EN 12353:2013

Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity

This European Standard specifies methods for keeping test organisms used and defined in European Standards for the determination of bactericidal (incl. Legionella pneumophila), mycobactericidal, sporicidal, fungicidal and virucidal (incl. bacteriophages) activity of chemical disinfectants and antiseptics drawn up by CEN/TC 216. These methods for keeping test organisms can only be carried out in connection with at least one of those standards where a reference to this European Standard is established.
NOTE 1   Annex A (informative) contains a non-exhaustive list of test organisms for which this standard can be applied.
NOTE 2   European Standards (EN and prEN) where this European Standard is referenced are listed in the Bibliography.
NOTE 3   A specific part on the preservation of bacterial spores may be added once the results of the ongoing ring trials are available.

Chemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden (einschließlich Legionella), mykobakteriziden, sporiziden, fungiziden und viruziden (einschlißlich Bakteriophagen) Wirkung

Diese Europäische Norm legt Verfahren zur Haltung von Prüfkeimen fest, wie sie in den Europäischen Normen des CEN/TC 216 zur Bestimmung der bakteriziden (einschließlich Legionella pneumophila), mykobakteriziden, sporiziden, fungiziden und viruziden (einschließlich Bakteriophagen) Wirkung chemischer Desinfektionsmittel und Antiseptika verwendet und festgelegt werden. Diese Verfahren zur Haltung von Prüfkeimen können nur in Verbindung mit mindestens einer der Normen erfolgen, in denen auf diese Norm verwiesen wird.
ANMERKUNG 1   Anhang A (informativ) enthält eine nicht abschließende Liste von Prüfkeimen, auf die diese Norm angewendet werden kann.
ANMERKUNG 2   Europäische Normen (EN und prEN), die auf diese Europäischen Normen verweisen, sind in den Literaturhinweisen aufgelistet.
ANMERKUNG 3   Ein spezifischer Teil über die Aufbewahrung von bakteriellen Sporen kann hinzugefügt werden, sobald die Ergebnisse der laufenden Ringversuche vorliegen.

Antiseptiques et désinfectants chimiques - Conservation des microorganismes d'essai utilisés pour la détermination de l’activité bactéricide (Legionella incluses), mycobactéricide, sporicide, fongicide et virucide (bactériophages inclus)

La présente Norme européenne spécifie les méthodes de conservation des micro-organismes d’essai utilisées et définies dans les Normes européennes relatives à la détermination de l’activité bactéricide (Legionella pneumophila incluse), mycobactéricide, sporicide, fongicide et virucide (bactériophages inclus) des désinfectants et antiseptiques chimiques établies par le CEN/TC 216. Ces méthodes peuvent être mises en œuvre uniquement en association avec au moins une des normes citant en référence la présente Norme Européenne.
NOTE 1   L’Annexe A (informative) dresse une liste non exhaustive des microorganismes d’essai auxquels s’applique la présente norme.
NOTE 2   Les Normes européennes (EN et prEN) faisant référence à la présente Norme européenne sont indiquées dans la Bibliographie.
NOTE 3   Une partie spécifique sur la conservation des spores bactériennes pourra être ajoutée dès que les résultats des essais interlaboratoires en cours seront disponibles.

Kemična razkužila in antiseptiki - Shranjevanje preskusnih organizmov za določanje baktericidnega (vključno Legionella), mikobaktericidnega, sporocidnega, fungicidnega in virucidnega (vključno bakteriofagi) delovanja

Ta tehnična specifikacija določa metodo za zagotavljanje informacij o storitvah in omrežjih v storitvi TPEG. Aplikacija TPEG-SNI je zasnovana za omogočanje učinkovite in jezikovno neodvisne dostave informacij o razpoložljivosti enake storitve na drugem nosilnem kanalu ali podobnih podatkov o storitvah drugega ponudnika storitev, in sicer neposredno od ponudnika storitev do končnih uporabnikov. Izraz »aplikacija« se v specifikacijah TPEG uporablja za opis posebnih aplikacij, ki so na najvišji plasti sklada protokolov ISO/OSI (ISO/IEC 7498-1). Vsaki aplikaciji TPEG (npr. TPEG-RTM) se dodeli enolična številka, tj. identifikacija aplikacije (AID). AID se opredeli ob razvoju nove aplikacije. AID se v aplikaciji TPEG za informacije o storitvah in omrežjih (ta dokument) uporablja za določanje načina obdelave vsebine TPEG in omogoča usmerjanje podatkov v ustrezen aplikacijski odkodirnik. AID = 0000 se dodeli aplikaciji TPEG-SNI, opisani v tej tehnični specifikaciji. Opisanih je več preglednic z informacijami, ki zagotavljajo izčrpne možnosti za opis storitev, njihovega časovnega razporeda, vsebine, geografske pokritosti itn. V vseh tokovih TPEG je obvezna dostava do t. i. GST. Poleg tega je možno signaliziranje povezav vsebine med različnimi nosilci in storitvami.

