatible with the apparatus, especially the membrane filter and should not
dissolve the particles.
3.12
statistical counting
counting and sizing particles using a proportion of the membrane filter’s surface, whereby at least
150 particles are counted over a total of at least 10 separate fields of view.
Note 1 to entry: Statistical counting requires an even distribution of particles over the complete surface and membrane
filters should be rejected for counting if this is not achieved.
3.13
threshold setting
level at which the grayscale value of an image pixel is determined by an automatic image analysing software
to belong to a particle or to the background
3.14
card insert
piece of card that is place inside of the slide mount to ensure the membrane filter is held flat
3.15
slide mount
two-piece vessel to secure the membrane filter in position and to prevent cross contamination
3.16
unit area
proportion of membrane filter that is counted for statistical purposes (manual counting)
Note 1 to entry: For manual counting the unit area is defined as the area of the membrane filter bound in the horizontal
plane by two adjacent vertical membrane filter grid lines and in the vertical plane by two parallel lines either on the
ocular micrometre eyepiece or drawn on a projection screen. Examples are given in Figure 1. For image analysis this is
a fixed field of view defined by the optical and electronic systems.
ISO/FDIS 4407:2025(en)
Key
1 grid square
2 width of grid square (mm)
3 length of grid square (mm)
4 full grid square
5 unit area on a gridded membrane
6 graticule height used for defining unit area (µm)
7 unit area
8 width of un-gridded unit area on membrane
9 effective filtration diameter of membrane
Figure 2 — Examples of unit areas
ISO/FDIS 4407:2025(en)
4 Apparatus
4.1 Sample preparation equipment.
Key
1 laminar flow cabinet
2 waste container
3 sample bottle
4 vacuum pump
5 filter housing
6 filter manifold
7 fluid funnel
8 filter membrane housing box
9 clamp
10 tweezers
11 card insert
12 filter membranes
13 filter membrane housing (2 parts)
Figure 3 — Recommended equipment for capturing the particulate contamination on the
membrane filter
4.1.1 Filtration system, consisting of
— a vacuum filtration manifold or filtration flask to support the membrane holder funnel. In relation to
the vacuum manifold, this may be singular or multiple dependent upon the number of fluid samples
required.
— a membrane holder funnel relevant to the size of membrane filters used for vacuum filtration of the
particulate contamination (typically 25 mm or 47 mm). This supports the membrane filter ensuring that
it is sealed correctly and provides an even, flat support structure for the sample fluid to pass through the
membrane filter.
ISO/FDIS 4407:2025(en)
— a funnel to retain the sample fluid. The volume capacity is dependent upon the volume of fluid required
for analysis.
— a clamp to secure the membrane holder funnel to the funnel providing a seal so as the sample fluid is
retained in the funnel until filtration commences.
— every effort shall be made to ensure that the filtration system is bonded to earth to prevent static build up.
4.1.2 Graduated vessel, for collecting the volume of fluid that is required for analysis. The accuracy of the
graduations should be ±2 %.
4.1.3 Membrane filters.
— They shall be compatible with the sample fluid and any solvents/chemicals used in the processes.
— They shall be non-gridded for automatic counting and gridded for manual counting with the pore size
that is approximately equal to the minimum particle size divided by 1,5 that is required to be counted
and sized (e.g. if 5 µm size particles are the smallest particles required to be counted, membrane filters
with a pore size of <3,3 µm should be used). The maximum membrane filter pore size that is to be used
is 3 µm.
— Various membrane filter diameters can be used. The choice of diameter shall be dependent upon the level
of expected particulate contamination within the fluid sample (as not to over-populate the membrane
filter resulting in overlapping particles) and the method of particle counting chosen (automatic via image
analysis software or manual counting).
— Membrane filters for manual counting have a printed grid square formation. For the image analysis
software method, a plain white membrane must be used. For both types of particle counting, appropriate
membrane filters should be selected which have a smooth and flat surface to ensure that the membrane
does not produce shadows which could affect the optical particle capture.
— The colour for the membrane filter chosen shall take into consideration the maximum contrast of
particulate contamination to be analysed. For example, if most of the particles are expected to be dark,
a white membrane filter should be used.
4.1.4 Membrane filter slide mounts (optional for manual counting).
A method of encasing the membrane filter can be used, where viable. This provides two key advantages:
— they ensure that no further cross contamination from airborne particulate contamination is possible;
— they aid in holding the membrane filter flat; this is crucial for image analysis software to ensure that the
EFA remains in focus.
4.1.5 Suitable sample bottles, that includes, as a minimum, the nominal capacity with the known volume
of sample required for analysis and include a screw cap. Every precaution shall be taken to avoid using a seal
on the cap that is not compatible with the fluid sample that is to be analysed. These can breakdown during
agitation of the sample and cause undue cross contamination of the sample fluid.
4.1.6 Stage micrometer, graduated in 0,1 mm and 0,01 mm scale divisions, calibrated and traceable to
national standards.
4.1.7 Tweezers, flat-bladed and, ideally, manufactured from stainless steel where possible.
4.1.8 Vacuum pump, able to establish a vacuum required to draw the fluid through the membrane filter
at a sufficient rate.
ISO/FDIS 4407:2025(en)
4.1.9 Oven (optional), may be required if the membrane filter appears to be translucent/transparent.
Every precaution is to be made to ensure that it is safe to place the fluid into the oven and expose it to the
temperatures and environment of the oven.
4.1.10 Ultrasonic bath (optional), may be required in certain fluid types to breakdown agglomerates.
4.2 Microscopes.
4.2.1 Microscope for automatic counting
It shall be able to accurately identify particles of the minimum size required by the chosen reporting
standard. The microscope shall be equipped with a minimum:
— That the optical microscope can accept the attachment of a camera. The camera must have an optical
pixel resolution of 1 µm per pixel (5 µm per 5 pixels).
— The optical microscope shall be incidental lighting with a polarisation filter to ensure uniform illumination
of the field of view of the camera and optics. The field of view is the entire image of the camera displayed
on the monitor/screen.
— The camera shall be a minimum of 8 bit to ensure the depth of the illumination intensity is sufficient.
— An X-Y stage with a range of movement to view the entire membrane filter diameter. It is mandatory that
the X-Y movement be motorised for automatic counting and controlled via the image analysis software.
Z axis movement is required to ensure that the membrane filter is in focus for an accurate analysis.
— A stage holder with adapter (i.e. card insert or drum adapter) to keep the membrane flat and in focus.
— A laptop/PC to run the automatic image analysis software and to view the live feed of the optical
microscope camera
4.2.1 Microscope for manual particle counting
— fine and course focus control on the Z axis;
— sufficient lighting to ensure uniform illumination of the field of view of the camera and/or optics;
— a mechanical stage capable of scanning the complete area of the membrane filter being used for the
analysis. The stage must also have a method for holding the membrane filter slide mount in a secure
position;
— a calibrated graticule of which the smallest division shall not sub-tend a distance larger than the smallest
particle to be counted at a particular magnification, and with suitable graduations.
5 Rinsing and cleaning fluids
5.1 General
— Propan-2-ol (isopropyl alcohol) or equivalent for use with water-based fluids for the rinsing of the
membrane filter preparation equipment and dilution of the fluid sample, if required. Alternatively,
distilled or de-mineralised water is used, when the use of solvents is not suitable.
— Petroleum spirit or equivalent for use with oil based fluids for the rinsing of the membrane filter
preparation equipment and the dilution of the fluid, if required.
WARNING — Exercise all necessary precautions when using solvents as detailed in their respective
safety data sheets. This includes following all company and local requirements for use and disposal.
Test conditions and cleanliness
ISO/FDIS 4407:2025(en)
5.2 Glassware cleaning procedure
Clean and validate the cleanliness level of the filtration apparatus, graduated cylinders, bottles, glass slides,
with cover slips and membrane filter holders in accordance with ISO 3722. The final liquid used for the flush
should be either filtered petroleum spirit (or similar solvent) for petroleum based or synthetic oils, propan-
2-ol or demineralized water for water-based liquids
The required cleanliness level (RCL) of the volumetric glassware with cleanliness levels lower than 0,5 % of
the number of concentration of particles (larger than the smallest particle size of interest) expected to be
observed in the samples. All liquids used for rinsing shall be filtered through a 1 µm or finer membrane filter.
