ISO 16604:2004

Clothing for protection against contact with blood and body fluids — Determination of resistance of protective clothing materials to penetration by blood-borne pathogens — Test method using Phi-X 174 bacteriophage

ISO 16604:2004 describes a laboratory test method for measuring the resistance of materials used in protective clothing to penetration by blood-borne pathogens. This test method uses a surrogate microbe under conditions of continuous liquid contact. Protective clothing "pass/fail" determinations are based on the detection of viral penetration at a specific hydrostatic pressure using the ISO 13994 test apparatus. This test method is not always effective in testing protective clothing materials having thick, inner liners which readily absorbs the challenge fluid. This test method involves a sensitive assay procedure. Because of the length of time required to complete this test method, it might not be suitable for use as a material or protective clothing quality control or assurance procedure.

Vêtements de protection contre les contacts avec le sang et les fluides corporels — Détermination de la résistance à la pénétration par des pathogènes véhiculés par le sang des matériaux entrant dans la fabrication des vêtements de protection — Méthode d'essai utilisant le bactériophage Phi-X 174

General Information

• Status: Published
• Publication Date: 29-Mar-2004
• ICS: 13.340.10 - Protective clothing
• Technical Committee: ISO/TC 94/SC 13 - Protective clothing
• Drafting Committee: ISO/TC 94/SC 13/WG 3 - Protective clothing against chemicals agents
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 10-Jun-2020

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 16604
First edition
2004-04-01


Clothing for protection against contact
with blood and body fluids —
Determination of resistance of protective
clothing materials to penetration by
blood-borne pathogens — Test method
using Phi-X174 bacteriophage
Vêtements de protection contre les contacts avec le sang et les fluides
corporels — Détermination de la résistance à la pénétration par des
pathogènes véhiculés par le sang des matériaux entrant dans la
fabrication des vêtements de protection — Méthode d'essai utilisant le
bactériophage Phi-X174





Reference number
ISO 16604:2004(E)
©
ISO 2004

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ISO 16604:2004(E)
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ISO 16604:2004(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 4
5 Microorganisms and reagents. 4
6 Apparatus and materials . 5
7 Test specimens . 6
7.1 Selection . 6
7.2 Preparation of test specimens. 6
8 Procedure. 6
8.1 Preparation of test media. 6
8.2 Preparation of controls. 7
8.3 Determination of material compatibility . 8
8.4 Procedure for preparation of bacteriophage suspension . 8
8.5 Preparation of settle plates. 9
8.6 Preliminary material measures. 9
8.7 Preparation of test cell . 9
8.8 Exposure of material to bacteriophage challenge suspension. 10
8.9 Procedure for quantification of assay fluid. 11
8.10 Interpretation of tests results . 12
9 Test report. 12
Annex A (informative) Sources of supplies and apparatus . 15
Bibliography . 16

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ISO 16604:2004(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 16604 was prepared by Technical Committee ISO/TC 94, Personal safety — Protective clothing and
equipment, Subcommittee SC 13, Protective clothing. It is based on ASTM F1671-97b.
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ISO 16604:2004(E)
Introduction
Workers, primarily those in the health care profession, involved in treating and caring for individuals injured or
sick, can be exposed to biological liquids capable of transmitting disease. These diseases, which may be
caused by a variety of microorganisms, can pose significant risks to life and health. This is especially true of
blood-borne viruses which cause hepatitis [hepatitis B virus (HBV) and hepatitis C virus (HCV)] and acquired
immune deficiency syndrome (AIDS) [human immunodeficiency viruses (HIV)]. Since engineering controls
cannot eliminate all possible exposures, attention is placed on reducing the potential of direct skin contact
through the use of protective clothing.
This International Standard is concerned with protective clothing and related protective devices designed to
protect against the penetration of blood or body fluids.
Given the variety of health care settings, activities, and the potential for exposure to blood or body fluids, the
barrier requirements for protective clothing materials will change with the application.
This International Standard describes a hydrostatic pressure test for measuring the viral penetration
resistance of clothing materials to a surrogate virus. The choice of an appropriate test method depends on the
specific application of protective clothing and its intended use. A risk assessment should be performed to

