Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli

This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge, treated biowaste and soil:
- Method A - Membrane filtration method for quantification (see Clause 6);
- Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see Clause 7);
- Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8).

Schlamm, behandelter Bioabfall und Boden - Nachweis und Zählung von Escherichia coli

Dieser Fachbericht beschreibt drei Verfahren zur Bestimmung und Auszählung von Escherichia coli in Schlamm, behandeltem Bioabfall und Boden:
   Verfahren A - Membranfiltrationsverfahren zur quantitativen Bestimmung (siehe Abschnitt 6);
   Verfahren B - Miniaturisiertes Verfahren durch Animpfen in Flüssigmedium (MPN-Verfahren) (siehe Abschnitt 7);
   Verfahren C - Makroverfahren (MPN) in Flüssigmedium (siehe Abschnitt 8).

Boue, biodéchet traité et sol - Recherche et dénombrement des Escherichia coli

Le présent Rapport technique spécifie trois méthodes de recherche et de dénombrement des Escherichia coli
dans les boues, les biodéchets traités et les sols :
 Méthode A - Méthode par filtration sur membrane pour le dénombrement (voir Article 6) ;
 Méthode B - Méthode miniaturisée (nombre le plus probable, NPP) pour ensemencent en milieu liquide
(voir Article 7) ;
 Méthode C - Macrométhode (nombre le plus probable) en milieu liquide (voir Article 8).

Blato, obdelani biološki odpadki in tla - Ugotavljanje prisotnosti in števila Escherichia coli

To tehnično poročilo določa tri metode za ugotavljanje prisotnosti in števila Escherichia coli v blatu, obdelanih bioloških odpadkih in tleh:
– metoda A – metoda filtriranja z membrano za kvantifikacijo (glej točko 6);
– metoda B – miniaturizirana metoda (najbolj verjetno število (MPN)) z inokulacijo v tekoči medij (glej točko 7);
– metoda C – makrometoda (najbolj verjetno število) v tekočem mediju (glej točko 8).

General Information

Status
Published
Public Enquiry End Date
31-Jan-2011
Publication Date
11-Jun-2013
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-Jun-2013
Due Date
09-Aug-2013
Completion Date
12-Jun-2013

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SLOVENSKI STANDARD
SIST-TP CEN/TR 16193:2013
01-julij-2013
Blato, obdelani biološki odpadki in tla - Ugotavljanje prisotnosti in števila
Escherichia coli

Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli

Schlamm, behandelter Bioabfall und Boden - Nachweis und Zählung von Escherichia coli

Boue, biodéchet traité et sol - Recherche et dénombrement des Escherichia coli
Ta slovenski standard je istoveten z: CEN/TR 16193:2013
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST-TP CEN/TR 16193:2013 en,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TP CEN/TR 16193:2013
TECHNICAL REPORT
CEN/TR 16193
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
May 2013
ICS 13.030.01
English Version
Sludge, treated biowaste and soil - Detection and enumeration
of Escherichia coli

Boue, biodéchet traité et sol - Recherche et dénombrement Schlamm, behandelter Bioabfall und Boden - Nachweis und

des Escherichia coli Zählung von Escherichia coli

This Technical Report was approved by CEN on 1 March 2011. It has been drawn up by the Technical Committee CEN/TC 400.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 16193:2013: E

worldwide for CEN national Members.
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Contents Page

Foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................. 5

1 Scope ................................................................................................................................................ 6

2 Normative references ....................................................................................................................... 6

3 Terms and definitions ...................................................................................................................... 6

4 Abbreviations ................................................................................................................................... 7

5 Quality assurance ............................................................................................................................ 7

6 Method A — Membrane filtration method for quantification .......................................................... 7

6.1 Scope ................................................................................................................................................ 7

6.2 Principle............................................................................................................................................ 8

6.3 Reagents, diluents and culture media ............................................................................................. 8

6.4 Apparatus ......................................................................................................................................... 9

6.5 Sampling ......................................................................................................................................... 10

6.5.1 General ........................................................................................................................................... 10

6.5.2 Storage ........................................................................................................................................... 11

6.5.3 Handling ......................................................................................................................................... 11

6.6 Procedure ....................................................................................................................................... 11

6.6.1 Sample preparation ........................................................................................................................ 11

6.6.2 Sample dilution .............................................................................................................................. 12