5.3 Environmental conditions
Every precaution should be taken not to introduce any form of cross contamination. This includes airborne
and particulate contamination from the individual carrying out the sample analysis preparation (clothing,
hands, PPE etc.). If feasible, the sample preparation and analysis should be carried out with a laminar flow
cabinet.
Ensure all apparatus for the filter membrane patch preparation is cleaned and, should it be required, that a
blank analysis has been carried out.
5.4 Solvent cleaning
Pass a known volume of compatible solvent through a membrane filter, that is at least the same micron pore
size as the filter membrane that is to be used for the sample analysis and place into a known clean container.
Use this solvent to perform the blank analysis flushing of glass hardware und dilution
6 Calibration
6.1 Microscope calibration for automatic counting
A calibration is recommended to be carried out at least annually or whenever any adjustment or movement
has been made to the system in relation to major components including the stage, camera, optics or a
relocation of the system. The stage micrometer (4.1.6) used to carry out the calibration must have a
recalibration certification every five years.
For systems utilising image analysing software, refer to the manufacturer’s recommendations for calibration.
The calibration shall verify:
— Measurement of the scale divisions on the graticule (for correct particle sizing).
— Stage alignment of the autom
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Hydraulic fluid power — — Fluid contamination — Determination of
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particulate contamination by the counting method using an optical
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microscope
Transmissions hydrauliques — Pollution des fluides — Détermination de la pollution particulaire par comptage
au microscope optique
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ii
ISO/FDIS 4407:2025(en)
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Contents
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Apparatus . 5
5 Rinsing and cleaning fluids . 8
5.1 General . 8
5.2 Glassware cleaning procedure . 8
5.3 Environmental conditions . 8
5.4 Solvent cleaning . 8
6 Calibration . 8
6.1 Microscope calibration for automatic counting . 8
6.2 Validation of system for automatic counting . 9
6.3 Microscope calibration for manual counting . 9
6.4 Determination of the EFA (effective filtration area) . 10
7 Membrane filter preparation . 11
7.1 Blank analysis . 11
7.2 Sample preparation . 11
7.3 Capturing the particulate contamination on the filter membrane . 11
7.4 Mounting of membrane filters for observation under transmitted light . 13
8 Particle sizing and counting procedure . 13
8.1 Automated counting . 13
8.2 Procedure for manual counting with statistical counting and extrapolation . 15
8.3 Calculation of total count . 17
9 Verification of data . 17
10 Identification statement (reference to this document) . 17
Annex A (normative) Report for automated counting . 19
Annex B (informative) Report for manual counting . 21
Annex C (informative) Binary pictures with different threshold settings. 23
Annex D (informative) Membrane pictures where counting is impossible . 24
Annex E (informative) Reference slides for system validation and adjustment . 26
Bibliography . 27
Foreword . v
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Introduction . vi
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1 Scope . 1
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2 Normative references . 1
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3 Terms and definitions . 2
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3.1 blank count . 2
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3.2 calculation factor . 2
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3.3 effective filtration area (EFA) . 2
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3.4 effective filtration diameter (EFD) . 2
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3.5 measured filtration area (MFA). . 2
3.6 fibre . 2
3.7 fixative fluid . 2
3.8 grid square . 2
3.9 image analyser . 2
3.10 mountant fluid . 3
3.11 particle size . 3
3.12 solvent . 3
3.13 statistical counting . 3
3.14 threshold setting . 3
3.15 card inserts . 3
3.16 slide mount . 3
3.17 unit area . 4
4 Apparatus . 5
4.1 Sample Preparation Equipment . 5
4.2 Microscopes . 7
5 Rinsing and cleaning fluids . 8
6 Test conditions and cleanliness . 8
6.1 Glassware cleaning procedure . 8
6.2 Environmental conditions . 8
6.3 Solvent cleaning . 8
7 Calibration . 8
7.1 Microscope calibration for automatic counting . 8
7.2 Validation of system for automatic counting . 9
7.3 Microscope calibration for manual counting . 9
7.4 Determination of the EFA (Effective Filtration Area) . 10
8 Membrane filter preparation . 10
8.1 Blank analysis . 10
8.2 Sample preparation . 11
8.3 Capturing the particulate contamination on the filter membrane . 11
8.4 Mounting of membrane filters for observation under transmitted light . 13
9 Particle sizing and counting procedure . 13
9.1 Automated Counting . 13
9.2 Procedure for manual counting with statistical counting and extrapolation . 15
9.3 Calculation of total count . 18
10 Verification of data . 18
11 Identification statement (reference to this document) . 18
Annex A1 Report for automated counting . 19
Annex A2 Report for manual counting . 21
Annex A3 Binary pictures with different threshold Settings . 22
Annex A4 Membrane pictures where counting is impossible . 23
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Annex A5 Reference slides for system validation and adjustment. 24
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Bibliography . 25
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iv © ISO 4407 2025 – All rights reserved
iv
ISO/FDIS 4407:2025(en)
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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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of
ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents.www.iso.org/patents. ISO shall not be held responsible for identifying any or all such
patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.htmlwww.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 131, Fluid power systems and components,
Subcommittee SC 6, Contamination control.
This fourththird edition cancels and replaces the thirdsecond edition (ISO 4407:20212002) which has been
Commented [eXtyles2]: Not found, but similar references
technically revised. exist
The main changes are as follows:
contamination — Determination of particulate contamination
by the counting method using an optical microscope
— — Moremore detailed procedure for automatic particle counting by image analysing software.
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Any feedback or questions on this document should be directed to the user’s national standards body. A Formatted: Default Paragraph Font
complete listing of these bodies can be found at www.iso.org/members.html.www.iso.org/members.html.
Commented [eXtyles3]: The URL
https://www.iso.org/members.html has been redirected to
http://www.iso.org/about/members. Please verify the URL.
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v
ISO /FDIS 4407: 20242025(en) Formatted: Font: 11 pt, Bold
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Introduction
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Fluids are used for a multitude of reasons over an array of industries. Whether they are used for hydraulic
power, lubricating or operational fluids, the presence of particulate contamination adversely affects the fluids
properties. This reduces the fluids capabilities and performance that can lead to damage of components,
equipment and eventual system failure.
The level of contamination in a fluid has a direct impact upon its performance and reliability.
Quantitative determination of particulate contamination requires precision in obtaining a representative
sample of the fluid to accurately ascertain the level of contamination. The method of particle counting using
an optical microscope is an accepted means of determining the extent of contamination. The accuracy of
particle counting can be affected by the different techniques and methods used. The accuracy when using the
automatic method described in ISO 4407 is typically in a range of +/- one ISO Codecode according to ISO 4406.
Commented [eXtyles4]: ISO 4407: current stage is 50.00
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This document details procedures that are acceptable methods for each step of the process of removing
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particulate contamination from a fluid for analysis to achieve a uniform method, both manual and automatic,
for particle counting. These steps include sample preparation, vacuum filtration, filter membrane preparation
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and, both manual and automatic counting methods.
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vi © ISO 4407 2025 – All rights reserved
vi
FINAL DRAFT International Standard ISO/FDIS 4407:2025(en)
Hydraulic fluid power — — Fluid contamination — Determination of
particulate contamination by the counting method using an optical
microscope
WARNING — The use of this document may involve hazardous materials, operations, and equipment.
This document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulative limitations prior to use.
1 Scope
This document specifies acceptable methods for determining the level of particulate contamination in a known
volume of fluid used in power systems by counting the number of particles captured on the surface of a filter
membrane using an optical microscope. It includes accepted methods of particle counting utilising image
analysis software or manual counting, see Annex B.Annex B.
The aim of this document is to regulate a uniform, non-subjective filter membrane method of particle counting
that will ensure that the classification standard result is achieved by analysing, where feasible, the entire
effective filtration area (EFA) of the filter membrane.
The resolution and accuracy of the results will be dependent upon the optical system used, whether image
capture analysis software is used, and when opting for the manual counting method, the capabilities of the
operator.
All fluids that can be successfully vacuumed through the appropriate pore sized filter membrane will be able
to use the following 2 methods of membrane filter analysis:
— — Automatic counting: automatic detection, size classification and counting of particles on a membrane
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filter by using an optical microscope and image analysing software.
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
— — Manual counting: manual detection, size classification and counting of particles on a membrane filter
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
using an optical microscope.