[1]
determine the level of risk for determining the appropriate test method.
This test method does not apply to all forms or conditions of blood-borne pathogen exposure. Users of this
test method should review modes for worker/clothing exposure and assess the appropriateness of this test
method for their specific applications. This test method has been specifically defined for modelling the viral
penetration of hepatitis (B and C) and human immunodeficiency viruses transmitted in blood and other
potentially infectious body fluids. The surrogate microbe, Phi-X174 bacteriophage, used in this test method, is
similar to HCV in size and shape but also serves as a surrogate for HBV and HIV. Inferences for protection
from other pathogens should be assessed on a case-by-case basis.
This test method addresses only the performance of materials or certain material constructions (e.g. seams)
used in protective clothing. This test method does not address the design, overall construction and
components, or interfaces of garments or other factors which may affect the overall protection offered by the
protective clothing. It is emphasized that the test does not necessarily simulate conditions that clothing
materials are likely to be exposed to in practice. The use of test data should therefore be restricted to broad
comparative assessment of such material according to their viral penetration resistance characteristics.
Testing prior to degradation by physical, chemical, and thermal stresses which could negatively impact the
performance of the protective barrier, could lead to a false sense of security. Consider tests which assess the
impact of sterilization, storage conditions, and shelf life on the penetration resistance for disposable products,
and the effects of laundering and sterilization on the penetration resistance for reusable products. The integrity
[1]
of the protective barrier can also be compromised during use by such effects as flexing and abrasion. It is
also possible that pre-wetting by contaminating materials such as alcohol and perspiration also compromises
the integrity of the protective barrier. If these conditions are of concern, evaluate the performance of protective
clothing materials for Phi-X174 bacteriophage penetration following an appropriate preconditioning technique
representative of the expected conditions of use.
Medical protective clothing materials are intended to be a barrier to blood, body fluids, and other potentially
infectious materials. Many factors can affect the wetting and penetration characteristics of body fluids, such as
surface tension, viscosity, and polarity of the fluid, as well as the structure and relative hydrophilicity or
hydrophobicity of the materials. The surface tension range for blood and body fluids (excluding saliva) is
[2]
approximately 0,042 N/m to 0,060 N/m. In order to help simulate the wetting characteristics of blood and
body fluids, the surface tension of the Phi-X174 bacteriophage challenge suspension is adjusted to
approximate the lower end of this surface tension range. The resulting surface tension of the Phi-X174
bacteriophage challenge suspension is (0,042 ± 0,002) N/m.
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ISO 16604:2004(E)
Part of this method for exposing the protective clothing material specimens with Phi-X174 bacteriophage
challenge suspension involves pressurization of the test cell to 14,0 kPa (in Procedures A and B). This
hydrostatic pressure has been documented to produce test results that correlate with visual penetration
[3]
results that are obtained with a human factors validation. Some studies, however, suggest that mechanical
[4] [5]
pressures exceeding 345 kPa can occur during clinical use. Therefore, it is important to understand that
this test method does not simulate all the physical stresses and pressures that are exerted on protective
clothing garments during actual use. Procedures C and D use a stepped pressurization approach with
pressures up to 20,0 kPa. These procedures simulate a range of possible pressures for ranking material
performance.
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INTERNATIONAL STANDARD ISO 16604:2004(E)

