ISO 20304-2:2024

International
Standard
ISO 20304-2
First edition
Fine bubble technology — Water
2024-01
treatment applications —
Part 2:
Test methods using Escherichia coli
as a test micro-organism
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Safety precautions. 2
5 Requirements . 2
6 Test methods . 2
6.1 Principle .2
6.2 Test facility .2
6.3 Culture media and reagents.2
6.3.1 General .2
6.3.2 Water .2
6.3.3 Tryptone soya agar (TSA) .3
6.3.4 Tryptone soya broth (TSB) .3
6.3.5 Lysogeny broth (LB) .3
6.3.6 Cryoprotective solution for bacterial species .3
6.3.7 Physiological saline .4
6.3.8 Tryptone sodium chloride solution (TSCS) .4
6.4 Laboratory apparatus and glassware .4
6.5 Preparation of bacterial test suspension .5
6.5.1 Storage of strains .5
6.5.2 Working culture of test bacteria .5
6.5.3 Bacterial test suspension . .6
6.6 Procedure .6
6.6.1 Choice of experimental conditions .7
6.6.2 Test procedure for antibacterial activity .8
6.7 Calculation and expression of results .9
Annex A (normative) Strain numbers . 10
Annex B (normative) Shaking method .11
Annex C (normative) Luminescence photometers and RLU .12
Annex D (informative) Test report of interlaboratory tests for disinfection efficiency of fine
bubbles .13
Bibliography .21

iii
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
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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).
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This document was prepared by Technical Committee ISO/TC 281, Fine bubble technology.
A list of all parts in the ISO 20304 series can be found on the ISO website.
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
Introduction
In the last decade, fine bubble technology generated by hydrodynamic cavitation methods has been applied
as part of water treatment facilities for water disinfection purposes. However, absence of International
Standards in relation to the efficiency of water disinfectant properties on fine bubbles has been an obstacle
to trading and technology transfer. This document describes a method for the determination of water
disinfection efficiency of fine bubbles generated by a hydrodynamic cavitation.
Fine bubbles act as a disinfectant through chemical (i.e. generation of OH radicals) and physical mechanisms
caused by presence of shock waves, pressure gradients and shear forces. The function as a disinfectant of
fine bubbles was also found in that fine bubbles provide a more effective means for cleaning and disinfecting
[1]
both, the bath and the reservoir, than traditional ultrasonic vibrator.
Properties of fine bubbles and their disinfection mechanisms differ according to applied generation processes,
bubble size distributions and bubble densities. However existing studies showed that fine bubbles generated
have a significant water disinfection effect. Particularly, high deactivation efficiency of Escherichia coli (E.
coli) has been achieved in water disinfection by microbubbles generated using hydrodynamic cavitation as
[2]-[4]
well.
Generation of highly reactive free radicals and turbulence associated with collapsing micro bubbles provides
great potential for water disinfection. The effect of ozone microbubble on E. coli has often been found more
effective and with faster disinfection kinetics of E. coli, when the ozone gas is activated as the micro or
[5],[6]
ultrafine bubble forms.
The test methods on the antibacterial activity of textile products have been standardized by ISO/TC 38,
textiles. The present standard operation procedure (SOP) for the evaluation of disinfection efficiency is
based on the quantitative measurement by ATP luminescence method for the evaluation of antibacterial
activity.
Therefore, under the test conditions, establishment of International Standards to measure the water
disinfection efficiency of fine bubbles is essential to promote the relevant trading technology transfer.

v
International Standard ISO 20304-2:2024(en)
Fine bubble technology — Water treatment applications —
Part 2:
Test methods using Escherichia coli as a test micro-organism
1 Scope
This document specifies a test method for assessing bactericidal viability of Escherichia coli as a test micro-
organism, in dispersions of various fine bubbles generated by the hydrodynamic cavitation of water medium.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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/
3.1
hydrodynamic cavitation
fine bubble generation method used by physical hydrodynamic structures and pressures
3.2
disinfection efficiency
ratio of viable cell count to the initial number of the test bacteria
3.3
control suspension
suspension used to validate the growth condition of test bacteria and validate the test, i.e. bacterial test
suspension produced without using fine bubble generation
3.4
test solution
solution samples obtained for the analysis of antibacterial activity after operating the test facility
3.5
test gas
gas mixed in the middle of operating the cavitation unit
3.6
relative light unit
RLU
unit used for adenosine triphosphate (ATP) luminescence measurement