A known volume of sample fluid is vacuum filtered through a membrane filter to capture the particulate
contamination onto the surface of the membrane. The captured particles are analysed utilising an optical
microscope and, where possible, image analysis software to size, count and tabulate the particles according to
the particles longest chord.
2 Normative references
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The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 3722, Hydraulic fluid power — Fluid sample containers — Qualifying and controlling cleaning
methods
ISO 5598, Fluid power systems and components — Vocabulary
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ISO /FDIS 4407: 20242025(en) Formatted: Font: 11 pt, Bold
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ISO 5598, Fluid power systems and components — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 5598 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— — ISO Online browsing platform: available at https://www.iso.org/obphttps://www.iso.org/obp
— — IEC Electropedia: available at https://www.electropedia.org/https://www.electropedia.org/
3.1 3.1
blank countanalysis
Commented [eXtyles5]: The term "blank count" has not
been used anywhere in this document
counting resulting from contaminants introduced from other sources, such as reagents, cleaning of glassware
and preparation of the membrane filter when performing a blank analysis. (see 6.1)
3.2
3.3Note 1 to entry: See 5.2 for the glassware cleaning procedure.
3.2
effective filtration area
EFA
circular area of the membrane filter open to flow during filtration of liquid
3.3 3.4
effective filtration diameter
EFD
longest chord length of the membrane filter open to flow during filtration of liquid
Note 1 to entry: Both the EFA and the effective filtration diameter (EFD) are determined in 8.2.8.2.
3.4 3.5
measured filtration area
MFA
when using the automated counting method, it is the area that the software has analysed
3.5 3.6
fibre
particle longer than 100 µm with a length-to-width ratio greater than or equal to 10:1
3.6 3.7
fixative fluid
fluid that, as a result of a heat curing process, causes a membrane filter to adhere to a glass base slide, can
generate an opaque residue
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3.7 3.8
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grid square
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square with side of nominally 3,1 mm printed on membrane filters
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2 © ISO 4407 2025 – All rights reserved
ISO/FDIS 4407:2025(en)
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Note 1 to entry: Gridded membrane filters cannot be used in combination with automatic image analysis
techniques.
3.8 3.9
image analyseranalysis system
Commented [eXtyles6]: The term "image analyser" has
not been used anywhere in this document
image analysis system with integrated software to automatically size, count and tabulate the particulate
contamination captured on membrane filter
Note 1 to entry: A video image of the particle is digitally recreated based upon the difference in the grey scale
contrast of the particle and background. The sizing is either automatically determined or manually on the video screen.
3.9 3.10
mountant fluid
fluid that when heated, causes a membrane filter, previously treated with fixative liquid, to become
transparent and to adhere to the cover slip
3.10 3.11
particle size
size of particle as defined by the particle’s maximum feretferret diameter
4407_ed3fig1.EPS
Figure 1 — Particle indicating the maximum ferret diameter
3.11 3.12
solvent
fluid that is physically and chemically compatible with and miscible in the sample fluid
Note 1 to entry: A solvent is used for diluting the sample fluid and can be used for cleaning and rinsing the
apparatus. The solvent should be chemically compatible with the apparatus, especially the membrane filter and should
not dissolve the particles.
3.12 3.13
statistical counting
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counting and sizing particles using a proportion of the membrane filter’s surface, whereby at least
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150 particles are counted over a total of at least 10 separate fields of view.
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Note 1 to entry: Statistical counting requires an even distribution of particles over the complete surface and
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membrane filters should be rejected for counting if this is not achieved.
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3.13 3.14
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threshold setting
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level at which the grayscale value of an image pixel is determined by an automatic image analysing software
to belong to a particle or to the background
3.14 3.15
card insert
piece of card that is place inside of the slide mount to ensure the membrane filter is held flat
3.15 3.16
slide mount
two-piece vessel to secure the membrane filter in position and to prevent cross contamination
3.16 3.17
unit area
proportion of membrane filter that is counted for statistical purposes (manual counting)
Note 1 to entry: For manual counting the unit area is defined as the area of the membrane filter bound in the
horizontal plane by two adjacent vertical membrane filter grid lines and in the vertical plane by two parallel lines either
on the ocular micrometre eyepiece or drawn on a projection screen. Examples are given in Figure 1.Figure 1. For image
Formatted Table
analysis this is a fixed field of view defined by the optical and electronic systems.
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4407_ed3fig2.EPS
stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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Key
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1 grid square Adjust space between Asian text and numbers, Tab
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2 width of grid square (mm)
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
3 length of grid square (mm)
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4 full grid square
5 unit area on a gridded membrane
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6 graticule height used for defining unit area (µm)
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7 unit area
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8 width of un-gridded unit area on membrane
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4 © ISO 4407 2025 – All rights reserved
ISO/FDIS 4407:2025(en)
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9 effective filtration diameter of membrane
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Figure 2 — Examples of unit areas
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3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
4 Apparatus
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4.1 4.1 Sample preparation equipment.
Asian text and numbers
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4407_ed3fig3.EPS
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
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Formatted Table
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stops: Not at 0.7 cm + 1.4 cm + 2.1 cm + 2.8 cm +
3.5 cm + 4.2 cm + 4.9 cm + 5.6 cm + 6.3 cm + 7 cm
Formatted
...
Key
Formatted
...
1 laminar flow cabinet
Formatted
...
2 waste container
3 sample bottle
Formatted
...
4 vacuum pump
Formatted
...
5 filter housing
Formatted
...
6 filter manifold
Formatted
...
7 fluid funnel
8 filter membrane housing box Formatted
...
9 clamp
Formatted
...
10 tweezers
Formatted
...
11 card insert
Formatted
...
12 filter membranes
13 filter membrane housing (2 parts) Formatted
...
Formatted
...
Figure 3 — Recommended equipment for capturing the particulate contamination on the membrane
filter Formatted: Font: Not Bold
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4.1.1 4.1.1 Filtration system, consisting of
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— — a vacuum filtration manifold or filtration flask to support the membrane holder funnel. In relation to
the vacuum manifold, this may be singular or multiple dependent upon the number of fluid samples
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required.
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...
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— — a membrane holder funnel relevant to the size of membrane filters used for vacuum filtration of the
spacing: single
particulate contamination (typically 25 mm or 47 mm). This supports the membrane filter ensuring that
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it is sealed correctly and provides an even, flat support structure for the sample fluid to pass through the
membrane filter.
— — a funnel to retain the sample fluid. The volume capacity is dependent upon the volume of fluid required
for analysis.
— — a clamp to secure the membrane holder funnel to the funnel providing a seal so as the sample fluid is
retained in the funnel until filtration commences.
— — every effort shall be made to ensure that the filtration system is bonded to earth to prevent static build
up.
4.1.2 4.1.2 Graduated vessel, for collecting the volume of fluid that is required for analysis. The accuracy
of the graduations should be ±2 %.
4.1.3 4.1.3 Membrane filters.
— — ShallThey shall be compatible with the sample fluid and any solvents/chemicals used in the processes.
— — They shall be non-gridded for automatic counting and gridded for manual counting with the pore size
that is approximately equal to the minimum particle size divided by 1,5 that is required to be counted and
sized (e.g. if 5 µm size particles are the smallest particles required to be counted, membrane filters with a
pore size of <3,3 µm should be used). The maximum membrane filter pore size that is to be used is 3 µm.
— — Various membrane filter diameters can be used. The choice of diameter shall be dependent upon the
level of expected particulate contamination within the fluid sample (as not to over-populate the membrane
filter resulting in overlapping particles) and the method of particle counting chosen (automatic via image
analysis software or manual counting).
— — Membrane filters for manual counting have a printed grid square formation. For the image analysis
software method, a plain white membrane must be used. For both types of particle counting, appropriate
membrane filters should be selected which have a smooth and flat surface to ensure that the membrane
does not produce shadows which could affect the optical particle capture.
— — The colour for the membrane filter chosen shall take into consideration the maximum contrast of
particulate contamination to be analysed. For example, if most of the particles are expected to be dark, a
white membrane filter should be used.
4.1.4 4.1.4 Membrane filter slide mounts (optional for manual counting).
A method of encasing the membrane filter can be used, where viable. This provides two key advantages:
— — they ensure that no further cross contamination from airborne particulate contamination is possible,
and;
— — they aid in holding the membrane filter flat. This; this is crucial for image analysis software to ensure
that the EFA remains in focus.
