Water quality -- Determination of the estrogenic potential of water and waste water

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay with a genetically modified yeast strain Arxula adeninivorans. This reporter gene assay is based on the activation of the human estrogen receptor alpha. Arxula adeninivorans is a highly robust and salt- and temperature-tolerant test organism and is especially suitable for the analysis of samples with high salinity (conductivity up to 70 mS/cm). The test organism can be cultivated in medium with sodium chloride content up to 20 %. This method is applicable to: — fresh water; — waste water; — sea water; — brackish water; — aqueous extracts and leachates; — eluates of sediments (fresh water); — pore water; — aqueous solutions of single substances or of chemical mixtures; — drinking water. The limit of quantification (LOQ) of this method for the direct analysis of water samples is between 1,5 ng/l and 3 ng/l 17β-estradiol equivalents (EEQ). The upper threshold of the dynamic range for this test is between 25 ng/l and 40 ng/l 17β-estradiol equivalents (EEQ). Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre-concentration of water samples can prove necessary, if their estrogenic potential is below the given LOQ. An international interlaboratory trial for the validation of this document has been carried out. The results are summarized in Annex F. NOTE Extraction and pre-concentration of water samples can prove necessary.

Qualité de l'eau -- Détermination du potentiel oestrogène de l'eau et des eaux résiduaires

General Information

Status
Published
Publication Date
06-Aug-2018
Current Stage
6060 - International Standard published
Start Date
12-May-2018
Completion Date
07-Aug-2018
Ref Project

Buy Standard

Standard
ISO 19040-2:2018 - Water quality -- Determination of the estrogenic potential of water and waste water
English language
55 pages
sale 15% off
Preview
sale 15% off
Preview

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 19040-2
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 2:
Yeast estrogen screen (A-YES, Arxula
adeninivorans)
Qualité de l'eau — Détermination du potentiel oestrogène de l'eau et
des eaux résiduaires —
Partie 2: Test d'oestrogénicité (A-YES, Arxula adeninivorans)
Reference number
ISO 19040-2:2018(E)
ISO 2018
---------------------- Page: 1 ----------------------
ISO 19040-2:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19040-2:2018(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 2

3 Terms and definitions ..................................................................................................................................................................................... 2

4 Principle ........................................................................................................................................................................................................................ 4

5 Interferences ............................................................................................................................................................................................................ 4

6 Apparatus and materials.............................................................................................................................................................................. 4

7 Reagents, media and test strains ......................................................................................................................................................... 5

8 Sampling and samples .................................................................................................................................................................................... 9

8.1 General ........................................................................................................................................................................................................... 9

8.2 Bottles and material for sampling .......................................................................................................................................... 9

8.3 Bottles and material pre-cleaning .......................................................................................................................................10

8.4 Sampling procedure ........................................................................................................................................................................10

8.5 Transport of samples ......................................................................................................................................................................10

8.6 Pretreatment of samples .............................................................................................................................................................10

8.7 Storage of samples ............................................................................................................................................................................11

9 Procedure..................................................................................................................................................................................................................11

9.1 Test set up ................................................................................................................................................................................................11

9.1.1 Preparation of the reference dilution series .........................................................................................11

9.1.2 Reactivation of the yeast ........................................................................................................................................11

9.1.3 Negative control ............................................................................................................................................................12

9.1.4 Blank replicate ................................................................................................................................................................12

9.1.5 Sample dilution ..............................................................................................................................................................12

9.1.6 Field blank ..........................................................................................................................................................................13

9.1.7 Plate set up .........................................................................................................................................................................13

9.1.8 Inoculation of the test plate .................................................................................................................................13

9.2 Measurement .........................................................................................................................................................................................14

9.2.1 Measurement of the reporter gene activity ...........................................................................................14

9.2.2 Measurement of the cell density .....................................................................................................................15

9.3 Calculations .............................................................................................................................................................................................15

9.3.1 Background correction ............................................................................................................................................15

9.3.2 Calculation of the relative growth ..................................................................................................................16

9.3.3 Calculations for assessment of sample blanks ....................................................................................17

9.3.4 Calculation of the reporter gene induction ............................................................................................19

10 Validity criteria ...................................................................................................................................................................................................22