General Information

• Status: Withdrawn
• Publication Date: 05-Feb-2013
• Withdrawal Date: 13-Apr-2025
• ICS: 07.100.99 - Other standards related to microbiology
• ICS: 11.080.20 - Disinfectants and antiseptics
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: CEN/TC 216 - Chemical disinfectants and antiseptics
• Drafting Committee: CEN/TC 216 - Chemical disinfectants and antiseptics
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 01-Sep-2021
• Completion Date: 14-Apr-2025
• Ref Project: SIST EN 12353:2013 - Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity

Relations

• Replaces: EN 12353:2006 - Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity
• Effective Date: 13-Feb-2013
• Replaced By: EN 12353:2021 - Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity
• Effective Date: 08-Sep-2021

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.DChemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden (einschließlich Legionella), mykobakteriziden, sporiziden, fungiziden und viruziden (einschlißlich Bakteriophagen) WirkungAntiseptiques et désinfectants chimiques - Conservation des organismes test utilisés pour la détermination de l’activité bactéricide (Legionella inclus), mycobactéricide, sporicide, fongicide et virucide (bacteriophages inclus)Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposes07.100.99Drugi standardi v zvezi z mikrobiologijoOther standards related to microbiologyICS:Ta slovenski standard je istoveten z:EN 12353:2013SIST EN 12353:2013en,fr,de01-julij-2013SIST EN 12353:2013SLOVENSKI
STANDARDSIST EN 12353:20061DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 12353
February 2013 ICS 11.080.20; 71.100.35 Supersedes EN 12353:2006English Version
Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity
Antiseptiques et désinfectants chimiques - Conservation des organismes test utilisés pour la détermination de l'activité bactéricide (Legionella inclus), mycobactéricide, sporicide, fongicide et virucide (bacteriophages inclus)
Chemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden (einschließlich Legionella), mykobakteriziden, sporiziden, fungiziden und viruziden (einschlißlich Bakteriophagen) Wirkung This European Standard was approved by CEN on 14 December 2012.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
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 worldwide for CEN national Members. Ref. No. EN 12353:2013: ESIST EN 12353:2013

Test organisms – Culture collection references and relation to
CEN/TC 216 standards and prENs . 23 A.1 Bacteria (except mycobacteria and spore-forming bacteria) . 23 A.2 Mycobacteria . 24 A.3 Spore-forming bacteria . 24 A.4 Fungi (moulds and yeasts) . 25 A.5 Viruses . 25 A.6 Bacteriophages . 26 Annex B (informative)
Graphical representations . 27 Bibliography . 32
Introduction Standardized tests for the determination of bactericidal (incl. Legionella pneumophila), mycobactericidal, sporicidal, fungicidal and virucidal (incl. bacteriophages) activity of chemical disinfectants and antiseptics necessitate the use of test organisms whose purity and identity have been verified and whose biological behaviour remains stable. Therefore it is essential to specify the storage requirements.
This European Standard aims at describing methods for preservation of test organisms used for such purposes. SIST EN 12353:2013