4.1.5 4.1.5 Suitable sample bottles, that includes, as a minimum, the nominal capacity with the known
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volume of sample required for analysis and include a screw cap. Every precaution shall is to betakenbe taken
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to avoid using a seal on the cap that is not compatible with the fluid sample that is to be analysed. These can
breakdown during agitation of the sample and cause undue cross contamination of the sample fluid.
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6 © ISO 4407 2025 – All rights reserved
ISO/FDIS 4407:2025(en)
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4.1.6 4.1.6 Stage micrometer, graduated in 0,1 mm and 0,01 mm scale divisions, calibrated and traceable
to national standards.
4.1.7 4.1.7 Tweezers, flat-bladed and, ideally, manufactured from stainless steel where possible.
4.1.8 4.1.8 Vacuum pump, able to establish a vacuum required to draw the fluid through the membrane
filter at a sufficient rate.
4.1.9 4.1.9 Oven (optional), may be required if the membrane filter appears to be
translucent/transparent. Every precaution is to be made to ensure that it is safe to place the fluid into the oven
and expose it to the temperatures and environment of the oven.
4.1.10 4.1.10 Ultrasonic bath (optional), may be required in certain fluid types to breakdown agglomerates.
4.2 4.2 Microscopes.
4.2.1 4.2.1 Microscope for automatic counting
ShallIt shall be able to accurately identify particles of the minimum size required by the chosen reporting
standard. The microscope shall be equipped with a minimum:
— — That the optical microscope can accept the attachment of a camera. The camera must have an optical
pixel resolution of 1 µm per pixel (5 µm per 5 pixels).
— — The optical microscope shall be incidental lighting with a polarisation filter to ensure uniform
illumination of the field of view of the camera and optics. The field of view is the entire image of the camera
displayed on the monitor/screen.
— — The camera shall be a minimum of 8 bit to ensure the depth of the illumination intensity is sufficient.
— — An X-Y stage with a range of movement to view the entire membrane filter diameter. It is mandatory
that the X-Y movement be motorised for automatic counting and controlled via the image analysis
software. Z axis movement is required to ensure that the membrane filter is in focus for an accurate
analysis.
— — A stage holder with adapter (i.e. card insert or drum adapter) to keep the membrane flat and in focus.
— — A laptop/PC to run the automatic image analysis software and to view the live feed of the optical
microscope camera
4.2.2 4.2.1 Microscope for manual particle counting
— — fine and course focus control on the Z axis;
— — sufficient lighting to ensure uniform illumination of the field of view of the camera and/or optics;
— — a mechanical stage capable of scanning the complete area of the membrane filter being used for the
analysis. The stage must also have a method for holding the membrane filter slide mount in a secure
position;
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— — a calibrated graticule of which the smallest division shall not sub-tend a distance larger than the
smallest particle to be counted at a particular magnification, and with suitable graduations.
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© ISO 2025 – All rights reserved
ISO /FDIS 4407: 20242025(en) Formatted: Font: 11 pt, Bold
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5 Rinsing and cleaning fluids
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5.1 — General
— Propan-2-ol (Isopropyl Alcoholisopropyl alcohol) or equivalent for use with water-based fluids for the
rinsing of the membrane filter preparation equipment and dilution of the fluid sample, if required.
Alternatively, distilled or de-mineralised water is used, when the use of solvents is not suitable.
— — Petroleum spirit or equivalent for use with oil based fluids for the rinsing of the membrane filter
preparation equipment and the dilution of the fluid, if required.
WARNING — Exercise all necessary precautions when using solvents as detailed in their respective
Safety Data Sheets.safety data sheets. This includes following all company and local requirements for
use and disposal. Test conditions and cleanliness
5.15.2 Glassware cleaning procedure
Clean and validate the cleanliness level of the filtration apparatus, graduated cylinders, bottles, glass slides,
with cover slips and membrane filter holders in accordance with ISO 3722. The final liquid used for the flush
should be either filtered petroleum spirit (or similar solvent) for petroleum based or synthetic oils, propan-2-
ol or demineralized water for water-based liquids
The required cleanliness level (RCL) of the volumetric glassware with cleanliness levels lower than 0,5 % of
the number of concentration of particles (larger than the smallest particle size of interest) expected to be
observed in the samples. All liquids used for rinsing shall be filtered through a 1 µm or finer membrane filter.
5.25.3 Environmental conditions
Every precaution should be taken not to introduce any form of cross contamination. This includes airborne
and particulate contamination from the individual carrying out the sample analysis preparation (clothing,
hands, PPE etc.). If feasible, the sample preparation and analysis should be carried out with a laminar flow
cabinet.
Ensure all apparatus for the filter membrane patch preparation is cleaned and, should it be required, that a
blank analysis has been carried out.
5.35.4 Solvent cleaning
Pass a known volume of compatible solvent through a membrane filter, that is at least the same micron pore
size as the filter membrane that is to be used for the sample analysis and place into a known clean container.
Use this solvent to perform the blank analysis flushing of glass hardware und dilution
6 Calibration
6.1 Microscope calibration for automatic counting
A calibration is recommended to be carried out at least annually or whenever any adjustment or movement
has been made to the system in relation to major components including the stage, camera, optics or a
relocation of the system. The stage micrometer (4.1.6)(4.1.6) used to carry out the calibration must have a
recalibration certification every five years.
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For systems utilising image analysing software, refer to the manufacturer’s recommendations for calibration.
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The calibration shall verify:
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...
FINAL DRAFT
International
Standard
ISO/FDIS 4407.2
ISO/TC 131/SC 6
Hydraulic fluid power — Fluid
Secretariat: BSI
contamination — Determination of
Voting begins on:
particulate contamination by the
2025-09-23
counting method using an optical
Voting terminates on:
microscope
2025-11-18
Transmissions hydrauliques — Pollution des fluides —
Détermination de la pollution particulaire par comptage au
microscope optique
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/FDIS 4407.2:2025(en) © ISO 2025
FINAL DRAFT
ISO/FDIS 4407.2:2025(en)
International
Standard
ISO/FDIS 4407.2
ISO/TC 131/SC 6
Hydraulic fluid power — Fluid
Secretariat: BSI
contamination — Determination of
Voting begins on:
particulate contamination by the
counting method using an optical
Voting terminates on:
microscope
Transmissions hydrauliques — Pollution des fluides —
Détermination de la pollution particulaire par comptage au
microscope optique
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© ISO 2025
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
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Phone: +41 22 749 01 11
Email: copyright@iso.org
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Published in Switzerland Reference number
ISO/FDIS 4407.2:2025(en) © ISO 2025
ii
ISO/FDIS 4407.2:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Apparatus . 5
4.1 Sample preparation equipment (see Figure 3). .5
4.2 Microscopes. .7
4.2.1 Microscope for automatic counting .7
4.2.2 Microscope for manual particle counting .7
5 Rinsing and cleaning fluids . 7
5.1 General .7
5.2 Glassware cleaning procedure .7
5.3 Environmental conditions .8
5.4 Solvent cleaning .8
6 Calibration . 8
6.1 Microscope calibration for automatic counting .8
6.2 Validation of system for automatic counting .9
6.3 Microscope calibration for manual counting .9
6.4 Determination of the EFA (effective filtration area) .9
7 Membrane filter preparation . 10
7.1 Blank analysis.10
7.2 Sample preparation .11
7.3 Capturing the particulate contamination on the filter membrane .11
7.4 Mounting of membrane filters for observation under transmitted light . 12
8 Particle sizing and counting procedure .13
8.1 Automated counting . 13
8.1.1 Evaluation of suitability for counting . 13
8.1.2 Image capturing . 13
8.1.3 Sequentiation . . .14
8.1.4 Particle counting .14
8.1.5 Validation of results.14
8.2 Procedure for manual counting with statistical counting and extrapolation .14
8.2.1 Evaluation of suitability for manual counting .14
8.2.2 Particle sizing and manual, statistical counting procedure . 15
8.3 Calculation of total count .16
9 Verification of data . 17
10 Identification statement (reference to this document) . 17
Annex A (informative) Report for automated counting .18
Annex B (informative) Report for manual counting .20
Annex C (informative) Binary pictures with different threshold settings .21
Annex D (informative) Membrane pictures where counting is impossible .22
Annex E (informative) Reference slides for system validation and adjustment.24
Bibliography .25
iii
ISO/FDIS 4407.2:2025(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 131, Fluid power system, Subcommittee SC 6
Contamination control.
