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 genetically modified yeast strains Saccharomyces cerevisiae. This reporter gene assay is based on the activation of the human estrogen receptor alpha. This method is applicable to: — fresh water; — waste 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 8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between 120 ng/l and 160 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.

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

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Status
Published
Publication Date
06-Aug-2018
Current Stage
6060 - International Standard published
Start Date
12-May-2018
Completion Date
07-Aug-2018
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INTERNATIONAL ISO
STANDARD 19040-1
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 1:
Yeast estrogen screen (Saccharomyces
cerevisiae)
Qualité de l'eau — Détermination du potentiel oestrogénique de l'eau
et des eaux résiduaires —
Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces
cerevisiae)
Reference number
ISO 19040-1:2018(E)
ISO 2018
---------------------- Page: 1 ----------------------
ISO 19040-1:2018(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

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Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19040-1:2018(E)
Contents Page

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

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

2 Normative references ...................................................................................................................................................................................... 1

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

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

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

6 Apparatus and materials.............................................................................................................................................................................. 5

7 Reagents, media and test strain ............................................................................................................................................................ 6

8 Sampling and samples .................................................................................................................................................................................10

8.1 General ........................................................................................................................................................................................................10

8.2 Bottles and material for sampling .......................................................................................................................................10

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

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

8.5 Transport of samples ......................................................................................................................................................................11

8.6 Pretreatment of samples .............................................................................................................................................................11

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

9 Procedure..................................................................................................................................................................................................................12

9.1 Preparation of cryo-cultures for long-term storage.............................................................................................12

9.2 Overnight culture ...............................................................................................................................................................................12

9.3 Test set up for aqueous samples ...........................................................................................................................................12

9.3.1 Preparation ........................................................................................................................................................................12

9.3.2 Preparation of the reference dilution series .........................................................................................12

9.3.3 Negative control ............................................................................................................................................................13

9.3.4 Blank replicate ................................................................................................................................................................14

9.3.5 Sample dilution ..............................................................................................................................................................14

9.3.6 Field blank ..........................................................................................................................................................................14

9.3.7 Plate setup ..........................................................................................................................................................................14

9.3.8 Inoculation of the test plate .................................................................................................................................14

9.4 Measurement .........................................................................................................................................................................................15

9.4.1 Measurement of the cell density .....................................................................................................................15

9.4.2 Measurement of the reporter gene activity ...........................................................................................16

9.5 Calculation of the corrected absorbance and the reporter gene induction .....................................16

9.6 Calculation of the relative growth .......................................................................................................................................17

9.7 Estimation of the EC of the reference compound by linear interpolation ...................................17

10 Validity criteria ...................................................................................................................................................................................................17

11 Assessment criteria ........................................................................................................................................................................................18

12 Test report ................................................................................................................................................................................................................18

Annex A (normative) Strain selection ..............................................................................................................................................................19

Annex B (informative) Plate set up ......................................................................................................................................................................20

Annex C (informative) Scheme of test principle ....................................................................................................................................21

Annex D (informative) Test set up for chemicals and extracts ...............................................................................................22

Annex E (informative) Preparation of dilution series .....................................................................................................................26

Annex F (informative) Performance data .....................................................................................................................................................27

Annex G (informative) Use of other yeast strains based on Saccharomyces cerevisiae .................................40

Annex H (informative) Statistical assessment .........................................................................................................................................43

© ISO 2018 – All rights reserved iii
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ISO 19040-1:2018(E)

Annex I (informative) Calculation of 17β-estradiol equivalents ..........................................................................................45

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

water — A simplified evaluation for testing of waste water ................................................................................48

Bibliography .............................................................................................................................................................................................................................50

iv © ISO 2018 – All rights reserved
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ISO 19040-1:2018(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
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INTERNATIONAL STANDARD ISO 19040-1:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 1:
Yeast estrogen screen (Saccharomyces cerevisiae)

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 genetically modified yeast strains Saccharomyces

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

This method is applicable to:
— fresh water;
— waste 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

8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international

interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between

120 ng/l and 160 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.