1 Scope This European Standard specifies methods for keeping test organisms used and defined in European Standards for the determination of bactericidal (incl. Legionella pneumophila), mycobactericidal, sporicidal, fungicidal and virucidal (incl. bacteriophages) activity of chemical disinfectants and antiseptics drawn up by CEN/TC 216. These methods for keeping test organisms can only be carried out in connection with at least one of those standards where a reference to this European Standard is established. NOTE 1 Annex A (informative) contains a non-exhaustive list of test organisms for which this standard can be applied. NOTE 2 European Standards (EN and prEN) where this European Standard is referenced are listed in the Bibliography. NOTE 3 A specific part on the preservation of bacterial spores may be added once the results of the ongoing ring trials are available. 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 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 apply. 4 Requirements Each test organism specified in a CEN/TC 216 European Standard and referred to in this European Standard shall be handled as described in this European Standard. The purity and identity of the preserved test organism shall be verified during the preparation and regularly during the storage, except for viruses where only the identity is checked before the stock virus suspension is stored. The preserved test organism – except viruses - should be checked at regular intervals (at least in the case of longer storage than 14 months) to ensure that its susceptibility to products has not changed. As long as CEN/TC 216 has not developed specific tests for this purpose any suitable method can be used e.g. EN 1040 for bacteria, EN 1275 for fungi, EN 14348 for mycobacteria, EN 13623 for Legionella pneumophila, EN 14476 for viruses or EN 13610 for dairy bacteriophages. 5 Methods 5.1 Principle A sample of the test organism – in general in freeze dried form - is obtained from a culture collection. This sample is cultured, prepared for storage, filled into storage vessels and placed in the deep freeze.
The origin (culture collection), taxonomic name and reference number, date of receipt and batch number of the freeze dried test organisms shall be recorded (5.11.2). 5.2.2 Culture media and reagents 5.2.2.1 General The formulas of all media and reagents are given in case commercial ready-to-use material is not used. It is to be checked that each commercial supplier has established an appropriate quality control system.
All weights of chemical substances given in this European Standard refer to the anhydrous salts unless otherwise stated. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. To improve reproducibility, it is recommended that whenever possible, commercially available dehydrated material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed.
All specified pH values are measured at (20 ± 1) °C. For each culture medium, cell culture and reagent a limitation for use should be fixed. 5.2.2.2 Water The water shall be freshly glass distilled water and not demineralized water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. Sterilize in the autoclave (5.3.2.1a)). Sterilization is not necessary if the water is used for e.g. preparation of culture media and subsequently sterilized. 5.2.2.3 Tryptone Soya Broth (TSB) for bacteria, except Legionella Tryptone soya broth, consisting of: Tryptone, pancreatic digest of casein 17,0 g Soya peptone, papaic digest of soybean meal 3,0 g Sodium chloride (NaCl) 5,0 g Water (5.2.2.2) 800,0 ml
Dipotassium phosphate (K2HPO4) 2,5 g Glucose 2,5 g Water (5.2.2.2)
to 1 000,0 ml Sterilize in the autoclave (5.3.2.1a)). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2.
from Difal or equivalent that is not highly
purified and not only based on maltose, e.g. malt extract from OXOID)1 20,0 g Water (5.2.2.2)
to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1a)). After sterilization the pH of the medium shall be equivalent to 5,6 ± 0,2.
5.2.2.5 Cryoprotectant solution for bacteria, spore-forming bacteria, fungi Cryoprotectant solution, consisting of: Beef extract 3,0 g Tryptone, pancreatic digest of casein 5,0 g Glycerol (C3H8O3) [2] 150,0 g Water (5.2.2.2)
to 1 000,0 ml Dissolve the constituents in boiling water. Sterilize in the autoclave (5.3.2.1a)). After sterilization the pH of the solution shall be equivalent to 6,9 ± 0,2.
Any commercially available cryoprotectant containing glycerol for preservation of test organisms equivalent to the solution described above may be used. If justified, any other equivalent cryoprotectant solution may be used, e.g. for Legionella (5.5.2). 5.2.2.6 Middlebrook 7 H 9 broth with 10 % ADC enrichment and glycerol as reconstituent and cryoprotectant solution for mycobacteria (MADC) Middlebrook 7 H 9 broth, consisting of: Middlebrook 7 H 9 broth powder 4,7 g Glycerol (C3H8O3) [2] 100,0 ml Water (5.2.2.2) 800,0 ml Treat in the autoclave (5.3.2.1a)) for a holding time of only 10 min and cool to 45 °C. Add under aseptic conditions 100 ml Middlebrook ADC enrichment to obtain approximately 1 000,0 ml. The pH of the medium shall be equivalent to 6,6 ± 0,2.
5.2.2.7 Polysorbate 80 solution Polysorbate 80 solution, consisting of: Polysorbate 80 0,5 g Water (5.2.2.2)
to 1 000,0 ml Sterilize in the autoclave (5.3.2.1a)).
This information is given for the information of users of this standard and does not constitute an endorsement of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. SIST EN 12353:2013