11 Assessment criteria ........................................................................................................................................................................................23

12 Test report ................................................................................................................................................................................................................23

13 Verification ..............................................................................................................................................................................................................23

Annex A (informative) Plate set up ......................................................................................................................................................................25

Annex B (informative) Lyophilization of Arxula adeninivorans cells ...............................................................................26

Annex C (informative) Scheme of test principle ....................................................................................................................................28

Annex D (informative) Test set up for chemicals and extracts ...............................................................................................29

Annex E (informative) Preparation of dilution series .....................................................................................................................30

Annex F (informative) Performance data .....................................................................................................................................................31

Annex G (informative) Statistical assessment .........................................................................................................................................41

© ISO 2018 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 19040-2:2018(E)

Annex H (informative) Calculation of estradiol equivalents.....................................................................................................42

Annex I (informative) Alternative test design for EEQ determination ...........................................................................45

Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water

— A simplified evaluation for testing of waste water .................................................................................................46

Annex K (informative) Example for statistical evaluation ..........................................................................................................48

Bibliography .............................................................................................................................................................................................................................55

iv © ISO 2018 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 19040-2:2018(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.

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 documents 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).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on 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 the following

URL: www .iso .org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.
A list of all parts in the ISO 19040 series can be found on the ISO website.
© ISO 2018 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 19040-2:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 2:
Yeast estrogen screen (A-YES, Arxula adeninivorans)

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
1 Scope

This document specifies a method for the determination of the estrogenic potential of water and waste

water by means of a reporter gene assay with a genetically modified yeast strain Arxula adeninivorans.

This reporter gene assay is based on the activation of the human estrogen receptor alpha.

Arxula adeninivorans is a highly robust and salt- and temperature-tolerant test organism and is

especially suitable for the analysis of samples with high salinity (conductivity up to 70 mS/cm). The test

organism can be cultivated in medium with sodium chloride content up to 20 %.
This method is applicable to:
— fresh water;
— waste water;
— sea water;
— brackish water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.

The limit of quantification (LOQ) of this method for the direct analysis of water samples is between

1,5 ng/l and 3 ng/l 17β-estradiol equivalents (EEQ). The upper threshold of the dynamic range for

this test is between 25 ng/l and 40 ng/l 17β-estradiol equivalents (EEQ). Samples showing estrogenic

potencies above this threshold have to be diluted for a valid quantification. Extraction and pre-

concentration of water samples can prove necessary, if their estrogenic potential is below the given LOQ.

An international interlaboratory trial for the validation of this document has been carried out. The

results are summarized in Annex F.
NOTE Extraction and pre-concentration of water samples can prove necessary.
© ISO 2018 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 19040-2:2018(E)
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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org ./obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
blank replicate

additional replicate that contains no test organism, but is treated in the same way as the other replicates

of a sample
[SOURCE: ISO 10872:2010, 3.5]
3.2
culture medium

nutrients presented in a form and phase (liquid or solidified) which support microbiological growth

3.3
dilution level

denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water

with dilution water as integral number

Note 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. The corresponding and

smallest possible value of D is 1. In this document, the arrow indicates the transition from initial total volume to

final total volume.
[SOURCE: ISO 6107-6:2004, 28]
3.4
dilution water
water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.5
50 % effect concentration
concentration of a compound which causes 50 % of an effect

Note 1 to entry: In the sense of this document the EC is the concentration of a compound which induces 50 % of

the maximal reporter gene activity which can be achieved by this compound.
3.6
field blank

container prepared in the laboratory, using reagent water or other blank matrix, and sent with the

sampling personnel for exposure to the sampling environment to verify possible contamination during

sampling
[SOURCE: ISO 11074:2015, 4.5.3]
2 © ISO 2018 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 19040-2:2018(E)
3.7
growth rate
proportional rate of increase in cell density
[SOURCE: ISO 10253:2006, 3.2]
3.8
induction rate

quotient of the mean value of wells with enhanced reporter gene activity measured on the plates

treated with a dose of the test sample, and the mean value of the corresponding wells treated with the

negative control using the same strain under identical conditions

Note 1 to entry: Instead of the negative control, the estimated parameter A of the four-parameter model, which

describes the dose response relationship between reference compound and the induction rate, can be used.