Clothing for protection against contact with blood and body
fluids — Determination of resistance of protective clothing
materials to penetration by blood-borne pathogens — Test
method using Phi-X174 bacteriophage
1 Scope
This International Standard describes a laboratory test method for measuring the resistance of materials used
in protective clothing to penetration by blood-borne pathogens. This test method uses a surrogate microbe
under conditions of continuous liquid contact. Protective clothing “pass/fail” determinations are based on the
detection of viral penetration at a specific hydrostatic pressure using the ISO 13994 test apparatus.
This test method is not always effective in testing protective clothing materials having thick, inner liners which
readily absorb the challenge fluid.
This test method involves a sensitive assay procedure. Because of the length of time required to complete this
test method, it might not be suitable for use as a material or protective clothing quality control or assurance
procedure.
2 Normative references
The following referenced documents are indispensable for the application 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 304, Surface active agents — Determination of surface tension by drawing up liquid films
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 3801, Textiles — Woven fabrics — Determination of mass per unit length and mass per unit area
ISO 5084, Textiles — Determination of thickness of textiles and textile products
ISO 13994, Clothing for protection against liquid chemicals — Determination of the resistance of protective
clothing materials to penetration by liquids under pressure
ISO 16603, Clothing for protection against contact with blood and body fluids — Determination of the
resistance of protective clothing materials to penetration by blood and body fluids — Test method using
synthetic blood
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
agar
semi-solid culture medium used to support the growth of bacteria and other microorganisms
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ISO 16604:2004(E)
3.2
assay
analysis of a mixture to determine the presence of or concentration of a particular component
NOTE In this test method, the component being analysed is a microorganism, Phi-X174 bacteriophage.
3.3
assay fluid
sterile liquid used to wash the test material surface to determine microbiological penetration
NOTE In this test method, the assay fluid is nutrient broth and the bacterial virus is the Phi-X174 bacteriophage. The
assay fluid is used to wash the Phi-X174 bacteriophage from the normal inside material surface of the test specimen.
3.4
bacteriophage
type of virus which infects bacteria
NOTE In this test method, the bacteriophage is Phi-X174. The Phi-X174 bacteriophage is not pathogenic to humans,
but serves to simulate viruses that are pathogenic to humans.
3.5
blood-borne pathogen
infectious secreted or excreted bacterium, virus, or other disease-inducing microbe carried in blood or other
body fluids
NOTE For the purpose of this International Standard, the primary blood-borne pathogens include hepatitis B virus
(HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Other microorganisms should be considered on
a case-by-case basis.
3.6
body fluid
any liquid produced (secreted or excreted) by the body
NOTE For the purpose of this International Standard, body fluids include those liquids potentially infected with blood-
borne pathogens, including, but are not limited to, blood, semen, vaginal secretions, cerebrospinal fluid, synovial fluid,
peritoneal fluid, amniotic fluid, saliva in dental procedures, and any body fluid that is visibly contaminated with blood, and
all body fluids in situation where it is difficult or impossible to differentiate between body fluids.
3.7
body fluid simulant
liquid which is used to act as a model for human body liquids
NOTE In this test method, the body fluid simulant is bacteriophage nutrient broth, which is intended as a model for
human body liquids as its approximates the lower end of the surface tension range for blood and body fluids (excluding
saliva), (0,042 ± 0,002) N/m.
3.8
challenge suspension
liquid containing an agent that is used to test the penetration resistance of materials
NOTE In this test method, the challenge suspension is the bacteriophage challenge suspension; a nutrient broth
containing the Phi-X174 bacteriophage.
3.9
lawn
〈microbiology〉 cloudy, uniform growth of bacteria in a thin layer of top agar in a petri dish
NOTE In this test method, Escherichia coli C. (E. coli C) has been selected as the bacterium used to produce the
lawn.
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ISO 16604:2004(E)
3.10
lysis
disintegration or destruction of whole bacterial cells
NOTE In this test method, the lysis of the host bacteria, E. coli C., is caused by Phi-X174 bacteriophage.
3.11
medium
media
nutrient system for the cultivation of cells or organisms
NOTE In this test method, the term media is used to describe mixtures compounded to support the growth of specific
microorganisms, for example, bacteriophage nutrient broth and top agar.
3.12
morphology
form and structure of a particular organism
3.13
nutrient broth
liquid medium