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
ISO 7027, Water quality — Determination of turbidity
© ISO 2018 – All rights reserved 1
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ISO 19040-1:2018(E)
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 http: //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

[SOURCE: ISO 6107-6:2004, 24]
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]
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ISO 19040-1:2018(E)
3.7
induction rate

quotient of the mean signal measured after exposure to a dose of the test sample or with a positive

control, and the mean signal measured for the negative control using the same experimental conditions

[SOURCE: ISO 6107-6:2004, 43, modified — “corrected absorbance” replaces “mutant colonies”; “wells”

replaces “corresponding plates”, “quotient” replaces “difference”.]
3.8
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]
3.9
limit of quantification
LOQ

lowest value of a determinant that can be determined with an acceptable level of accuracy and precision

[SOURCE: ISO 15839:2003, 3.18]
3.10
lowest ineffective dilution
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
overnight culture

culture started late in the afternoon and incubated overnight to be ready during the following morning

for purposes such as the inoculation of a preculture
Note 1 to entry: The procedure for the overnight culture is described in 9.2.
[SOURCE: ISO 6107-6:2004, 54, modified — deleted: "usually about 16 h".]
3.13
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”.]

3.14
reporter gene activity

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

© ISO 2018 – All rights reserved 3
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ISO 19040-1:2018(E)
3.15
stock culture

culture of a strain of organisms maintained under conditions to preserve original features such as

nucleotide sequences
[SOURCE: ISO 6107-6:2004, 87]
3.16
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]
4 Principle

The Yeast Estrogen Screen (YES) is a reporter gene assay which can be used for the measurement of

the activation of the human estrogen receptor alpha (hERα) in the presence of a sample containing

compounds which activate the estrogen receptor (ER).

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 on the whole

process of the reporter gene expression.

The basic concept of such assays is explained in References [10] and [11]. The hERα is heterologously

expressed in the yeast cell under control of a copper dependent 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 which translocates to the nucleus. This activation of the

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

β-galactosidase. The lacZ is fused to a promoter containing estrogen responsive elements (ERE) and

is thus controlled by the activity of the estrogen receptor. The ER-dimer binds to the promoter and

by this activates the expression of the β-galactosidase. Finally, the activity of the β-galactosidase 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 cell density and/or the

detection of the reaction product of the reporter enzyme β-Galactosidase (see Clause 10 for further

information).

Toxic effects of the test sample may lead to a reduction of viable cells and to a reduction of the

measurable signal. Consequently, estrogenic effects of a sample may be masked by acute toxic effects

leading to false negative test results (see Clause 10 for further information).

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

sample is a measure for its salinity. The yeast strain constructed by McDonnell et al. (Reference [10])

tolerates a conductivity of the sample up to 34 000 µS/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.
4 © ISO 2018 – All rights reserved
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ISO 19040-1:2018(E)

Due to the high sensitivity of this test avoid any contamination of buffer, media and all reagents used

with compounds exhibiting estrogenic activity to avoid false positive test results.

6 Apparatus and materials

For suitable sampling devices see Clause 8. Usual laboratory apparatus and glassware is required. In

particular, the following material is needed:

6.1 Incubator shaker, temperature- and time-controlled, 30 °C ± 1 °C and 37 °C ± 1 °C.

6.2 pH meter.
6.3 Steam sterilizer.
6.4 Dry sterilizer.

6.5 Centrifuge, with a rotor for 15 ml and 50 ml tubes up to 2 500 g and with a rotor for 96-well plates

up to 2 500 g.
6.6 Rotary mixer.
6.7 Freezer, at least ≤−18 °C and ≤−70 °C.
6.8 Sterile filter, cellulose acetate, 0,2 µm pore size.
6.9 Inoculation loops.
6.10 Multi-channel multistepper pipette (repeater pipette).
6.11 Multi-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
6.12 Spectrophotometer.