Sigma Cat : 565012) 25 × 500,000 units Mycostatin (eg
Nystatin : E R Squibb 591502) Water (5.2.2.2) to 2,5 l.
Preparation Dissolve vial contents of antibiotics in water (5.2.2.2) and fill up to 2,5 l.
Dispense aseptically into 50 ml and 5 ml aliquots. Store at − 20 °C. Shake the bottle after thawing. Use 5 ml per litre of medium to give a final concentration of: Penicillin 100 units/ml Streptomycin 100 µg/ml Mycostatin
25 units/ml
5.2.2.12 Phosphate-buffered saline solution (PBS) Sodium chloride (NaCl)
8,00 g
Potassium chloride (KCl) 0,20 g Disodium hydrogen phosphate, 12-hydrate (Na2HPO4 x 12H2O) 2,89 g Potassium phosphate, monobasic (KH2PO4) 0,20 g Water (5.2.2.2)
to 1 000,0 ml
5.2.2.13 Foetal calf serum (FCS) FCS has to be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may interfere with cell and virus growth resulting in false results. 5.2.2.14 Earle’s BSS Sodium chloride (NaCl)
68,0 g Potassium chloride (KCl)
4,0 g
2 This information is given for the information of users of this standard and does not constitute an endorsement of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. SIST EN 12353:2013

2,0 g
Magnesium sulphate, 7-hydrate (MgSO4 x 7H2O)
2,0 g Sodium hydrogenphosphate, 2-hydrate (NaH2PO2 x 2H2O) 1,4 g Glucose
10,0 g
Phenol red, 1 % (5.2.2.15)
20,0 ml
Water (5.2.2.2)
to 1 000,0 ml CaCl2 should be dissolved separately in 100 ml of water (5.2.2.2) and added to the other dissolved reagents just before the solution is brought to its final volume. The solution is 10-fold concentrated. It is sterilized by membrane filtration through a 0,22 µm Millipore or Seitz-type filter3 and can be stored at (4 ± 1) °C for 4 weeks.
For use the solution is diluted 10-fold with water (5.2.2.2) and buffered by the addition of 2,5 % of an 8,8 % Sodium hydrogen carbonate (NaHCO3) solution.
5.2.2.15 Phenol red, 1 % solution a) A 1,0 N Sodium hydroxide (NaOH) solution is prepared. b) 10 g of alcohol soluble Phenol red, European Pharmacopeia [2] are placed in a 100 ml flask (5.3.2.12); 20 ml of the NaOH solution are added, mixed and allowed to stand for a few minutes. c) The dissolved dye is transferred in a 1 000 ml volumetric flask (5.3.2.12). d) Additional 10 ml amounts of the NaOH solution are added to the flask and the dissolved material is added to the volumetric flask. No more than a total of 70 ml of the NaOH solution should be used. e) The solution is brought to a final volume of 1 000 ml with water (5.2.2.2) and stored at room temperature. 5.2.2.16 Sodium bicarbonate (8,8 % w/v solution) Dissolve 8,8 g sodium bicarbonate in water (5.2.2.2) to 100 ml and sterilize by autoclaving (5.3.2.1a)).
Store at (4 ±1) °C. 5.2.2.17 Eagle’s minimum essential medium (MEM) for cell cultures MEM is used for growth and maintenance of cell cultures. First prepare a stock solution. For use, the stock solution is diluted 10-fold with water (5.2.2.2). 1 % of the 3 % Glutamine solution (= 0,03 %) (5.2.2.9), Antibiotic suspension (5.2.2.11), and 2,5 % of a 8,8 % Sodium bicarbonate solution (5.2.2.16) are added. An appropriate concentration of foetal calf serum (FCS, (5.2.2.13); 10 % for growth, 2 % for maintenance) is added before use.
The following solutions are prepared:
Solution A per litre stock solution L-Arginine HCl
1,05
g L-Histidine HCl 0,31
g Lysine HCl 0,58
g Tryptophane 0,10
g L-Phenylalanine 0,32
g L-Threonine 0,48
g L-Leucine 0,52
g
Millipore® and Seitz® are examples of suitable products available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product.
g L-Isoleucine 0,52
g L-Methionine
0,15
g
These amino acids are dissolved with gentle heating (80 °C) in 200 ml of 0,075 N HCl. 0,075 N HCl is prepared by adding 1,5 ml of commercial C.P. HCl (11.9 N) to 236,6 ml water. Take 200 ml from the prepared 238,1 ml.
Solution B
per litre stock solution L-Tyrosine
0,36
g L-Cysteine
0,24
g
These two amino acids are dissolved in 200 ml of 0,075 N hydrochloric acid (HCl) by heating up to 80 °C for 2 h and subsequently cooling to 20 °C.
Solution C
per litre stock solution Nìcotinamide
0,20 g Pyridoxal
0,20 g Thiamine
0,20 g Pantothenic acid
0,20 g Choline
0,20 g Inositol
0,40 g Riboflavin
0,02 g
These reagents are dissolved in approximately 175 ml of water (5.2.2.2) then brought to a final volume of 200 ml with water (5.2.2.2). The solution is dispensed in 10 ml volumes. NOTE The solutions A, B and C are 10-fold concentrated preparations and can be stored in the refrigerator (5.3.2.8). Solution D Dissolve 200 mg of Biotin in 150 ml of water (5.2.2.2). To increase stability upon storage, 1 ml of 1 N hydrochloric acid (HCl) is added. The total volume is brought to 200 ml with water (5.2.2.2) and the solution is dispensed in 10 ml aliquots and stored at (-20 ± 1) °C.
Solution E Dissolve 200 mg Folic acid (crystalline) in 200 ml of 10 fold diluted Earle’s BSS (5.2.2.14), pH = 7,4. The solution is dispensed in 10 ml amounts and stored at (-20 ± 1) °C.
Preparation of the final mixture of Eagle’s MEM a) The following reagents are dissolved in 200 ml Solution B : Sodium chloride (NaCl) 68,0 g Potassium chloride (KCl) 4,0 g Magnesium sulphate heptahydrate (MgSO4x 7H2O) 2,0 g b) 1,4 g of Sodium dihydrogen orthophosphate monohydrate (NaH2PO4 x H20) are dissolved in 55 ml of water (5.2.2.2) and added to the solution a). c) 10 g of Glucose dissolved in 50 ml of water (5.2.2.2) and 20 ml of a 1 % Phenol red solution (5.2.2.15) are added to the solution b). SIST EN 12353:2013