This third edition cancels and replaces the second edition (ISO 4407:2002) which has been technically
revised.
The main changes are as follows:
— more detailed procedure for automatic particle counting by image analysing software.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO/FDIS 4407.2:2025(en)
Introduction
Fluids are used for a multitude of reasons over an array of industries. Whether they are used for hydraulic
power, lubricating or operational fluids, the presence of particulate contamination adversely affects
the fluids properties. This reduces the fluids capabilities and performance that can lead to damage of
components, equipment and eventual system failure.
The level of contamination in a fluid has a direct impact upon its performance and reliability.
Quantitative determination of particulate contamination requires precision in obtaining a representative
sample of the fluid to accurately ascertain the level of contamination. The method of particle counting using
an optical microscope is an accepted means of determining the extent of contamination. The accuracy of
particle counting can be affected by the different techniques and methods used. The accuracy when using
the automatic method described in this document is typically in a range of +/- one ISO code according to
ISO 4406.
This document details procedures that are acceptable methods for each step of the process of removing
particulate contamination from a fluid for analysis to achieve a uniform method, both manual and
automatic, for particle counting. These steps include sample preparation, vacuum filtration, filter membrane
preparation and, both manual and automatic counting methods.
v
FINAL DRAFT International Standard ISO/FDIS 4407.2:2025(en)
Hydraulic fluid power — Fluid contamination —
Determination of particulate contamination by the counting
method using an optical microscope
WARNING — The use of this document may involve hazardous materials, operations, and equipment.
This document does not purport to address all the safety issues associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulative limitations prior to use.
1 Scope
This document specifies acceptable methods for determining the level of particulate contamination in a
known volume of fluid used in power systems by counting the number of particles captured on the surface
of a filter membrane using an optical microscope. It includes accepted methods of particle counting utilising
image analysis software or manual counting.
The aim of this document is to regulate a uniform, non-subjective filter membrane method of particle
counting that will ensure that the classification standard result is achieved by analysing, where feasible, the
entire effective filtration area (EFA) of the filter membrane.
The resolution and accuracy of the results will be dependent upon the optical system used, whether image
capture analysis software is used, and when opting for the manual counting method, the capabilities of the
operator.
All fluids that can be successfully vacuumed through the appropriate pore sized filter membrane will be
able to use the following 2 methods of membrane filter analysis:
— Automatic counting (see Annex A): automatic detection, size classification and counting of particles on a
membrane filter by using an optical microscope and image analysing software.
— Manual counting (see Annex B): manual detection, size classification and counting of particles on a
membrane filter using an optical microscope.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 3722, Hydraulic fluid power — Fluid sample containers — Qualifying and controlling cleaning methods
ISO 5598, Fluid power systems and components — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 5598 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
ISO/FDIS 4407.2:2025(en)
3.1
blank analysis
counting resulting from contaminants introduced from other sources, such as reagents, cleaning of
glassware and preparation of the membrane filter when performing a blank analysis
Note 1 to entry: See 7.1).
3.2
effective filtration area
EFA
circular area of the membrane filter open to flow during filtration of liquid
3.3
effective filtration diameter
EFD
longest chord length of the membrane filter open to flow during filtration of liquid
Note 1 to entry: Both the EFA and the effective filtration diameter (EFD) are determined in 8.2.
3.4
measured filtration area
MFA
area that the software has analysed when using the automated counting method
3.5
fibre
particle longer than 100 µm with a length-to-width ratio greater than or equal to 10:1
3.6
fixative fluid
fluid that, as a result of a heat curing process, causes a membrane filter to adhere to a glass base slide, can
generate an opaque residue
3.7
grid square
square with side of nominally 3,1 mm printed on membrane filters
Note 1 to entry: Gridded membrane filters cannot be used in combination with automatic image analysis techniques.
3.8
image analysis software
image analysis system with integrated software to automatically size, count and tabulate the particulate
contamination captured on membrane filter
Note 1 to entry: A video image of the particle is digitally recreated based upon the difference in the grey scale contrast
of the particle and background. The sizing is either automatically determined or manually on the video screen.
3.9
mountant fluid
fluid that when heated, causes a membrane filter, previously treated with fixative liquid, to become
transparent and to adhere to the cover slip
3.10
particle size
size of particle as defined by the particle’s maximum Feret diameter
ISO/FDIS 4407.2:2025(en)
Figure 1 — Particle indicating the maximum Feret diameter
3.11
required cleanliness level
RCL
permitted level of particulate contamination within a given area on the surface of the membrane filter
3.12
solvent
fluid that is physically and chemically compatible with and miscible in the sample fluid
Note 1 to entry: A solvent is used for diluting the sample fluid and can be used for cleaning and rinsing the apparatus.
The solvent should be chemically compatible with the apparatus, especially the membrane filter and should not
dissolve the particles.
3.13
statistical counting
counting and sizing particles using a proportion of the membrane filter’s surface, whereby at least
150 particles are counted over a total of at least 10 separate fields of view.
Note 1 to entry: Statistical counting requires an even distribution of particles over the complete surface and membrane
filters should be rejected for counting if this is not achieved.
3.14
threshold setting
level at which the grayscale value of an image pixel is determined by an automatic image analysing software
to belong to a particle or to the background
3.15
card insert
piece of card that is place inside of the slide mount to ensure the membrane filter is held flat
3.16
slide mount
two-piece vessel to secure the membrane filter in position and to prevent cross contamination
3.17
unit area
proportion of membrane filter that is counted for statistical purposes (manual counting)
Note 1 to entry: For manual counting the unit area is defined as the area of the membrane filter bound in the horizontal
plane by two adjacent vertical membrane filter grid lines and in the vertical plane by two parallel lines either on the
ocular micrometre eyepiece or drawn on a projection screen. Examples are given in Figure 2. For image analysis this is
a fixed field of view defined by the optical and electronic systems.
ISO/FDIS 4407.2:2025(en)
Key
1 grid square
2 width of grid square (mm)
3 length of grid square (mm)
4 full grid square
5 unit area on a gridded membrane
6 graticule height used for defining unit area (µm)
7 unit area
8 width of un-gridded unit area on membrane
9 effective filtration diameter of membrane
Figure 2 — Examples of unit areas
ISO/FDIS 4407.2:2025(en)
4 Apparatus
4.1 Sample preparation equipment (see Figure 3).
Key
1 laminar flow cabinet
2 waste container
3 sample bottle
4 vacuum pump
5 filter housing
6 vacuum manifold
7 fluid funnel
8 filter membrane housing box
9 clamp
10 tweezers
11 card insert
12 filter membranes
13 filter membrane housing (2 parts)
Figure 3 — Recommended equipment for capturing the particulate contamination on the
membrane filter
4.1.1 Filtration system, consisting of
— a vacuum filtration manifold or filtration flask to support the membrane holder funnel. In relation to
the vacuum manifold, this may be singular or multiple dependent upon the number of fluid samples
required.
— a membrane holder funnel relevant to the size of membrane filters used for vacuum filtration of the
particulate contamination (typically 25 mm or 47 mm). This supports the membrane filter ensuring that
it is sealed correctly and provides an even, flat support structure for the sample fluid to pass through the
membrane filter.
— a funnel to retain the sample fluid. The volume capacity is dependent upon the volume of fluid required
for analysis.
ISO/FDIS 4407.2:2025(en)
— a clamp to secure the membrane holder funnel to the funnel providing a seal so as the sample fluid is
retained in the funnel until filtration commences.
— every effort shall be made to ensure that the filtration system is bonded to earth to prevent static build up.
4.1.2 Graduated vessel, for collecting the volume of fluid that is required for analysis. The accuracy of the
graduations should be ±2 %.
4.1.3 Membrane filters.
— Shall be compatible with the sample fluid and any solvents/chemicals used in the processes
— Shall be non-gridded for automatic counting and gridded for manual counting with a pore size ≤ 3 µm.
— Various membrane filter diameters can be used. The choice of diameter shall be dependent upon the level
of expected particulate contamination within the fluid sample (as not to over-populate the membrane
filter resulting in overlapping particles) and the method of particle counting chosen (automatic via image
analysis software or manual counting).