6.6.3 Membrane filtration ........................................................................................................................ 12

6.6.4 Resuscitation and enumeration of colonies on chromogenic agar ............................................. 13

6.6.5 Confirmation of colony identity ..................................................................................................... 13

6.6.6 Determination of the dry residue content ..................................................................................... 13

6.7 Calculation and expression of results .......................................................................................... 13

6.8 Performance data of the interlaboratory comparison — Method A ............................................. 14

6.8.1 Material used in the interlaboratory comparison study ............................................................... 14

6.8.2 First assessment of the precision of the method ......................................................................... 15

6.8.3 Interlaboratory comparison results ............................................................................................... 16

6.9 Pre-filtration and centrifugation — Comparison tests .................................................................. 17

7 Method B — Miniaturised method (Most Probable Number) by inoculation in liquid

medium ........................................................................................................................................... 19

7.1 Scope .............................................................................................................................................. 19

7.2 Principle.......................................................................................................................................... 19

7.3 Reagents, diluents and culture media ........................................................................................... 19

7.4 Apparatus ....................................................................................................................................... 21

7.5 Sampling ......................................................................................................................................... 22

7.5.1 General ........................................................................................................................................... 22

7.5.2 Storage ........................................................................................................................................... 22

7.5.3 Handling ......................................................................................................................................... 22

7.6 Procedure ....................................................................................................................................... 22

7.6.1 Sample preparation ........................................................................................................................ 22

7.6.2 Analysis .......................................................................................................................................... 23

7.6.3 Determination of the dry residue content ..................................................................................... 24

7.7 Expression of results ..................................................................................................................... 24

7.7.1 Determination of the characteristic number ................................................................................. 24

7.7.2 Calculation of the MPN and its confidence interval ...................................................................... 25

7.8 Performance data ........................................................................................................................... 27

7.8.1 MPN Statistical table ...................................................................................................................... 27

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7.8.2 Performance data of the interlaboratory comparison ...................................................................3 5

7.8.3 First assessment of the precision of the method .........................................................................3 6

7.8.4 Interlaboratory comparison results ...............................................................................................3 7

7.9 Preparation of synthetic sea salt ...................................................................................................3 9

7.9.1 Major ion composition of a convenient ocean synthetic sea salt ................................................ 39

7.9.2 Example for preparation from defined substances ......................................................................3 9

7.10 Quality criteria for the manufacturing of the medium in microtitre plates (E. coli) ..................... 40

8 Method C — Macromethod (Most Probable Number) in liquid medium ....................................... 41

8.1 Scope ..............................................................................................................................................4 1

8.2 Principle ..........................................................................................................................................4 1

8.3 Reagents, diluents and culture media ...........................................................................................4 1

8.4 Apparatus .......................................................................................................................................4 2

8.5 Sampling .........................................................................................................................................4 3

8.5.1 General ...........................................................................................................................................4 3

8.5.2 Sample storage...............................................................................................................................4 3

8.5.3 Sample handling .............................................................................................................................4 3

8.6 Procedure .......................................................................................................................................4 3

8.6.1 Sample preparation ........................................................................................................................4 3

8.6.2 Analysis ..........................................................................................................................................4 4

8.6.3 Determination of the dry residue content .....................................................................................4 4

8.7 Expression of the results ...............................................................................................................4 4

8.8 Performance data ...........................................................................................................................4 6

8.8.1 MPN Statistical table for 3-tubes MPN procedure .........................................................................4 6

8.8.2 Repeatability and reproducibility ...................................................................................................4 7

8.8.3 First assessment of the precision of the method .........................................................................4 7

8.8.4 Interlaboratory comparison results ...............................................................................................4 8

9 Test report ......................................................................................................................................5 0

Bibliography ................................................................................................................................................5 1

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Foreword

This document (CEN/TR 16193:2013) has been prepared by Technical Committee CEN/TC 400 “Project

Committee - Horizontal standards in in the fields of sludge, biowaste and soil”, the secretariat of which is held

by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.

This document is part of a modular horizontal approach in which this document belongs to the analytical step.

The preparation of this document by CEN is based on a mandate by the European Commission (Mandate

M/330). The mandate considers standards on sampling and analytical methods for hygienic and biological

parameters as well as inorganic and organic determinants. It was the aim of the mandate to develop

standards that are applicable to sludge, treated biowaste and soil and lead to equivalent results as far as this

is technically feasible.