6.13 Transparent sterile polystyrene 96-well plates, for suspension cultures with flat bottom and lid.

6.14 Microplate photometer for 96-well plates, for absorbance measurement at 540 nm ± 20 nm or

580 nm ± 20 nm and at 600 nm ± 20 nm.
6.15 Clean bench.
6.16 Petri dishes, diameter approximately 94 mm, height approximately 16 mm.
6.17 Cryogenic vials, sterile, 1 ml, 10 ml.
6.18 Disposable nitrile gloves.
6.19 Air-permeable sealing membranes for 96-well plates.
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ISO 19040-1:2018(E)
7 Reagents, media and test strain
7.1 Reagents
As far as possible, use "reagent grade" chemicals.
7.1.1 Yeast nitrogen base without amino acids .

7.1.2 α-D-Glucose, anhydrous, C H O , molecular weight 180,15 g/mol, CAS: 50-99-7.

6 12 6
7.1.3 Adenine, C H N , molecular weight 135,13 g/mol, CAS: 73-24-5.
5 5 5
7.1.4 l-arginine, C H N O , molecular weight 174,20 g/mol, CAS: 74-79-3.
6 14 4 2
7.1.5 l-aspartic acid, C H NO , molecular weight 133,10 g/mol, CAS: 56-84-8.
4 7 4

7.1.6 l-glutamic acid monosodium salt hydrate, C H NNaO ·H O, molecular weight (anhydrous)

5 8 4 2
169,11 g/mol, CAS: 142-47-2 (anhydrous basis).

7.1.7 l-histidine-HCl, C H N O ·HCl·H O, molecular weight 209,6 g/mol, CAS: 5934-29-2.

6 9 3 2 2
7.1.8 l-isoleucine, C H NO , molecular weight 131,17 g/mol, CAS: 73-32-5.
6 13 2
7.1.9 l-leucine, C H NO , molecular weight 131,17 g/mol, CAS: 61-90-5.
6 13 2
7.1.10 l-lysine-HCl, C H N O ·HCl, molecular weight 182,65 g/mol, CAS: 657-27-2.
6 14 2 2
7.1.11 l-methionine, C H NO S, molecular weight 149,21 g/mol, CAS: 63-68-3.
5 11 2
7.1.12 l-phenylalanine, C H NO , molecular weight 165,19 g/mol, CAS: 63-91-2.
9 11 2
7.1.13 l-serine, C H NO , molecular weight 105,09 g/mol, CAS: 56-45-1.
3 7 3
7.1.14 l-threonine, C H NO , molecular weight 119,12 g/mol, CAS: 72-19-5.
4 9 3
7.1.15 l-tyrosine, C H NO , molecular weight 181,19 g/mol, CAS: 60-18-4.
9 11 3
7.1.16 l-valine, C H NO , molecular weight 117,15 g/mol, CAS: 72-18-4.
5 11 2

7.1.17 Copper(II) sulfate pentahydrate, CuSO ·5H O, molecular weight 249,69 g/mol, CAS: 7758-99-8.

4 2

7.1.18 Ampicillin sodium salt, C H N NaO S, molecular weight 371,39 g/mol, CAS: 69-52-3.

16 18 3 4

7.1.19 Streptomycin sulfate salt, C H N O ·1,5H SO , molecular weight 728,69 g/mol,

21 39 7 12 2 4
CAS: 3810-74-0.
7.1.20 Agar for microbiology, (C H O )n, CAS: 9002-18-0.
12 18 9

1) Yeast nitrogen base without amino acids contains a nitrogen source such as ammonium sulfate, vitamins and

trace elements which are required for growth of yeast cells. Yeast nitrogen base without amino acids is used for the

selection of yeast strains depending on requirements for carbon sources and amino acids.

6 © ISO 2018 – All rights reserved
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ISO 19040-1:2018(E)

7.1.21 Hydrochloric acid solution, 1 M (HCl), molecular weight 36,46 g/mol, CAS: 7647-01-0.

7.1.22 Sodium hydroxide, NaOH, molecular weight 40,00 g/mol, CAS: 1310-73-2.
7.1.23 Ethanol, ≥99,8 %, CH CH OH, molecular weight 46,07 g/mol, CAS: 64-17-5.
3 2

7.1.24 Glycerol for molecular biology, ≥99 %, HOCH CH(OH)CH OH, molecular weight 92,09 g/mol,

2 2
CAS: 56-81-5.