4 Safety precautions
The test methods specified in this document require the use of bacteria and conditions that promote bacterial
growth. Since the bacteria can be pathogenic, the tests should be carried out by persons with training
and experience in the use of microbiological techniques. The laboratory facilities should be designated as
[7]
appropriate biosafety level.
Appropriate safety precautions should be observed with due consideration given to country-specific.
5 Requirements
This document is to provide the measurement method of disinfection efficiency for fine bubbles enhanced
waters by test hydrodynamic cavitation units. The disinfection efficiency is measured by the test fine bubble
solution generated by each test hydrodynamic cavitation unit.
The antibacterial (bactericidal) activity shall be evaluated using the following test organism: E. coli (see
Table A.1). The test tank and test facility should be cleaned by appropriate methods like triple-wash with
sterilized water after chlorination before the test.
6 Test methods
6.1 Principle
The test tank is filled with a test suspension of bacteria and the test hydrodynamic cavitation (HC) unit is
operated under the required initial test condition (approximately 15 °C to 24 °C ± 1 °C). The fine bubbles-
enriched water will be contacted to the test suspension of bacteria in the test tank (see Figure 1).
At a specific contact time, an aliquot is taken; the bactericidal action in this portion is immediately measured
using the luminescence photometer (see 6.4.8). First, the relationship between the measurement unit of the
luminescence photometer, relative light unit (RLU) (see Annex C) and counted colonies forming unit (CFU)
of the initial density of the test microorganism is measured to check the initial cell density of the bacterial
test suspension.
The test is performed using E. coli (see Table A.1).
6.2 Test facility
Fine bubbles are generated by circulating the liquid in a fine bubble generating system with the following
characteristics: a 125-litre tank, a 0,4 kW centrifugal multistage pump made of stainless steel, PVC pipes
and a cavitation unit with the test fine bubble generating unit (see Figure 1). Test gas can be chosen for the
appropriate test purposes.
6.3 Culture media and reagents
6.3.1 General
The reagents shall be of analytical grade and/or appropriate for microbiological purposes.
To improve reproducibility, dehydrated material should be used for the preparation of culture media. The
manufacturer’s instructions relating to the preparation of these products should be rigorously followed.
6.3.2 Water
Water used in tests shall be analytical-grade water for microbiological media preparation, which is freshly
distilled and/or ion-exchanged and/or ultra-filtered and/or filtered with reverse osmosis (RO). It shall be
free from substances that are toxic or inhibiting to the bacteria.

6.3.3 Tryptone soya agar (TSA)
TSA used in the test is for maintenance of bacterial strains and performance of viable counts.
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of soybean meal 5,0 g
NaCl 5,0 g
Agar 5,0 g
Water (see 6.3.2) to 1,0 l
Sterilize in the autoclave (6.4.1). After sterilization, the pH of the medium shall be 7,2 ± 0,2 when measured
at 20 °C.
6.3.4 Tryptone soya broth (TSB)
Tryptone, pancreatic digest of casein 17 g
Soya peptone, papaic digest of soya 3 g
Sodium chloride (NaCl) 5 g
Glucose 2,5 g
Dipotassium hydrogen phosphate 2,5 g
Water 1 000 ml
Mix well and adjust pH, 7,2 ± 0,2, then sterilize by autoclave (6.4.1).
6.3.5 Lysogeny broth (LB)
Tryptone, pancreatic digest of casein 10 g
Yeast extract 5 g
NaCl 10 g
Water 1 000 ml
Mix well and adjust pH, 6,9 ± 0,2, then sterilize by autoclave (6.4.1).
6.3.6 Cryoprotective solution for bacterial species
For freezing, a cryoprotective solution containing 150 g/l of glycerol or 100 g/l of dimethylsulfoxide shall be
used and prepared as follows:
TSB (see 6.3.4) or LB (see 6.3.5): 1 000 ml
Add Glycerol: 150 g or dimethylsulfoxide: 100 g
Mix well and sterilize by autoclave (6.4.1).
For solutions containing glycerol, sterilize the mixed solution by autoclave (6.4.1). For solutions containing
dimethylsulfoxide, sterilize the mixed solution by using a 0,22 µm membrane filter.
NOTE Any commercially available product can be used as long as it is a cryoprotective solution or preserving
system that contains glycerol or dimethylsulfoxide.