NOTE In this test method, the nutrient broth is the bacteriophage nutrient broth which is used to culture the host
bacteria, E. coli C., and to aid in manipulating the Phi-X174 bacteriophage through the various stages of the procedure,
such as suspending the Phi-X174 bacteriophage for challenging the test material in the penetration cell, assaying the
normal inside test material surface, and if required, making dilutions of assay fluid for plating.
3.14
penetration
flow of a liquid through closures, porous materials, seams and holes or other imperfections in a protective
clothing material on a non-molecular level
3.15
plaque
〈virology〉 visible, clear area which is theoretically the result of the infection and lysis of host cells by a single
viable virus
NOTE In this test method, the term plaque is used to describe a visible, clear area, in the lawn of E. Coli C. in top
agar which is theoretically the result of a single viable Phi-X174, where the bacteria have been destroyed by
bacteriophage infection and lysis.
3.16
plaque-forming unit
PFU
virus particle capable of producing plaques by infecting and lysing bacteria in a lawn in top agar
3.17
plate
〈microbiology〉 Petri dish containing culture medium
3.18
protective clothing
item of clothing that is specifically designed and constructed for the intended purpose of isolating all or part of
the body from a potential hazard; or, isolating the external environment from contamination by the wearer of
the clothing.
3.19
surrogate microbe
microorganism which is used to act as a simulant for other microorganisms which are pathogenic to humans
NOTE In this test method, the surrogate microbe is the Phi-X174 bacteriophage, intended as a model for HCV and to
simulate both HBV and HIV.
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ISO 16604:2004(E)
3.20
titre
quantity of a substance required to react with, or to correspond to, a given amount of another substance
NOTE In this test method, titre is used to describe the concentration of viable bacteriophage as measured in plaque-
forming units per millilitre (PFU/ml).
3.21
virus
minute infectious agent, which lacks independent metabolism and is only able to replicate within a living host
cell
3.22
viral penetration
penetration of a material by a virus
NOTE In this test method, the physical translocation of Phi-X174 bacteriophage through closures, seams, pores, and
pinholes or other imperfections in materials used in protective clothing.
3.23
viral resistant
referring to materials which impede viral penetration under specified laboratory test conditions and detection
methods
NOTE In this test method, protective clothing materials which demonstrate “pass” results are considered to be
resistant to viral penetration.
4 Principle
A specimen is subjected to a nutrient broth containing a virus in a test apparatus as specified in ISO 13994 for
a specified time and pressure sequence. Visual detection of penetration is supplemented with an assay
procedure that will detect viable viruses which penetrate the material even when liquid penetration is not
visible. Any evidence of viral penetration for a test specimen constitutes failure.
This test method requires a working knowledge of basic microbiological techniques.
5 Microorganisms and reagents
8
5.1 Bacteriophage Phi-X174 (ATCC 13706-B1), used at a challenge titre of at least 1,0 × 10 PFU/ml
(plaque-forming units per millilitre).
NOTE The surrogate microbe, Phi-X174 bacteriophage, was selected as the most appropriate model for blood-borne
pathogens because of its small size, spherical (icosahedral) morphology, environmental stability, non-human infectivity,
high assay sensitivity, rapid assay, and high titre. The Phi-X174 bacteriophage has no envelope and is one of the smallest
known viruses (0,027 µm in diameter).
5.2 Bacteria E. coli (ATCC 13706).
5.3 Purified water, grade 3 in accordance with ISO 3696:1987.
5.4 Nutrient broth.
5.5 Calcium chloride (CaCl ).
2
5.6 Potassium chloride (KCl).
5.7 Sodium hydroxide (NaOH), 2,5 mol/l solution.
5.8 Surfactant, Polysorbate 80.
5.9 Bacto-agar.
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ISO 16604:2004(E)
6 Apparatus and materials
6.1 Penetration test cell, as specified in ISO 13994, to restrain the specimen during contact with the
pressurized challenge fluid.
In the test cell, the specimen acts as a partition separating Phi-X174 bacteriophage challenge suspension
from the view side of the test cell. It consists of a cell body that is fastened to a cell support. The cell body has
a capacity of approximately 60 ml for the Phi-X174 bacteriophage challenge suspension. A flange cover, with
an open area to allow visual observation, and a transparent cover are included. The cell body has a top port
for filling and a drain valve for draining the penetration test cell. Other items, such as a fitting to allow
attachment of the air line to the top port in the cell body, gaskets, and the retaining screen are also required. A
diagram of the penetration test cell and apparatus are provided in Figures 1 and 2.
6.2 Other equipment
6.2.1 Thickness gauge, suitable for measuring thickness to the nearest 0,02 mm.
6.2.2 Retaining screen, comprising a smooth finish plastic or metal square mesh screen to support