per litre of stock solutions Solution C
10 ml Solution D
10 ml Solution E
10 ml e) In a separate flask containing 160 ml of water (5.2.2.2), 2 g calcium chloride CaCl2 are dissolved and then added to solution d) with vigorous shaking until complete dissolution. f) 200 ml of solution A is added to solution e); this mixture can be kept overnight in the refrigerator (5.3.2.8). g) The total volume of f) is brought to exactly 1 000 ml with water (5.2.2.2) and the solution is sterilised by membrane filtration (5.3.2.16). This 10-fold concentrated medium is stored at 4 °C. h) For use, the medium is diluted 10-fold with water (5.2.2.2). Add 1 % of the 3 % glutamine solution (5.2.2.9) and 2,5 % of the 8,8 % sodium bicarbonate solution (5.2.2.16). The resulting solution cannot be stored longer than 2 h.
5.2.2.18 M17-broth For maintenance of bacterial host strain (Lactococcus lactis) and propagation of dairy bacteriophages
Phytone peptone (from soya meal) 5,00 g Polypeptone peptone (from casein & animal tissue) 5,00 g Beef extract powder 5,00 g Yeast extract 2,50 g D(+)-lactose 5,00 g Ascorbic acid 0,50 g Sodium-ß-glycerophosphate 19,00 g Magnesium sulphate heptahydrate , 7 H2O 0,25 g Water (see 5.2.2.2) 1 000 ml
Sterilize in the autoclave (5.3.2.1a)). After sterilization the pH of the medium shall be equivalent to 7,0 ± 0,2.
5.2.2.19 M17-agar (bottom agar) Bottom agar for quantitative counting of lysis zones (plaques) derived from single infective bacteriophage particles in the bacterial lawn of the host bacteria. Add 15 g of agar to 1 000 ml of M17-broth (5.2.2.18). Dissolve the agar by boiling with constant stirring. Sterilize in the autoclave (5.3.2.1a)). After sterilization the pH of the medium shall be equivalent to 7,0 ± 0,2 when measured at 20 °C. When the agar is cooled down to (47 ± 1) °C, add 10 ml of a sterile 1 mol/l CaCl2-stock solution (5.2.2.21). Mix gently and pour 15 ml to 18 ml of agar into Petri dishes (5.3.2.10). 5.2.2.20 Overlay agar (top agar, soft agar) For counting bacteriophages: Dissolve 6,5 g agar in 1 000 ml M17-broth (5.2.2.18) and heat until boiling with constant stirring. Dispense the molten agar in test tubes (2,5 to 3 ml each). Sterilize in the autoclave (5.3.2.1a)). For achieving clear phage-derived lysis zones (plaques) in the lawn of host bacterial cells only well-defined agar should be used which is specified by the supplier for phage enumeration by the overlay technique. SIST EN 12353:2013