[SOURCE: ISO 6107-6:2004, 43, modified — "wells with enhanced reporter gene activity measured"

replaces "mutant colonies"; "corresponding wells" replaces "corresponding plates"; “quotient” replaces

“difference”.]
3.9
inoculum

fraction of a culture of microorganisms used to start a new culture, or an exponentially growing

preculture, in fresh medium
[SOURCE: ISO 6107-6:2004, 44]
3.10
lowest ineffective dilution value
LID

lowest dilution within a test batch which does not show any effect, i.e. no statistically significant

increase in the reporter gene activity compared with the negative control

[SOURCE: ISO 11350:2012, 3.4, modified — “increase in the reporter gene activity” replaces “increase

in the number of revertant wells”.]
3.11
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.12
reference compound

compound with one or more property values that are sufficiently reproducible and well established to

enable the calibration of the measurement method

[SOURCE: ISO 7405:2008, 3.6, modified — “compound” replaces “material”; “the calibration of the

measurement method” replaces “use of the material or substance for the calibration of an apparatus,

the assessment of a measurement method or for the assignment of values to materials”.]

3.13
reporter gene activity

quantitative activity of a gene attached to the promoter sequence of another gene

3.14
test sample

undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all

preparation steps such as centrifugation, filtration, homogenization, pH adjustment and determination

of ionic strength
[SOURCE: ISO 6107-6:2004, 92]
© ISO 2018 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 19040-2:2018(E)
4 Principle

The A-YES (Arxula Yeast Estrogen Screen) is a reporter gene assay which can be used for the

measurement of the activation of the human estrogen receptor alpha (ERα) in the presence of a sample

containing compounds which cause estrogenic effects. By this means the assay detects the estrogenic

activity of the whole sample in its actual state as an integral measure including possible additive,

synergistic and antagonistic mixture-effects.

The human estrogen receptor α is constitutively expressed in the yeast cell under control of a TEF1

promoter. The estrogen receptor belongs to the family of nuclear hormone receptors. If agonists of the

estrogen receptor enter the yeast cell, they bind to the estrogen receptor protein and thus induce its

conformational change. As a consequence two receptor proteins form a receptor dimer. This activation

of the estrogen receptor is measured by the induction of the reporter gene phyK which encodes the

enzyme phytase. The phyK is fused to an estrogen dependent promoter which contains estrogen

responsive elements (ERE). The ER-dimer binds to the promoter and by this activates the expression

and secretion of the phytase. Finally, the activity of the phytase as a measure for the estrogenic

potential of the sample is determined using an appropriate substrate which is cleaved to a coloured

reaction product. The reaction product can be measured photometrically. See Annex C for a scheme of

the test principle.
5 Interferences

Coloured or turbid samples might interfere with the photometric detection of the cell density and/

or the detection of the cleaved substrate of the reporter enzyme phytase (see Clause 10 for further

information).

Effects of the sample matrix may lead to a reduction or increase of viable cells and to a reduction or

increase of the measurable signal. Estrogenic effects of a sample may be masked by matrix effects

leading to false negative or false positive test results.

High salinity can cause toxic effects due to the resulting osmotic pressure. The conductivity of a sample

is a measure for its salinity. The Arxula adeninivorans yeast tolerates a conductivity of the sample up to

20 % sodium chloride which meets a conductivity of 180 mS/cm.

Bacterial growth in the test wells is assessed by the blank replicate (3.1). See Clause 10 for further

information.

If filtered samples are tested in order to remove bacteria from the sample solid particles are separated

from the sample also. Thus, substances with estrogenic activity which are adsorbed on particles might

not be detected.

For detailed information about appropriate sampling material that does not influence the test result

see Clause 8.
6 Apparatus and materials

For suitable sampling devices see Clause 8. Use usual laboratory apparatus and glassware if required.

In particular, the following material is needed:

6.1 Temperature- and time-controlled incubator shaker, shaker orbit at least 3 mm, 30 °C to

37 °C with an accuracy of ±1 °C.

If the shaker has no incubation function use a lab shaker with a shaker orbit of at least 3 mm in

combination with an incubator (6.17).
6.2 Lab mini shaker.
4 © ISO 2018 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 19040-2:2018(E)

6.3 Multi-parameter measurement device for pH and conductivity or separate devices for each

parameter.
6.4 Steam sterilizer.