— Membrane filters for manual counting have a printed grid square formation. For the image analysis
software method, a plain white membrane must be used. For both types of particle counting, appropriate
membrane filters should be selected which have a smooth and flat surface to ensure that the membrane
does not produce shadows which could affect the optical particle capture.
— The colour for the membrane filter chosen shall take into consideration the maximum contrast of
particulate contamination to be analysed. For example, if most of the particles are expected to be dark,
a white membrane filter should be used.
— Only measurements with the same membrane pore size shall be used for comparison.
4.1.4 Membrane filter slide mounts (optional for manual counting).
A method of encasing the membrane filter can be used, where viable. This provides two key advantages:
— they ensure that no further cross contamination from airborne particulate contamination is possible;
— they aid in holding the membrane filter flat. This is crucial for image analysis software to ensure that the
EFA remains in focus.
4.1.5 Suitable sample bottles, that includes, as a minimum, the nominal capacity with the known volume
of sample required for analysis and include a screw cap. Every precaution shall be taken to avoid using a seal
on the cap that is not compatible with the fluid sample that is to be analysed. These can breakdown during
agitation of the sample and cause undue cross contamination of the sample fluid.
4.1.6 Stage micrometer, graduated in 0,1 mm and 0,01 mm scale divisions, calibrated and traceable to
national standards.
4.1.7 Tweezers, flat-bladed and, ideally, manufactured from stainless steel where possible.
4.1.8 Vacuum pump, able to establish a vacuum required to draw the fluid through the membrane filter
at a sufficient rate.
4.1.9 Oven (optional), may be required if the membrane filter appears to be translucent/transparent.
Every precaution is to be made to ensure that it is safe to place the fluid into the oven and expose it to the
temperatures and environment of the oven.
4.1.10 Ultrasonic bath (optional), may be required in certain fluid types to breakdown agglomerates.
ISO/FDIS 4407.2:2025(en)
4.2 Microscopes.
4.2.1 Microscope for automatic counting
It shall be able to accurately identify particles of the minimum size required by the chosen reporting
standard. The microscope shall be equipped with at least the following:
— The optical microscope can accept the attachment of a camera. The camera must have an optical pixel
resolution of 1 µm per pixel (5 µm per 5 pixels). The reference for optical resolution is to be at least 5 µm.
— The optical microscope shall include incidental lighting with a polarisation filter to ensure uniform
illumination of the field of view of the camera and optics. The field of view is the entire image of the
camera displayed on the monitor/screen.
— The camera shall be a minimum of 8 bit to ensure the depth of the illumination intensity is sufficient.
— An X-Y stage with a range of movement to view the entire membrane filter diameter. It is mandatory that
the X-Y movement be motorised for automatic counting and controlled via the image analysis software.
Z axis movement is required to ensure that the membrane filter is in focus for an accurate analysis.
— A stage holder with adapter (i.e. card insert or drum adapter) to keep the membrane flat and in focus.
— A laptop/PC to run the automatic image analysis software and to view the live feed of the optical
microscope camera
4.2.2 Microscope for manual particle counting
It shall include:
— fine and course focus control on the Z axis;
— sufficient lighting to ensure uniform illumination of the field of view of the camera and/or optics;
— a mechanical stage capable of scanning the complete area of the membrane filter being used for the
analysis. The stage must also have a method for holding the membrane filter slide mount in a secure
position;
— a calibrated graticule of which the smallest division shall not sub-tend a distance larger than the smallest
particle to be counted at a particular magnification, and with suitable graduations.
5 Rinsing and cleaning fluids
5.1 General
— Propan-2-ol (Isopropyl Alcohol) or equivalent for use with water-based fluids for the rinsing of the
membrane filter preparation equipment and dilution of the fluid sample, if required. Alternatively,
distilled or de-mineralised water is used, when the use of solvents is not suitable.
— Petroleum spirit or equivalent for use with oil based fluids for the rinsing of the membrane filter
preparation equipment and the dilution of the fluid, if required.
WARNING — Exercise all necessary precautions when using solvents as detailed in their respective
safety data sheets (SDS). This includes following all company and local requirements for use and
disposal.
5.2 Glassware cleaning procedure
Clean and validate the cleanliness level of the filtration apparatus, graduated cylinders, bottles, glass slides,
with cover slips and membrane filter holders in accordance with ISO 3722. The final liquid used for the flush
ISO/FDIS 4407.2:2025(en)
should be either filtered petroleum spirit (or similar solvent) for petroleum based or synthetic oils, propan-
2-ol or demineralized water for water-based liquids
The required cleanliness level (RCL) of the volumetric glassware with cleanliness levels lower than 0,5 % of
the number of concentration of particles (larger than the smallest particle size of interest) expected to be
observed in the samples. All liquids used for rinsing shall be filtered through a 1 µm or finer membrane filter.
5.3 Environmental conditions
Every precaution should be taken not to introduce any form of cross contamination. This includes airborne
and particulate contamination from the individual carrying out the sample analysis preparation (clothing,
hands, PPE). If feasible, the sample preparation and analysis should be carried out with a laminar flow
cabinet.
Ensure all apparatus for the filter membrane patch preparation is cleaned and, should it be required, that a
blank analysis has been carried out.
5.4 Solvent cleaning
Pass a known volume of compatible solvent through a membrane filter, that is at least the same micron pore
size as the filter membrane that is to be used for the sample analysis and place into a known clean container.
Use this solvent to perform the blank analysis flushing of glass hardware und dilution.
6 Calibration
6.1 Microscope calibration for automatic counting
A calibration is recommended to be carried out at least annually or whenever any adjustment or movement
has been made to the system in relation to major components including the stage, camera, optics or a
relocation of the system. The stage micrometer (4.1.6) used to carry out the calibration must have a
recalibration's certification every five years.
For systems utilising image analysing software, refer to the manufacturer’s recommendations for calibration.
The calibration shall verify:
— Measurement of the scale divisions on the graticule (for correct particle sizing).
— Stage alignment of the automatic stage. This stage alignment verification ensures that the automatic
analysis does not split particles when crossing over mo
...
ISO/FDIS 4407.2:2025(en)
ISO /TC 131/SC 6
Secretariat: BSI
Date: 2025-08-01xx
Hydraulic fluid power — — Fluid contamination — Determination of
particulate contamination by the counting method using an optical
microscope
Transmissions hydrauliques — Pollution des fluides — Détermination de la pollution particulaire par comptage
au microscope optique
FDIS stage
ISO/FDIS 4407.2:2025(en)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
EmailE-mail: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO/FDIS 4407.2:2025(en)
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Apparatus . 5
4.1 Sample preparation equipment (see Figure 3). . 5
4.2 Microscopes. . 7
5 Rinsing and cleaning fluids . 7
5.1 General. 7
5.2 Glassware cleaning procedure . 8
5.3 Environmental conditions . 8
5.4 Solvent cleaning . 8
6 Calibration . 8
6.1 Microscope calibration for automatic counting . 8
6.2 Validation of system for automatic counting . 9
6.3 Microscope calibration for manual counting . 9
6.4 Determination of the EFA (effective filtration area) . 9
7 Membrane filter preparation . 10
7.1 Blank analysis . 10
7.2 Sample preparation . 11
7.3 Capturing the particulate contamination on the filter membrane . 11
7.4 Mounting of membrane filters for observation under transmitted light . 13
8 Particle sizing and counting procedure . 13
8.1 Automated counting . 13
8.2 Procedure for manual counting with statistical counting and extrapolation . 15
8.3 Calculation of total count . 17
9 Verification of data . 18
10 Identification statement (reference to this document) . 18
Annex A (informative) Report for automated counting . 19
Annex B (informative) Report for manual counting . 21
Annex C (informative) Binary pictures with different threshold settings . 22
Annex D (informative) Membrane pictures where counting is impossible . 23
Annex E (informative) Reference slides for system validation and adjustment. 25
Bibliography . 26
Bibliography . 25
iii
ISO/FDIS 4407.2:2025(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of
ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 131, Fluid power systems and components,
Subcommittee SC 6 Contamination control.
This fourththird edition cancels and replaces the thirdsecond edition (ISO 4407:20212002) which has been
technically revised.
The main changes are as follows:
— — more detailed procedure for automatic particle counting by image analysing software.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO/FDIS 4407.2:2025(en)
Introduction
Fluids are used for a multitude of reasons over an array of industries. Whether they are used for hydraulic
power, lubricating or operational fluids, the presence of particulate contamination adversely affects the fluids
properties. This reduces the fluids capabilities and performance that can lead to damage of components,
equipment and eventual system failure.