(5.2.2.2);  treat by steaming at 100 °C on 3 successive days (30 min each) and leave between steam treatments at room temperature. Do not leave between subsequent steamings in the refrigerator! NOTE Undiluted skim milk is used for maintenance of the bacterial host strain (5.4.1). Alternatively, treat at a reduced temperature of (115 ± 3) °C for 15 min in an autoclave (5.3.2.1a)). To obtain a volume fraction of 10 % working solution, dilute 1 part of skim milk with 9 parts of sterile water (5.2.2.2) which is required as an optional interfering substance for the phage suspension test (5.7.2). Store the volume fraction of 10 % skim milk at 4 °C to 8 °C. The final concentration of the skim milk in the test procedure (5.7.1) shall be a volume fraction of 1 %. 5.2.2.23 Buffered Charcoal Yeast Extract (BCYE) Broth, for Legionella BCYE agar, consisting of 
yeast extract (bacteriological grade)
10,0 g; 
Activated charcoal
2,0 g; 
.-ketoglutarate, monopotassium salt
1,0 g; 
ACES buffer (N-2-acetamido-2-aminoethanesulfonic acid)
10,0 g; 
Potassium hydroxide (KOH) (pellets)
2,8 g; 
L-cysteine hydrochloride monohydrate
0,4 g; 
Iron(III) pyrophosphate [Fe4 (P2O7) 3]
0,25 g; 
distilled water
to 1 000,0 ml. Preparation of BCYE a) Cysteine and iron solutions Prepare fresh solutions of L-cysteine hydrochloride and iron (III) pyrophosphate by adding 0,4 g and 0,25 g respectively to 10-ml-volumes of water (5.2.2.2). Sterilize each solution by membrane filtration (5.3.2.16).
Store in clean sterile containers at (20
± 3) °C for not more than three months. b) ACES buffer SIST EN 12353:2013

To prepare the ACES buffer, mix the two solutions. NOTE ACES buffer can cause denaturation of the yeast extract if the following sequence is not followed. c)
Final medium Add sequentially to the 980 ml of ACES buffer, the charcoal, yeast extract and α-ketoglutarate. Prepare a 0,1 mol/l solution of potassium hydroxide (KOH) by dissolving 5,6 g in 1 l of water (5.2.2.2). Prepare a 0,1 mol/l solution of sulphuric acid (H2SO4) by carefully adding 5,3 ml of H2SO4 to 1 l of water (5.2.2.2). Use the solutions of 0,1 mol/l potassium hydroxide or 0,1 mol/l sulphuric acid as appropriate to adjust the pH to 6,9 ± 0,2. Add the agar, mix and autoclave (5.3.2.1a)). After autoclaving, allow to cool to (47 ± 2) °C in a water bath (5.3.2.2). Add the L-cysteine and the iron(III) pyrophosphate solutions aseptically, mixing well between additions. The pH of the final medium is 6,9 ± 0,2 at 25 °C.
Prolonged heating during sterilisation or heating at too high a temperature shall be avoided, as it can affect the nutritional qualities of BCYE medium. Batch-to-batch variation of the ingredients of the medium (particularly α-ketoglurarate) can also affect its performance. Therefore it is essential to check the quality of each newly prepared batch of media for its ability to support the growth of the test organism within three days of incubation. This is assessed quantitatively using either known quantities of the obligatory Legionella strain or by direct comparison to previous batches. 5.2.2.24 Page’s saline Saline solution, consisting of:  Sodium chloride (NaCl)
0,120 g;  Magnesium sulphate (MgSO4 x 7H20)
0,004 g;  Calcium chloride (CaCl2 x 2H20)
0,004 g;  Disodium hydrogen phosphate (Na2HPO4) 0,142 g;  Potassium dihydrogenphosphate (KH2PO4) 0,136 g;  Water (5.2.2.2)
to 1 000,0 ml. Sterilize in the autoclave (5.3.2.1a)). To aid accurate preparation, it is recommended that a 10 l volume of Page’s Saline is prepared and dispensed in smaller volumes as required for autoclaving. Alternatively the salt solutions may be made up individually in concentrated form for dilution when the product is required. 5.2.3 Cell cultures 5.2.3.1 Storage of cell cultures a) Actively growing cell cultures (log phase) shall be used for storage. Grow cell culture in MEM (5.2.2.17) supplemented with 10 % FCS (5.2.2.13). Aspirate the growth medium from the flask and rinse the cells with PBS - for a 75 cm2 flask, use 5 ml to 10 ml. Aspirate PBS (5.2.2.12) and pipette 5 ml of
ATV (5.2.2.10) (at 37 °C) onto the monolayer in the flask. Incubate at 37 °C for 2-5 min. Bump the side of the flask against the palm of the hand to help detach the cells. Wait until the cells are detached.
Add 5 ml of MEM (5.2.2.17) + 10 % FCS (5.2.2.13) with a 10 ml pipette and vigorously wash to detach any remaining SIST EN 12353:2013