7.1.25 17β-Estradiol, ≥98 %, C H O , molecular weight 272,38 g/mol, CAS: 50-28-2.

18 24 2

7.1.26 Disodium hydrogen phosphate dihydrate, Na HPO ·2H O, molecular weight 177,99 g/mol,

2 4 2
CAS: 10028-24-7.

7.1.27 Sodiumdihydrogen phosphate monohydrate, NaH PO ⋅H O, molecular weight 137,99 g/mol,

2 4 2
CAS: 10049-21-5.
7.1.28 Potassium chloride, KCl, molecular weight 74,55 g/mol, CAS: 7447-40-7.

7.1.29 Magnesium sulfate heptahydrate, MgSO ·7H O, molecular weight 246,47 g/mol,

4 2
CAS: 10034-99-8.

7.1.30 Chlorophenolred-β-D-galactopyranoside (CPRG), C H Cl O S, molecular weight 585,41 g/

25 22 2 10
mol, CAS: 99792-79-7.

7.1.31 Lyticase from Arthrobacter luteus lyophilized powder, ≥2 000 units/mg protein,

CAS: 37340-57-1.

7.1.32 DL-Dithiothreitol, HSCH CH(OH)CH(OH)CH SH, molecular weight 154,25 g/mol, CAS: 3483-

2 2
12-3.

7.1.33 Sodium dodecyl sulfate, CH (CH ) OSO Na, molecular weight 288,38 g/mol, CAS: 151-21-3.

3 2 11 3

7.1.34 Aceton (puriss p.a.), CH COCH , molecular weight 58,08 g/mol, CAS: 67-64-1.

3 3

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

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

specified here is also used for the stepwise dilution of the test sample.
7.3 Test strain.

The generation of the test strain is described in References [10] and [11]. It is derived from the strain

Saccharomyces cerevisiae BJ3505 (protease deficient, MATα, PEP4::HIS3, prb-1-delta1.6R, HIS3-delta200,

lys2-801, trp1-delta101, ura3-52gal2can1). This strain harbours two plasmids. The construction of

these plasmids is described in Reference [10]. The plasmid YEPE10 contains the CUP1::hER fusion which

encodes the human estrogen receptor α cloned from the MCF-7 human cell lineage under the control

of the metallothionein promoter CUP1. This plasmid is selected via the tryptophane auxothropy of

the parent strain. The second plasmid is the reporter plasmid YRPEG3 which contains the fusion gene

2ERE-CyC1::lacZ. This fusion gene expresses the β-galactosidase (encoded by lacZ) under the control

of the iso1cytochrom c promoter from S. cerevisiae which is fused to two copies of the vitellogenin A2-

gene from Xenopus laevis. This plasmid is selected via the uracil auxothropy of the parent strain.

© ISO 2018 – All rights reserved 7
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ISO 19040-1:2018(E)
7.4 Media.

If required autoclave for 20 min at 121 °C ± 2 °C. Cover the vessels loosely (e.g. with aluminium foil).

Never seal air-tight.
7.4.1 10x SD-Medium.

Dissolve 67,0 g yeast nitrogen base without amino acids (7.1.1) and 200 g glucose (7.1.2) in water (7.2)

and adjust the final volume with water (7.2) to 1 l. Sterilize the solution by filtration (cellulose acetate,

0,2 µm). Do not autoclave the solution because it contains vitamins such as Biotin. Store the solution in

50 ml aliquots in the dark at ≤ −18 °C no longer than 12 months.
7.4.2 10x McD-DO-Medium (McDonnell).
Dissolve
— 175 mg l-lysine-HCl (7.1.10);
— 120 mg l-histidine-HCl (7.1.7);
in water (7.2) and adjust the final volume with water (7.2) to 500 ml.

Sterilize by filtration (cellulose acetate, 0,2 µm) and store aliquots of 40 ml in the dark at ≤−18 °C no

longer than six months.
7.4.3
...

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