6.3.7 Physiological saline
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml
Mix well, then sterilize by autoclave (6.4.1).
6.3.8 Tryptone sodium chloride solution (TSCS)
Tryptone, pancreatic digest of casein 1 g
NaCl 8,5 g
Water 1 000 ml
6.4 Laboratory apparatus and glassware
The usual laboratory apparatus and, in particular, the following shall be used.
6.4.1 Autoclave, capable of sterilizing at 121 °C ± 2 °C and 103 kPa ± 5 kPa.
6.4.2 Incubator, capable of being controlled at either 36 °C ± 1 °C or 37 °C ± 1 °C. An incubator at
37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not available.
6.4.3 pH meter, having an accuracy ±0,1 in calibration on pH units at 25 °C.
6.4.4 Stopwatch.
6.4.5 Container, test tubes or flasks of suitable capacity.
6.4.6 Pipettes, having the most suitable volume for each use, with a tip made of glass or plastic, and with
a tolerance of 0,5 % or less.
6.4.7 Petri dishes, that have been sterilized, made of glass or plastic, in diameter sizes of 90 mm to
100 mm.
−12
6.4.8 Luminescence photometer, with ATP kit for water test, capable of measuring ATP of 10 mol/l to
−7
10 mol/l at 300 nm to 650 nm with a luminescence-measuring reagent.
6.4.9 Vials, 250 ml plastic bottles, with screw openings, polytetrafluoroethylene or silicone packing and
caps made of polypropylene, polycarbonate or another suitable material.
6.4.10 Glass beads, with a diameter of 3 mm to 4 mm.
6.4.11 Freezers, one adjustable to a temperature below −70 °C and another to a temperature below −20 °C.
6.4.12 Homogenizer, capable of speeds of six blows per second to eight blows per second, with the
corresponding disposable containers.
6.4.13 Disposable plastic bags, sterile bags suitable for containing food products, to be used for one of the
shaking methods of the specimens.
6.4.14 Sterilized stirring rod, with a 6 mm (±2 mm) diameter and a 300 mm (±50 mm) length.

6.5 Preparation of bacterial test suspension
6.5.1 Storage of strains
6.5.1.1 General
The strains shall be stored in accordance with the supplier’s recommendations. The experiment instruments
should be sterilized by the autoclave (6.4.1) for a minimum of 15 min.
6.5.1.2 Preparation method
Obtain a sample of the freeze-dried bacterial strain in accordance with Annex A, following the
recommendations supplied with the culture and resuspend it in 5 ml of TSB (see 6.3.4). Obtain a sample of
the suspension and isolate it in a plastic bottle (6.4.10) containing LB (see 6.3.5). Incubate the cultures for
18 h to 24 h at 37 °C ± 2 °C.
After incubation, use the culture isolated in the Petri dish to verify the purity of the strain.
After verification, prepare the stock cultures to check the density of E. coli of the bacterial test suspension.
Sample 0,7 ml of the broth culture and spread it over the surface of the Petri dish containing the TSA.
Incubate the culture on plates for 18 h to 24 h at 37 °C ± 2 °C.
Add 10 ml of cryoprotective solution (see 6.3.6) to the surface of the TSA plate culture and resuspend the
cells in the solution using a sterile glass spreader. Sample the suspended cells from the surface of the agar,
dilute them in 100 ml of cryoprotective solution and incubate for 30 min at 20 °C.
Using a pipette (6.4.6), sample 1 ml of the suspension and transfer it to a cryogenic vial (6.4.9) containing the
beads (6.4.10). Shake the vial in order to spread the suspended cells around the beads.
— Where a cryoprotective solution containing dimethylsulfoxide is used, do not let it stand longer than
1 min at ambient temperature.
— Where a cryoprotective solution containing glycerol is used, let it stand for 30 min at 20 °C.
— Withdraw the excess cryoprotective solution with a sterile pipette. Place the cryogenic vials in a freezer
(6.4.11) set at −70 °C or lower.
−6 −7
Prepare 10 and 10 dilutions of the suspension using the serial dilution method. Take a 1,0 ml sample of
each dilution and transfer it to separate Petri dishes. Add 12 ml to 15 ml of nutritive solution
...