6.5 Centrifuge, with a rotor for 96-well plates up to 1 000 g and a rotor for 2 ml reaction tubes.

6.6 Sterile filters, cellulose acetat, 0,2 µm pore size.
6.7 Single-channel pipettes, nominal volume 10 µl up to 10 000 µl.
6.8 Multi-channel pipettes, nominal volume 100 µl and 300 µl.

6.9 Transparent polystyrene 96-well plates with flat bottom (F-profile, 300 µl) and lid.

6.10 96-deep well plates with at least 1 ml volume with round bottom and square wells.

6.11 Microplate photometer for 96-well plates, for absorbance measurement for wavelength

405 nm ± 20 nm and 630 nm ± 5 nm or alternatively 600 nm ± 20 nm.
6.12 Air-permeable adhesive foil for deep well plates.
6.13 Reaction tubes, 2 ml.
6.14 Test tubes, 15 ml and 50 ml.
6.15 Multi-channel pipette trough.
6.16 Balance, minimum load 1 mg, d = 0,1 mg.

6.17 Incubator, 30 °C to 37 °C with an accuracy of ±1 °C. For the purpose of using the incubator in

combination with a shaker a cooled incubator is required.
7 Reagents, media and test strains
7.1 Reagents
As far as possible, use “reagent grade“-chemicals.

7.1.1 Hydrochloric acid solution, c(HCl) = 1 mol/l, molecular weight 36,46 g/mol, CAS: 7647-01-0.

7.1.2 Sodium hydroxide solution, c(NaOH) = 1 mol/l, molecular weight 40,00 g/mol, CAS: 1310-73-2.

7.1.3 Ethanol, ≥99,8 %, C H OH, molecular weight 46,07 g/mol, CAS: 64-17-5.
2 5
7.1.4 17β-estradiol, ≥98 %, C H O , molecular weight 272,38 g/mol, CAS: 50-28-2.
18 24 2

7.1.5 Maltose monohydrate, >95 %, C H O ·H O, molecular weight 360,32 g/mol, CAS: 6363-53-7.

12 22 11 2

7.1.6 Sodium nitrate, >99 %, NaNO , molecular weight 84,98 g/mol, CAS: 7631-99-4.

© ISO 2018 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 19040-2:2018(E)

7.1.7 Potassium dihydrogen phosphate, ≥99 %, KH PO , molecular weight 136,09 g/mol,

2 4
CAS: 7778-77-0.

7.1.8 Magnesium sulfate pure, MgSO , molecular weight 120,37 g/mol (water free), CAS: 7487-88-9.

7.1.9 Iron(III) chloride hexahydrate, >97 %, FeCl ·6H O, molecular weight 270,29 g/mol,

3 2
CAS: 10025-77-1.

7.1.10 Calcium nitrate, >99 %, Ca(NO ) , molecular weight 164,09 g/mol, CAS: 10124-37-5.

3 2

7.1.11 Calcium D-pantothenate, >98 %, C H CaN O molecular weight 238,27 g/mol, CAS: 137-08-6.

18 32 2 10

7.1.12 Thiamine hydrochloride, >98,5 %, C H Cl N OS, molecular weight 337,27 g/mol,

12 18 2 4
CAS: 67-03-8.
7.1.13 Niacin, >99,5 %, C H NO , molecular weight 123,11 g/mol, CAS: 59-67-6.
6 5 2

7.1.14 Pyridoxine hydrochloride, >99 %, C H NO ·HCl, molecular weight 205,64 g/mol, CAS: 58-56-0.

8 11 3

7.1.15 D-(+)-Biotin, ≥98,5 %, C H N O S, molecular weight 244,31 g/mol, CAS: 58-85-5.

10 16 2 3
7.1.16 Inositol, ≥99 %, C H O , molecular weight 180,16 g/mol, CAS: 87-89-8.
6 12 6

7.1.17 Boric acid, >99,8 %, H BO , molecular weight 61,83 g/mol, CAS: 10043-35-3.

3 3

7.1.18 Copper(II) sulfate pentahydrate, >99,5 %, CuSO ·5H O, molecular weight 249,68 g/mol,

4 2
CAS: 7758-99-8.