The level of contamination in a fluid has a direct impact upon its performance and reliability.
Quantitative determination of particulate contamination requires precision in obtaining a representative
sample of the fluid to accurately ascertain the level of contamination. The method of particle counting using
an optical microscope is an accepted means of determining the extent of contamination. The accuracy of
particle counting can be affected by the different techniques and methods used. The accuracy when using the
automatic method described in ISO 4407this document is typically in a range of +/- one ISO code according to
ISO 4406.
This document details procedures that are acceptable methods for each step of the process of removing
particulate contamination from a fluid for analysis to achieve a uniform method, both manual and automatic,
for particle counting. These steps include sample preparation, vacuum filtration, filter membrane preparation
and, both manual and automatic counting methods.
v
ISO/FDIS 4407.2:2025(en)
Hydraulic fluid power — — Fluid contamination — Determination of
particulate contamination by the counting method using an optical
microscope
WARNING — The use of this document may involve hazardous materials, operations, and equipment.
This document does not purport to address all the safety problemsissues associated with its use. It is
the responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulative limitations prior to use.
1 Scope
This document specifies acceptable methods for determining the level of particulate contamination in a known
volume of fluid used in power systems by counting the number of particles captured on the surface of a filter
membrane using an optical microscope. It includes accepted methods of particle counting utilising image
analysis software or manual counting.
The aim of this document is to regulate a uniform, non-subjective filter membrane method of particle counting
that will ensure that the classification standard result is achieved by analysing, where feasible, the entire
effective filtration area (EFA) of the filter membrane.
The resolution and accuracy of the results will be dependent upon the optical system used, whether image
capture analysis software is used, and when opting for the manual counting method, the capabilities of the
operator.
All fluids that can be successfully vacuumed through the appropriate pore sized filter membrane will be able
to use the following 2 methods of membrane filter analysis:
— — Automatic counting (see Annex A:): automatic detection, size classification and counting of particles
on a membrane filter by using an optical microscope and image analysing software.
— — Manual counting (see Annex B:): manual detection, size classification and counting of particles on a
membrane filter using an optical microscope.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 3722, Hydraulic fluid power — Fluid sample containers — Qualifying and controlling cleaning methods
ISO 5598, Fluid power systems and components — Vocabulary
ISO 16232, Road vehicles — Cleanliness of components and systems
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 5598 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— — ISO Online browsing platform: available at https://www.iso.org/obp
ISO/FDIS 4407.2:2025(en)
— — IEC Electropedia: available at https://www.electropedia.org/
3.1 3.1
blank analysis
counting resulting from contaminants introduced from other sources, such as reagents, cleaning of glassware
and preparation of the membrane filter when performing a blank analysis. (see 6.1)
Note 1 to entry: See 7.13.2).
3.2
effective filtration area
EFA
circular area of the membrane filter open to flow during filtration of liquid
3.3 3.3
effective filtration diameter
EFD
longest chord length of the membrane filter open to flow during filtration of liquid
Note 1 to entry: Both the EFA and the effective filtration diameter (EFD) are determined in 8.28.2.
3.4 3.4
measured filtration area
MFA
area that the software has analysed when using the automated counting method, it is the area that the software
has analysed
3.5 3.5
fibre
particle longer than 100 µm with a length-to-width ratio greater than or equal to 10:1
3.6 3.6
fixative fluid
fluid that, as a result of a heat curing process, causes a membrane filter to adhere to a glass base slide, can
generate an opaque residue
3.7 3.7
grid square
square with side of nominally 3,1 mm printed on membrane filters
Note 1 to entry: Gridded membrane filters cannot be used in combination with automatic image
analysis techniques.
3.8 3.8
image analysis software
image analysis system with integrated software to automatically size, count and tabulate the particulate
contamination captured on membrane filter
Note 1 to entry: A video image of the particle is digitally recreated based upon the difference in the grey
scale contrast of the particle and background. The sizing is either automatically determined or manually on the video
screen.
3.9 3.9
mountant fluid
fluid that when heated, causes a membrane filter, previously treated with fixative liquid, to become
transparent and to adhere to the cover slip
ISO/FDIS 4407.2:2025(en)
3.10 3.10
particle size
size of particle as defined by the particle’s maximum Feret diameter
4407_ed3fig1.EPS
Figure 1 — Particle indicating the maximum Feret diameter
3.11
xxxx
3.11 RCL
required cleanliness level
3.12RCL
permitted level of particulate contamination within a given area on the surface of the membrane filter
3.12
solvent
fluid that is physically and chemically compatible with and miscible in the sample fluid
Note 1 to entry: A solvent is used for diluting the sample fluid and can be used for cleaning and rinsing
the apparatus. The solvent should be chemically compatible with the apparatus, especially the membrane filter and
should not dissolve the particles.
3.13 3.13
statistical counting
counting and sizing particles using a proportion of the membrane filter’s surface, whereby at least
150 particles are counted over a total of at least 10 separate fields of view.
Note 1 to entry: Statistical counting requires an even distribution of particles over the complete surface
and membrane filters should be rejected for counting if this is not achieved.
3.14 3.14
threshold setting
level at which the grayscale value of an image pixel is determined by an automatic image analysing software
to belong to a particle or to the background
3.15 3.15
card insert
piece of card that is place inside of the slide mount to ensure the membrane filter is held flat
ISO/FDIS 4407.2:2025(en)
3.16 3.16
slide mount
two-piece vessel to secure the membrane filter in position and to prevent cross contamination
3.17 3.17
unit area
proportion of membrane filter that is counted for statistical purposes (manual counting)
Note 1 to entry: For manual counting the unit area is defined as the area of the membrane filter bound
in the horizontal plane by two adjacent vertical membrane filter grid lines and in the vertical plane by two parallel lines
either on the ocular micrometre eyepiece or drawn on a projection screen. Examples are given in Figure 2Figure 1. For
image analysis this is a fixed field of view defined by the optical and electronic systems.
4407_ed3fig2.EPS
Key
1 grid square
2 width of grid square (mm)
3 length of grid square (mm)
4 full grid square
5 unit area on a gridded membrane
6 graticule height used for defining unit area (µm)
7 unit area
8 width of un-gridded unit area on membrane
9 effective filtration diameter of membrane
Figure 2 — Examples of unit areas
ISO/FDIS 4407.2:2025(en)
4 Apparatus
4.1 Sample preparation equipment (see Figure 3.).
4407_ed3fig3.EPS
Key
1 laminar flow cabinet
2 waste container
3 sample bottle
4 vacuum pump
5 filter housing
6 vacuum manifold
7 fluid funnel
8 filter membrane housing box
9 clamp
10 tweezers
11 card insert
12 filter membranes
13 filter membrane housing (2 parts)
Figure 3 — Recommended equipment for capturing the particulate contamination on the membrane
filter
4.1.1 4.1.1 Filtration system, consisting of
— — a vacuum filtration manifold or filtration flask to support the membrane holder funnel. In relation to
the vacuum manifold, this may be singular or multiple dependent upon the number of fluid samples
required.
— — a membrane holder funnel relevant to the size of membrane filters used for vacuum filtration of the
particulate contamination (typically 25 mm or 47 mm). This supports the membrane filter ensuring that
it is sealed correctly and provides an even, flat support structure for the sample fluid to pass through the
membrane filter.
ISO/FDIS 4407.2:2025(en)
— — a funnel to retain the sample fluid. The volume capacity is dependent upon the volume of fluid required
for analysis.
— — a clamp to secure the membrane holder funnel to the funnel providing a seal so as the sample fluid is
retained in the funnel until filtration commences.
— — every effort shall be made to ensure that the filtration system is bonded to earth to prevent static build
up.
4.1.2 4.1.2 Graduated vessel, for collecting the volume of fluid that is required for analysis. The accuracy
of the graduations should be ±2 %.
4.1.3 4.1.3 Membrane filters.
— — Shall be compatible with the sample fluid and any solvents/chemicals used in the processes
— — Shall be non-gridded for automatic counting and gridded for manual counting with a pore size ≤ 3 µm.
— — Various membrane filter diameters can be used. The choice of diameter shall be dependent upon the
level of expected particulate contamination within the fluid sample (as not to over-populate the membrane
filter resulting in overlapping particles) and the method of particle counting chosen (automatic via image
analysis software or manual counting).