Until now, test methods determining properties of materials within the environmental area were prepared in

Technical Committees (TCs) working on specific products/matrices (soil, waste, sludge etc). However, it is

recognised that many steps in test procedures can be used in test procedures for other products/matrices. By

careful determination of these steps and selection of specific questions within these steps, elements of the

test procedure can be described in a way that can be used for more matrices and materials with certain

specifications. This optimisation is in line with the development among end-users of standards. A majority of

routine environmental analyses are carried out by institutions and laboratories working under a scope which is

not limited to one single environmental matrix but covers a wide variety of matrices. Availability of standards

covering more matrices contributes to the optimisation of laboratory procedures and standard maintenance

costs, e.g. costs related to accreditation and recognition.

A horizontal modular approach was developed in the project 'Horizontal'. 'Modular' means that a test standard

developed in this approach concerns a specific step in assessing a property and not the whole "chain of

measurement” (from sampling to analyses). A beneficial feature of this approach is that “modules” can be

replaced by better ones without jeopardising the standard “chain”.

The results of the desk study as well as the evaluation and validation studies have been subject to

discussions with all parties concerned in the CEN structure during the development by project 'Horizontal'.

The results of these consultations with interested parties in the CEN structure have been presented to and

discussed in CEN/TC 400.

This Technical Report contains the most common detection and enumeration methods for the determination of

E. coli consolidated in one document. The individual methods are specified in the following clauses:

 Clause 6: Method A - Membrane filtration method for quantification;

 Clause 7: Method B - Miniaturised method (Most Probable Number) by inoculation in liquid medium;

 Clause 8: Method C - Macromethod (Most Probable Number) in liquid medium.
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Introduction

Escherichia coli is a non-pathogenic, Gram negative bacterium with a faecal origin. Consequently, it can be

used as an indicator of faecal contamination. It can also be used to monitor the effectiveness of pasteurisation

or disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the

behaviour of all pathogens in these materials.

This Technical Report contains three different methods for the detection and enumeration of Escherichia coli

which were included in a validation trial in 2007.

The results achieved in this validation trial have been judged differently by experts. Consequently, it was

decided by CEN/TC 400 to publish the methods as a Technical Report, aiming for further improvement of the

methods and a later publication as European Standard.

Table 1 — Matrices for which the methods described in this Technical Report are applicable and

tested in a validation trial
Matrix Method A Method B Method C
Mesophilic anaerobic Mesophilic anaerobic Mesophilic anaerobic
Sludge
digested sewage sludge digested sewage sludge digested sewage sludge

Pelletised air dried sludge Pelletised air dried sludge Pelletised air-dried sludge

Digested sewage sludge Digested sewage sludge Digested sewage sludge
presscake presscake presscake
Composted sewage sludge Composted sewage sludge Composted sewage sludge
Biowaste Composted biowaste Composted biowaste Anaerobic treated biowaste
Composted green waste Composted green waste Composted green waste
Anaerobic treated biowaste Anaerobic treated biowaste Composted biowaste

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

This Technical Report 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 and to

ensure compliance with any national regulatory conditions.

WARNING — Samples may contain hazardous and inflammable substances. They may contain

pathogens and be liable to biological action. Consequently, it is recommended that these samples be

handled with special care. The gases which can be produced by microbiological activity are

potentially inflammable and will pressurise sealed bottles. Exploding bottles are likely to result in

infectious shrapnel and/or pathogenic aerosols. Glass bottles should be avoided wherever possible.

National regulations should be followed with respect to microbiological hazards associated with this

method.

IMPORTANT — It is absolutely essential that tests conducted according to this Technical Report be

carried out by suitably trained staff.
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1 Scope

This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge,

treated biowaste and soil:
 Method A - Membrane filtration method for quantification (see Clause 6);

 Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see

Clause 7);
 Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8).
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated references,

the latest edition of the referenced document (including any amendments) applies.

EN 15934, Sludge, treated biowaste, soil and waste — Calculation of dry matter fraction after determination of

dry residue or water content

EN ISO 9308-3:1998, Water quality — Detection and enumeration of Escherichia coli and coliform bacteria in

surface and wastewater — Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid

medium (ISO 9308-3:1998)

ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Escherichia coli
E.coli

β-D-glucuronidase-positive microorganism growing at an incubation temperature of 44 °C in the specified

liquid medium containing 4-methylumbelliferyl-β-D-glucuronide (MUG)
[SOURCE: EN ISO 9308-3:1998]

Note 1 to entry: During growth, indole is produced from tryptophan and gas produced from lactose.