b) Transfer the suspension into a 15 ml centrifuge tube, add 5 ml of MEM (5.2.2.17) + 10 % FCS (5.2.2.13) and centrifuge at 100 gN for 5 min (5.3.2.14). Remove the supernatant fluid, resuspend the cells in 5 ml of MEM + 10 % FCS and inoculate in a new flask (5.3.2.15) containing 10 ml to 15 ml of MEM + 10 % FCS – for a 75 cm² flask use 15 ml to 22 ml of culture medium. Incubate at 37 °C (5.3.2.3). c) When the cell monolayer is confluent, the cell culture can be either infected (5.8) or used to prepare new subcultures. Cells can be subcultured at 1:3 or higher ratio for up to several passages (sometimes up to 40), before discarding them and taking a fresh aliquot from the liquid nitrogen stocks.
To subculture, aspirate the growth medium from the flask and rinse the cells with PBS (5.2.2.12) - for a 75 cm2 flask use 5 ml to 10 ml. Aspirate PBS and pipette 5 ml of
ATV (5.2.2.10) (at 37 °C) onto the monolayer in the flask. Incubate at 37 °C for 2 min to 5 min. Bump the side of the flask against the palm of the hand to help detach the cells. Wait until the cells are detached. Add 5 ml of MEM (5.2.2.17) + 10 % FCS (5.2.2.13) with a 10 ml pipette and vigorously wash to detach any remaining cell from the bottom of the flask and to separate any cell clumps. Take an aliquot of the cells, appropriate to the desired ratio, and add them into fresh MEM (5.2.2.17) + 10 % FCS (5.2.2.13) in a new flask (15 ml to 22 ml for a 75 cm2 flask). Cell cultures should be regularly checked for the absence of mycoplasma and contaminating viruses.
5.2.4 Host strains for dairy bacteriophages (Lactococcus lactis) 5.2.4.1 Stock culture of host bacteria Inoculate (1 % volume fraction) Lactococcus lactis subsp. lactis F 7/2 (DSM 4366) in M17-broth (5.2.2.18) and incubate for 24 h at 30 °C (5.3.2.3). Inoculate skim milk (5.2.2.22) with a volume fraction of 1 % liquid culture, incubate for 2 h at (30 ± 1) °C (5.3.2.3) and maintain this stock culture of the host strain in skim milk in a refrigerator (5.3.2.8). In 2-weeks-intervals, let these stock cultures grow overnight at (30 ± 1) °C and repeat the method to obtain a fresh stock culture. If prolonged storage is necessary, freeze the skim milk cultures at -18 °C to -20 °C or lower. Alternatively use lyophilized cultures and reactivate according to the supplier’s recommendation or inoculate Lactococcus lactis subsp. lactis F7/2 (DSM 4366) on an M17 agar (5.2.2.19) plate or slope and maintain this stock culture in a refrigerator (5.3.2.8)
5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in the autoclave (5.3.2.1 a)]; b) by dry heat, in the hot air oven (5.3.2.1 b)]. 5.3.2 Usual microbiological laboratory equipment4
and, in particular, the following: 5.3.2.1 Apparatus for sterilization (moist and dry heat) a) for moist heat sterilization, an autoclave capable of being maintained at (30121+) °C for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (170 50+) °C for a minimum holding time of 1 h or at (50160+) °C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, (37± 1) °C and at (45 ± 1) °C if pour plate technique is used. 5.3.2.3 Incubator, capable of being controlled at either (36 ± 1) °C or at (37± 1) °C (for bacteria (incl. Legionella), mycobacteria and viruses) or at (30 ± 1) °C (for fungi and Lactococcus lactis host strains for dairy bacteriophages). For mycobacteria and eukaryotic cell cultures a CO2 – incubator (95 % air, 5 % CO2) and a temperature of (36 ± 1) ºC is recommended. For mycobacteria, if a CO2 – incubator is not used, the inoculated plates should be protected from drying by sealing with insulating tape or packing them into polyethylene bags. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.4.2).
4 Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 12353:2013