7.1.19 Potassium iodide, >99 %, KI molecular weight 166,00 g/mol, CAS: 7681-11-0.

7.1.20 Manganese sulfate monohydrate, >99 %, MnSO ·H O, molecular weight 169,02 g/mol,

4 2
CAS: 10034-96-5.

7.1.21 Zinc sulfate heptahydrate, >99,5 %, ZnSO ·7H O, molecular weight 287,56 g/mol, CAS:

4 2
7446-20-0.

7.1.22 Sodium molybdate dihydrate, >99,5 %, Na MoO ·2H O, molecular weight 241,95 g/mol,

2 4 2
CAS: 10102-40-6.

7.1.23 Cobalt(II) chloride for synthesis, CoCl , molecular weight 129,84 g/mol, CAS: 7646-79-9.

7.1.24 Trisodium citrate dihydrate, >99 %, C H Na O ·2H O, molecular weight 294,10 g/mol,

6 5 3 7 2
CAS: 6132-04-3.

7.1.25 Citric acid, >99,5 %, C H O , molecular weight 192,12 g/mol, CAS: 77-92-9.

6 8 7

7.1.26 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP), C H NNa O P·6H O, molecular

6 4 2 6 2
weight 371,14 g/mol, CAS: 333338-18-4.

7.1.27 Sodium hydroxide, ≥99 %, NaOH, molecular weight 40,00 g/mol, CAS: 1310-73-2.

6 © ISO 2018 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 19040-2:2018(E)

7.1.28 Sea salt with the specifications: chloride (Cl) 19 290 mg/l, sodium 10 780 mg/l, sulfate

2 660 mg/l, potassium 420 mg/l, calcium 400 mg/l, carbonate (bicarbonate) 200 mg/l, strontium

8,8 mg/l, boron 5,6 mg/l, bromide 56 mg/l, iodide 0,24 mg/l, lithium 0,3 mg/l, fluoride 1,0 mg/l,

magnesium (Mg) 1 320 mg/l.

7.1.29 Sodium chloride, ≥99 %, NaCl, molecular weight 58,44 g/mol, CAS: 7647-14-5.

7.1.30 Aceton (purity p.a.), C H O, molecular weight 58,08 g/mol, CAS: 67-64-1.
3 6

7.2 Water, grade 3, as defined in ISO 3696; water with conductivity up to 5 µS/cm is acceptable, or

ultrapure water.

If sterile water is needed, autoclave or sterilize by filtration (cellulose acetate, 0,2 µm).

Water as specified here is also used for the preparation of dilution water which is used for the stepwise

dilution of the test sample.
7.3 Test strain

This test strain is derived from Blastobotrys adeninivorans G1214 Syn.: Arxula adeninivorans G1214

(aleu2 aura3::ALEU2), Reference [1]. This strain displays an uracil auxotrophy. To prevent any formations

of antibiotic resistances in environment and to increase acceptance regarding legal requirements the

test organism contains no antibiotic resistance markers.

Genetic modifications: Integration of the selection marker AURA3mm in the plasmid Xplor2-102-hERα-

GAA2(ERE107)-phyK after exchange of the marker ALEU2 promoter-ATRP1m. The selection marker

AURA3mm was isolated from the plasmid pCR4-AURA3mm-13. The sequences of E. coli and Kanamycin

resistant marker were eliminated through restriction digestion. Stable integration of Xplor2-102-hERα-

GAA2(ERE107)-phyK in Arxula adeninivorans genome was achieved by transformation the cassette in

uracil auxotroph mutant of A. adeninivorans G1214 (aleu2 aura3::ALEU2) through recombination with

25S-rDNA.

The yeast cell suspension for determination of the estrogenic potential of aqueous samples is prepared

from lyophilized yeast cells. As the Arxula adeninivorans cells are lyophilized the test can be conducted

under highly standardized conditions and no specific lab equipment for long-term cell cultivation is

required.

The yeast cells are available commercially. Store the lyophilized yeast cells between 4 °C and 8 °C and

follow the manufacturer’s recommendations. After reactivation, the yeast cells can directly be used for

the test, precultivation is not needed for testing.
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.