3.2
vegetative bacteria

bacteria which are capable of normal growth in broth or on agar media without pre-culture resuscitation

3.3
sub-lethally damaged bacteria

bacteria which have been stressed but not killed by storage or subsequent treatment by, e.g., mesophilic

anaerobic digestion, lime stabilisation or composting, and therefore may not be recovered

3.4
resuscitation

recovery to vegetative growth of sub-lethally damaged bacteria previously incapable of growth on agar media

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3.5
quantitative resuscitation

recovery to vegetative growth of sub-lethally damaged bacteria isolated discretely on a membrane filter, prior

to transfer to chromogenic medium for growth of individual colonies
3.6
colony forming unit
cfu

growth of individual bacterial cells into visible colonies on agar media, including on membrane filters

overlaying the agar media
3.7
Most probable number
MPN

every well whose inoculum contains even one viable organism will produce detectable growth or change

Note 1 to entry: The individual wells of the sample are independent.
4 Abbreviations
BCIG: 5-bromo-4-chloro-3-indolyl-β-glucuronide
CN: Characteristic number
DS: Dry solid
E. coli: Escherichia coli
MLGA: Membrane Lactose Glucuronide Agar
MPN: Most Probable Number
MUG: 4-methylumbelliferyl-β-D-glucuronide
SMD: Special Microplate Diluent
5 Quality assurance

Suitable quality control procedures, at least those described in ISO 8199, shall be applied.

6 Method A — Membrane filtration method for quantification
6.1 Scope

Method A specifies a membrane filtration procedure for the quantitative detection, by culture of individual

colonies on chromogenic agar media. It is not suitable for materials whose treatment will significantly reduce

bacterial levels to less than 10 viable E. coli per g wet weight, such as lime addition, drying or pasteurisation.

This membrane filtration method is not appropriate for enumeration and detection of other coliform bacteria

without modifications to the chromogenic agar medium.

It is suitable to evaluate the log reduction of E. coli through treatment, as well as the quality of the end

product.
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Method A has a limit of detection of approximately 27 E. coli cfu ∙ g wet weight according to ENV ISO 13843,

dependent on the solids content which at high concentrations (> 0,1 g/ml) may restrict filtration of the sample

volume through the membrane if not first diluted.
6.2 Principle

The homogenised diluted sample is filtered, the membrane filter recovered aseptically and incubated on

membrane lactose glucuronide agar (MLGA), initially at (30 ± 1) °C for (4,0 ± 0,5) h. Subsequently, the

temperature is increased to (44 ± 1) °C for (16 ± 2) h. The presence of E. coli is indicated by green colonies

resulting from the hydrolysis of BCIG.
6.3 Reagents, diluents and culture media
6.3.1 General instructions

To ensure reproducible results, prepare culture media and diluents using either constituents of uniform quality

and chemicals of recognised analytical grade, or a dehydrated diluent or complete medium prepared following

the manufacturer’s instructions. Prepare them with demineralised or distilled water free from substances

capable of inhibiting growth under the test conditions (see ISO 8199).

The use of chemicals of other grades is permissible provided that they are shown to be of equivalent

performance in the test.
6.3.2 Peptone saline solution
Bacteriological peptone 1,0 g
Sodium chloride 8,5 g
Distilled water 1 000 ml
Sodium hydroxide solution
Hydrochloric acid, 1mol/l

Dissolve the bacteriological peptone and sodium chloride into distilled water. Adjust the pH by adding sodium

hydroxide solution or hydrochloric acid so that, after sterilisation, it will correspond to (7,0 ± 0,5) at 25 °C.

Sterilise in the autoclave (6.4.1) at (121 ± 3) °C for (15 ± 1) min. Store at (5 ± 3) °C for a maximum of

3 months.
6.3.3 Membrane Lactose Glucuronide Agar (MLGA)
6.3.3.1 5-bromo-4-chloro-3-indolyl-β-glucuronide (BCIG) suspension
BCIG, monohexylammonium salt 0,2 g
Aqueous ethanol, 95 % 2,5 ml
Sodium hydroxide, 1 mol/l 0,5 ml

Dissolve 200 mg BCIG in a combined solution of 95 % aqueous ethanol and 1 mol/l sodium hydroxide.