— — Membrane filters for manual counting have a printed grid square formation. For the image analysis
software method, a plain white membrane must be used. For both types of particle counting, appropriate
membrane filters should be selected which have a smooth and flat surface to ensure that the membrane
does not produce shadows which could affect the optical particle capture.
— — The colour for the membrane filter chosen shall take into consideration the maximum contrast of
particulate contamination to be analysed. For example, if most of the particles are expected to be dark, a
white membrane filter should be used.
— — Only measurements with the same membrane pore size shall be used for comparison.
4.1.4 4.1.4 Membrane filter slide mounts (optional for manual counting).
A method of encasing the membrane filter can be used, where viable. This provides two key advantages:
— — they ensure that no further cross contamination from airborne particulate contamination is possible,
and;
— — they aid in holding the membrane filter flat. This is crucial for image analysis software to ensure that
the EFA remains in focus.
4.1.5 4.1.5 Suitable sample bottles, that includes, as a minimum, the nominal capacity with the known
volume of sample required for analysis and include a screw cap. Every precaution shall be taken to avoid using
a seal on the cap that is not compatible with the fluid sample that is to be analysed. These can breakdown
during agitation of the sample and cause undue cross contamination of the sample fluid.
4.1.6 4.1.6 Stage micrometer, graduated in 0,1 mm and 0,01 mm scale divisions, calibrated and traceable
to national standards.
4.1.7 4.1.7 Tweezers, flat-bladed and, ideally, manufactured from stainless steel where possible.
4.1.8 4.1.8 Vacuum pump, able to establish a vacuum required to draw the fluid through the membrane
filter at a sufficient rate.
ISO/FDIS 4407.2:2025(en)
4.1.9 4.1.9 Oven (optional), may be required if the membrane filter appears to be
translucent/transparent. Every precaution is to be made to ensure that it is safe to place the fluid into the oven
and expose it to the temperatures and environment of the oven.
4.1.10 4.1.10 Ultrasonic bath (optional), may be required in certain fluid types to breakdown agglomerates.
4.2 Microscopes.
4.2.1 Microscope for automatic counting
It shall be able to accurately identify particles of the minimum size required by the chosen reporting standard.
The microscope shall be equipped with a minimumat least the following:
— — That The optical microscope can accept the attachment of a camera. The camera must have an optical
pixel resolution of 1 µm per pixel (5 µm per 5 pixels). The reference for optical resolution is to be at least
5 micronsµm.
— — The optical microscope shall include incidental lighting with a polarisation filter to ensure uniform
illumination of the field of view of the camera and optics. The field of view is the entire image of the camera
displayed on the monitor/screen.
— — The camera shall be a minimum of 8 bit to ensure the depth of the illumination intensity is sufficient.
— — An X-Y stage with a range of movement to view the entire membrane filter diameter. It is mandatory
that the X-Y movement be motorised for automatic counting and controlled via the image analysis
software. Z axis movement is required to ensure that the membrane filter is in focus for an accurate
analysis.
— — A stage holder with adapter (i.e. card insert or drum adapter) to keep the membrane flat and in focus.
— — A laptop/PC to run the automatic image analysis software and to view the live feed of the optical
microscope camera
4.2.2 Microscope for manual particle counting
— It shall include:
— fine and course focus control on the Z axis;
— — sufficient lighting to ensure uniform illumination of the field of view of the camera and/or optics;
— — a mechanical stage capable of scanning the complete area of the membrane filter being used for the
analysis. The stage must also have a method for holding the membrane filter slide mount in a secure
position;
— — a calibrated graticule of which the smallest division shall not sub-tend a distance larger than the
smallest particle to be counted at a particular magnification, and with suitable graduations.
5 Rinsing and cleaning fluids
5.1 — General
— Propan-2-ol (Isopropyl Alcohol) or equivalent for use with water-based fluids for the rinsing of the
membrane filter preparation equipment and dilution of the fluid sample, if required. Alternatively,
distilled or de-mineralised water is used, when the use of solvents is not suitable.
ISO/FDIS 4407.2:2025(en)
— — Petroleum spirit or equivalent for use with oil based fluids for the rinsing of the membrane filter
preparation equipment and the dilution of the fluid, if required.
WARNING –— Exercise all necessary precautions when using solvents as detailed in their respective
safety data sheets. (SDS). This includes following all company and local requirements for use and
disposal.
5.15.2 Glassware cleaning procedure
Clean and validate the cleanliness level of the filtration apparatus, graduated cylinders, bottles, glass slides,
with cover slips and membrane filter holders in accordance with ISO 3722. The final liquid used for the flush
should be either filtered petroleum spirit (or similar solvent) for petroleum based or synthetic oils, propan-2-
ol or demineralized water for water-based liquids
The required cleanliness level (RCL) of the volumetric glassware with cleanliness levels lower than 0,5 % of
the number of concentration of particles (larger than the smallest particle size of interest) expected to be
observed in the samples. All liquids used for rinsing shall be filtered through a 1 µm or finer membrane filter.
5.25.3 Environmental conditions
Every precaution should be taken not to introduce any form of cross contamination. This includes airborne
and particulate contamination from the individual carrying out the sample analysis preparation (clothing,
hands, PPE etc.).). If feasible, the sample preparation and analysis should be carried out with a laminar flow
cabinet.
Ensure all apparatus for the filter membrane patch preparation is cleaned and, should it be required, that a
blank analysis has been carried out.
5.35.4 Solvent cleaning
Pass a known volume of compatible solvent through a membrane filter, that is at least the same micron pore
size as the filter membrane that is to be used for the sample analysis and place into a known clean container.
Use this solvent to perform the blank analysis flushing of glass hardware und dilution.
6 Calibration
6.1 Microscope calibration for automatic counting
A calibration is recommended to be carried out at least annually or whenever any adjustment or movement
has been made to the system in relation to major components including the stage, camera, optics or a
relocation of the system. The stage micrometer (4.1.6(4.1.6)) used to carry out the calibration must have a
recalibrationrecalibration's certification every five years.
For systems utilising image analysing software, refer to the manufacturer’s recommendations for calibration.
The calibration shall verify:
— — Measurement of the scale divisions on the graticule (for correct particle sizing).
— — Stage alignment of the automatic stage. This stage alignment verification ensures that the automatic
analysis does not split particles when crossing over more than one field of vision of the camera assembly.
— — Camera alignment with the stage axis and that optical axis is perpendicular to the level of the x-y axis
of the stage.
ISO/FDIS 4407.2:2025(en)
— — Camera orientation.
The camera measures the light intensity of each pixel of the captured image and converts it into digital values
i.e. 0 for no illumination (black) and at least 255 for the full illumination (white). The conversion process
should be linear. Depending on the manufacturing process and type of camera, the conversion calculation
process could drift, resulting in a lower resolution for dark or light areas of the image. Even the sensitivity and
converting process of each pixel of the camera could be different. As a result, a picture of a surface which has
a uniform grey level could have divergent grey levels between the pixels. The manufacturer should provide a
tool to ascertain that the grey value is evenly distributed over the entire image and that the converting process
of the light intensity is linear. In case that a colour camera is used, it can be used in black and white mode,
where the ratio between the three colours red, blue and green is equal. It is not recommended to use the
camera in colour mode. The manufacturer should provide a calibration graticule to cover all aspects
mentioned above.
6.2 Validation of system for automatic counting
The system for automatic counting needs, in addition to the calibration, a validation to ensure the system is
functioning accurately. For the validation process, a particle reference slide shall be used, ideally provided by
the manufacturer. A proposal for a suitable reference slide is shown in Annex EAnnex E.
As a minimum, the optical image analysing software must verify the following prior to analysis of the fluid
sample:
— — Particle sizing by way of certified scale divisions;
— — The relative threshold settings have been confirmed;
— — Confirmation of the stage alignment of the automatic stage;
— — The above 3 checks must be carried out periodically before performing the analysis. It is recommended
that these checks are carried out at least monthly or when the optical microscope system has been
disturbed, including inadvertent movement of any moving component or maintenance.
6.3 Microscope calibration for manual counting
Calibration procedure should be carried out at least annually or whenever any major adjustment or change
has been made to the optics. The stage micrometer used to carry out the calibration must have a
recalibrationrecalibration's certification every five years.
Select a magnification on the microscope an
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