5.3.2.14 Centrifuge (100 gN to 4,000 gN). 5.3.2.15 Flasks for cell culture use (e.g. 25 cm², 75 cm²) 5.3.2.16 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,22 µm pore size for sterilization of liquid materials and reagents. For bacteriophages disposable filter membrane units (pore size 0,45) µm are used The vacuum source used shall give an even filtration flow rate.
5.3.2.17 Biological safety cabinet, class II or higher, for growing cell cultures and viruses. 5.4 Procedure for preservation of test organisms – General
5.4.1 Handling of freeze dried / frozen test organisms from culture collections Follow for reconstitution of the freeze dried samples of the test organisms (viruses are shipped mostly in a frozen state) the procedures described in 5.5 to 5.9. If in special cases it is not possible or appropriate, follow the supplier's recommendations. 5.4.2 Choice of incubation procedure, agar medium, cell culture/cell line Apply always the same incubation procedure (temperature, incubation time and type of incubator (5.3.2.3)] and use the same agar, cell culture/cell line and/or host strain as prescribed for the preparation of the working cultures / test suspension (e.g. “N”) of test organisms in the corresponding European Standard. NOTE In most of the CEN/TC 216 Standards this information is given in “5.4.1”.
5 Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 12353:2013

5.5.1 Reconstitution of the freeze dried test organisms Rehydrate the pellet of the freeze dried sample of the test organism using about 1,0 ml TSB (5.2.2.3) for bacteria, BCYE broth (5.2.2.23) for Legionella or MEB (5.2.2.4) for yeasts as suspension medium and allow to swell 30 min. Dilute this suspension in about 5,0 ml of the suspension medium (TSB for bacteria, BCYE broth for Legionella or MEB for yeasts) and mix (5.3.2.6) till homogeneity for at least 30 s.
Inoculate two agar plates (5.4.2) with samples of this suspension – one plate with max 0,1 ml, the other with a loopful to achieve single colonies. Incubate (5.4.2). The rest of the suspension can be discarded. Take a sample from the agar plate with single colonies for verification of purity and identity of the test organism as described in 5.10. Simultaneously continue with 5.5.2. 5.5.2 Preparation for storage a) Add cryoprotectant solution (5.2.2.5) to the surface of one or both of the agar plates (10 ml per plate) and resuspend the cells in this solution using a glass spreader. Aspirate the cell suspension from the surface of the agar and dilute with cryoprotectant solution (5.2.2.5) to 10 ml or more – depending on the amount needed per year. Mix (5.3.2.6) for 30 s. For Legionella, either water (5.2.2.2) or Page’s saline (5.2.2.24) may be used instead of cryoprotectant solution. In case of no growth on the agar BCYE broth (5.2.2.23) may be inoculated and a subculture from this broth may be incubated (5.3.2.3) and prepared as described above. Instead of using 10 ml, 5 ml per plate for resuspending with subsequent aspiration may be used. It is possible then to add again 5 ml, resuspend and aspirate again. b) Immediately after mixing, pipette 0,5 ml quantities of the diluted suspension (a) into cryovials. An alternative way of storage is by coating of beads: Pipette 1 ml of suspension into a cryovial containing two beads (5.3.2.11). Shake the vial to distribute the suspension onto the beads. Allow to stand for 30 min at 20 °C. Remove the excess cryoprotectant solution with a pipette. It is recommended to prepare a sufficient number of cryovials. c) Place and store the cryovials at a temperature of –70 °C or less (5.3.2.13) for maximum 14 months. Longer periods are possible as long as sufficient viability can be established. The duration is also dependent on the results of susceptibility tests to be performed. Discard all cryovials if the purity or identity could not be verified in accordance with 5.5.1 and 5.11. 5.5.3 Preparation of stock culture / working cultures Defrost the cryovial (5.5.2c)) or a single bead of a cryovial (removed by using a wire or forceps (5.3.2.7)) at room temperature. Inoculate agar plate(s) or slope(s) (5.4.2) with this suspension / this coated bead and incubate (5.4.2). Use the culture(s) as stock culture(s) and store for no more than nine weeks in the refrigerator (5.3.2.8) (Pseudomonas aeruginosa for no more than six weeks). From the stock culture(s) working cultures can be prepared according to the corresponding standard.
6 For a graphical representation of this procedure see Figure B.1 for bacteria and Figure B.3 for yeasts. SIST EN 12353:2013

Instead of using 10 ml, 5 ml per plate for resuspending with subsequent aspiration may be used. It is possible then to add again 5 ml, resuspend and aspirate again. b) Immediately after mixing, pipette 0,5 ml quantities of the diluted suspension (a) into cryovials. The a
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