6.3.3.2 MLGA
Peptone 40,0 g
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Yeast extract 6,0 g
Lactose 30,0 g
Sodium lauryl sulphate 1,0 g
Phenol red 0,2 g
Sodium pyruvate 0,5 g
Bacteriological agar 10,0 g
Demineralised or distilled water 1 000 ml
Mix all ingredients and bring to the boil whilst stirring continuously.

Add the BCIG suspension to the molten base agar medium and mix thoroughly. Adjust the pH to (7,0 ± 0,5).

Sterilise by autoclaving at (121 ± 3) °C for (15 ± 1) min. Pour into 55 mm Petri dishes in volumes of

approximately 10 ml. Allow setting and store refrigerated at (5 ± 3) °C in the dark. Use within 7 days.

6.3.4 MacConkey Agar
Peptone 20,0 g
Lactose 10,0 g
Bile salts 5,0 g
Sodium chloride 5,0 g
Neutral red 0,075 g
Agar 12,0 g
Distilled water 1 000 ml

Suspend the ingredients in 1 000 ml of distilled water. Bring to the boil whilst stirring continuously to dissolve

all ingredients completely. Adjust the pH to (7,0 ± 0,5).

Sterilise in the autoclave (6.4.1) at (121 ± 3) °C for (15 ± 1) min. Store at (5 ± 3) °C for a maximum of 1 month.

Dry the surface of the agar before inoculation.
6.4 Apparatus

With the exception of equipment supplied sterile, the glassware shall be sterilised in accordance with the

instructions given in ISO 8199.
Usual microbiological laboratory equipment and in particular:
6.4.1 Apparatus for sterilisation – autoclave.
6.4.2 Thermostatic incubator(s) adjustable to (30 ± 1) °C and/or (44 ± 1) °C.
6.4.3 Homogeniser
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6.4.4 Centrifuge, capable of centrifuging 50 ml at 200 g to 300 g.
6.4.5 Membrane filters, 0,45 µm gridded, cellulose nitrate.
6.4.6 Glass fibre pre-filter discs, 47 mm diameter, pore size 2,7 µm.
6.4.7 Vacuum pump
6.4.8 Vacuum manifold – magnetic filter bases and cups.

6.4.9 Sterile homogeniser bags, 250 ml volume, with or without integrated mesh to exclude large

particulate matter.
6.4.10 Sterile Petri dishes, 50 mm in diameter, for holding MLGA medium.
6.4.11 Sterile universals of 20 ml volume, or containers with similar capacity.

6.4.12 Sterile pipettes, glass or disposable plastic ware, capable of dispensing 1 ml and 10 ml volumes.

6.4.13 Sterile conical centrifuge tubes, 50 ml volume, disposable plastic.

6.4.14 Tweezers, capable of sterilisation by immersion in ethanol and subsequent flaming.

6.4.15 Analytical balance
6.4.16 Refrigerator, capable of maintaining (5 ± 3) °C.
6.4.17 Vortex mixer
6.4.18 pH meter with an accuracy of ± 0,1.
6.4.19 Beakers or containers, 100 ml, 250 ml and 1 000 ml.
6.4.20 Laboratory spatula
6.4.21 Boiling bath
6.4.22 Bunsen burner
6.4.23 Sterile forceps
6.4.24 Filter funnels
6.5 Sampling
6.5.1 General

Take samples of at least 100 g wet weight and deliver them to the laboratory as quickly as possible (within

24 h). In order to prevent propagation or inactivation of E. coli during transport to the laboratory and

subsequent storage, refrigerate the sample at (5 ± 3) °C.

Samples are liable to ferment and may contain pathogenic micro-organisms. It is essential to keep them away

from any food or drink, and to protect any cuts. When transporting and handling samples, it is essential that

national and international regulations relating to bio-hazardous samples are followed.

1) g = 9,81 m∙s
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See also the Warning note in the introduction.
6.5.2 Storage

Do not store samples in the open laboratory. If samples are to be stored, store them at (5 ± 3) °C for no longer

than 72 h after receipt.
6.5.3 Handling

Cleanliness when working is essential. When handling sludge samples, it is necessary to wear gloves, face

and eye protection, and sufficient body protection to guard against bottles bursting. The gas evolved is usually

flammable, so all equipment u
...

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