extensible or elastomeric materials, meeting the following specifications:
a) open area of > 50 %,
b) deflection of the test specimen is limited to u 5,0 mm.
+2
6.2.3 Air pressure source, capable of providing air at 20,0 kPa.
( 0 )
6.2.4 Incubator, capable of sustaining a temperature range of (36 ± 1) °C.
6.2.5 Water bath, capable of achieving a temperature range of (45 ± 2) °C.
6.2.6 Balance, with a precision of 0,001 g.
6.2.7 Vortex mixer.
6.2.8 Refrigerator, capable of maintaining a temperature range of (5 ± 3) °C.
6.2.9 Autoclave, capable of maintaining (122 ± 1) °C and (214 ± 7) kPa absolute.
6.2.10 Stopwatch, or electronic timer.
6.2.11 Orbital shaker.
6.2.12 pH meter, sensitive to 0,1 pH units.
6.2.13 Inoculating loop.
6.2.14 Torque wrench, capable of applying a torque of 13,6 N·m.
6.2.15 Spectrophotometer, capable of measuring absorbed light at 640 nm.
6.2.16 Centrifuge, capable of an acceleration of 10 000 g.
6.3 Laboratory glassware
6.3.1 Petri dishes, sterile, 15 mm × 100 mm.
6.3.2 Pipettes, sterile, of 1 ml, 5 ml, 10 ml capacity.
6.3.3 Test tubes, 13 mm × 100 mm.
6.3.4 Test tube rack.
6.3.5 Glass bottles, sterile, with a capacity of 100 ml to 500 ml.
6.3.6 Micropipettes, capable of delivering 2 µl accurately and consistently.
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ISO 16604:2004(E)
7 Test specimens
7.1 Selection
7.1.1 Select specimens from single material samples or individual protective clothing items (following
sterilization, if applicable) consisting of either a single layer or a composite of multiple layers that is
representative of an actual protective clothing construction with all layers arranged in proper order.
If in the design of an item of protective clothing, different materials or thicknesses of material are specified at
different locations, select specimens from each location.
If in the design of an item of protective clothing, seams are claimed to offer the same protection as the base
materials, test additional specimens containing such seams.
Cut each material specimen into squares with a minimum dimension of 70 mm. A 75 mm square is preferred.
Test three specimens taken at random from each material, composite, area (in the case of heterogeneous
design), or other condition.
If this procedure is used for quality control or to support broad product claims concerning the viral-resistant
properties of materials used in protective clothing, proper statistical design and analysis of larger data sets
than those specified in this test method should be performed. Examples of acceptable sampling plans are
[8]
found in references such as ISO 2859-1 .
7.1.2 It is possible that protective clothing materials incorporating an impervious layer between two fabric
layers are sensitive to false positive failures by wicking at the edges. Seal the edges of the test specimens to
prevent “wicking” modes of failure using an adhesive, parafilm, paraffin wax, or adhesive-backed foam prior to
testing. Seal only the edges of the test specimens, leaving the centre 57 mm area open for testing. Do not
allow sealants to intrude, block, or occlude the structure of the test specimen in the test area, as this may
compromise the test procedure. Choose sealants and sealing methods that are compatible with the protective
clothing materials.
7.2 Preparation of test specimens
Condition each protective clothing specimen for a minimum of 24 h by exposure to a temperature of
(21 ± 5) °C and a relative humidity of (60 ± 10) %.
If warranted, use other preconditioning options, such as sterilization, to assess possible degradation
mechanisms of protective clothing.
8 Procedure
8.1 Preparation of test media
8.1.1 Bacteriophage nutrient broth (Phi-X)
Prepare bacteriophage nutrient broth using the following:
 Bacto-tryptone (8,0 ± 0,1) g
 Potassium chloride (5,0 ± 0,06) g
 Calcium chloride (0,2 ± 0,003) g
 Purified water (5.3) (1 000 ± 12,5) ml
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ISO 16604:2004(E)
 Surfactant (5.8) (0,1 ± 0,001 25) ml
Adjust pH of the bacteriophage nutrient broth to (7,3 ± 1) using 2,5 mol/l sodium hydroxide.
Dilute 1 volume of 0,1 % surfactant with 9 volumes of bacteriophage nutrient broth. To ensure adequate
mixing, prior to sterilization, heat the bacteriophage nutrient broth while stirring in the surfactant. A final
concentration of 0,01 % surfactant is recommended to adjust the surface tension to (0,042 ± 0,002) N/m.
Sterilize the bacteriophage nutrient broth in the autoclave.
Measure the resulting surface tension of the sterile solution in accordance with ISO 304. Do not use
bacteriophage nutrient broth unless the corrected surface tension is within the (0,042 ± 0,002) N/m range.
8.1.2 Bottom agar (Phi-X)
Prepare the bottom agar using the following:
 Bacto-agar (15,0 ± 0,19) g
 Nutrient broth (8,0 ± 0,1) g
 Potassium chloride (5,0 ± 0,06) g
 Purified water (5.3) (1 000 ± 12,5) ml
 Calcium chloride (1,0 ± 0,012 5) ml (to be added after autoclaving the bottom agar)
Prepare sterile calcium chloride by autoclaving a 1 mol/l solution of calcium chloride in purified water.
Adjust pH of the bottom agar to (7,3 ± 1) using 2,5 mol/l sodium hydroxide.
Sterilize the bottom agar in the autoclave.
8.1.3 Top agar (Phi-X)
Prepare the top agar using the following:
 Bac
...