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BS 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, 2021
- undefined
- 1 Scope
- 2 Normative references
- 3 Terms and definitions
- 4 Requirements
- 5 Methods [Go to Page]
- 5.1 Principle
- 5.2 Test organisms, culture media and reagents [Go to Page]
- 5.2.1 Test organisms
- 5.2.2 Culture media and reagents [Go to Page]
- 5.2.2.1 General
- 5.2.2.2 Water
- 5.2.2.3 Tryptone Soya Broth (TSB) for bacteria, except Legionella
- 5.2.2.4 Malt Extract Broth (MEB) for fungi
- 5.2.2.5 Cryoprotectant solution for bacteria and fungi
- 5.2.2.6 Middlebrook 7 H 9 broth with 10 % ADC enrichment and glycerol as reconstituent and cryoprotectant solution for mycobacteria (MADC)
- 5.2.2.7 Polysorbate 80 solution
- 5.2.2.8 DMSO as cryoprotectant for cell culture freezing
- 5.2.2.9 Glutamine solution (3 % m/V solution)
- 5.2.2.10 TV (Trypsin-Versene)
- 5.2.2.11 Antibiotic suspension
- 5.2.2.12 Phosphate-buffered saline solution (PBS)
- 5.2.2.13 Fetal calf serum (FCS)
- 5.2.2.14 Earle’s BSS
- 5.2.2.15 Phenol red (1 % m/V solution)
- 5.2.2.16 Sodium bicarbonate (8,8 % w/v solution)
- 5.2.2.17 Cell culture medium
- 5.2.2.18 M17-broth
- 5.2.2.19 M17-agar (bottom agar)
- 5.2.2.20 Overlay agar (top agar, soft agar)
- 5.2.2.21 CaCl2-stock solutions (1 mol/l and 0,05 mol/l)
- 5.2.2.22 Reconstituted skim milk
- 5.2.2.23 Buffered Charcoal Yeast Extract (BCYE) Broth, for Legionella
- 5.2.2.24 Buffered Charcoal Yeast Extract (BCYE) Agar, for Legionella
- 5.2.2.25 Page’s saline (cryoprotective solution for Legionella)
- 5.2.3 Cell cultures [Go to Page]
- 5.2.3.1 Storage of cell cultures
- 5.2.3.2 Preparation of new cell cultures
- 5.2.3.3 Passages
- 5.2.4 Host strains for dairy bacteriophages (Lactococcus lactis) [Go to Page]
- 5.2.4.1 Stock culture of host bacteria
- 5.2.4.2 Working culture of host bacteria
- 5.3 Apparatus and glassware [Go to Page]
- 5.3.1 General
- 5.3.2 Usual microbiological laboratory equipment
- 5.4 Procedure for preservation of test organisms – General [Go to Page]
- 5.4.1 Handling of freeze-dried / frozen test organisms from culture collections
- 5.4.2 Choice of incubation procedure, agar medium, cell culture/cell line
- 5.5 Procedure for preservation of bacteria (incl. Legionella, aerobic spore-forming bacteria, excl. mycobacteria and bacterial spores) and yeasts [Go to Page]
- 5.5.1 Reconstitution of the freeze-dried test organisms
- 5.5.2 Preparation for storage
- 5.5.3 Preparation of stock culture / working cultures
- 5.6 Procedure for preservation of mycobacteria [Go to Page]
- 5.6.1 Reconstitution of the freeze-dried test organisms
- 5.6.2 Preparation for storage
- 5.6.3 Preparation of working cultures
- 5.7 Procedure for preservation of moulds (e.g. Aspergillus brasiliensis) [Go to Page]
- 5.7.1 Reconstitution of the freeze-dried test organism
- 5.7.2 Preparation for storage
- 5.7.3 Preparation of stock culture / working cultures
- 5.8 Procedure for preservation of viruses (except dairy bacteriophages) [Go to Page]
- 5.8.1 Reconstitution of frozen virus
- 5.8.2 Preparation for storage of stock virus suspension
- 5.8.3 Preparation of test virus suspension
- 5.9 Procedure for preservation of bacteriophages [Go to Page]
- 5.9.1 Reconstitution of frozen bacteriophages
- 5.9.2 Preparation for storage
- 5.9.3 Preparation of bacteriophages working suspensions
- 5.10 Verification of the purity and identity of test organisms [Go to Page]
- 5.10.1 General
- 5.10.2 Information on source of strains
- 5.10.3 Purity
- 5.10.4 Identity
- 5.11 Documentation [Go to Page]
- 5.11.1 General
- 5.11.2 Freeze-dried test organism / frozen viruses
- 5.11.3 Cryovials of frozen test organism
- 5.11.4 Stock culture
- 5.11.5 Verification of purity and identity
- 5.11.6 Storage of documentation
- Annex A (informative)Test organisms – Culture collection references and relation to CEN/TC 216 standards [Go to Page]
- A.1 Bacteria (except mycobacteria and spore-forming bacteria) [Go to Page]
- A.1.1 Enterobacter cloacae
- A.1.2 Enterococcus hirae
- A.1.3 Enterococcus faecium
- A.1.4 Escherichia coli (1)
- A.1.5 Escherichia coli (2) K12
- A.1.6 Lactobacillus brevis
- A.1.7 Legionella pneumophila, subsp. Pneumophila
- A.1.8 Proteus hauseri
- A.1.9 Pseudomonas aeruginosa
- A.1.10 Salmonella enterica subsp. enterica, serotype Typhimurium
- A.1.11 Staphylococcus aureus subsp. aureus
- A.2 Mycobacteria [Go to Page]
- A.2.1 Mycobacterium avium subsp. avium
- A.2.2 Mycobacterium terrae
- A.3 Spore-forming bacteria [Go to Page]
- A.3.1 Bacillus cereus
- A.3.2 Bacillus subtilis subsp. spizizenii
- A.3.3 Clostridioides difficile (former “Clostridium difficile”) R027
- A.3.4 Clostridium sporogenes
- A.4 Fungi (moulds and yeasts) [Go to Page]
- A.4.1 Aspergillus brasiliensis (former “A. niger”) (mould)
- A.4.2 Candida albicans (yeast)
- A.4.3 Saccharomyces cerevisiae (1) (yeast)
- A.4.4 Saccharomyces cerevisiae (2) var. diastaticus (yeast)
- A.5 Viruses [Go to Page]
- A.5.1 Adenovirus type 5, strain Adenoid 75
- A.5.2 Enterovirus E (Enterovirus Bovine Type 1), strain LCR 4 (former “Bovine enterovirus Type 1(ECBO)”)
- A.5.3 Feline coronavirus (Alphacoronavirus 1), Strain Munich (FeCoV)
- A.5.4 Minute virus of mice (Rodent protoparvovirus 1) (former “Murine parvovirus, minute virus of mice, strain Crawford”)
- A.5.5 Murine norovirus, strain S99 Berlin
- A.5.6 Poliovirus type 1, Sabin strain LSc-2ab
- A.5.7 Porcine parvovirus (Ungulate protoparvovirus I), Strain NADL-2 (PPV)
- A.5.8 Vaccinia virus, strain modified vaccinia virus Ankara (MVA)
- A.5.9 Vaccinia virus, strain Elstree
- A.6 Bacteriophages [Go to Page]
- A.6.1 Lactococcus lactis subsp. lactis bacteriophage P008
- A.6.2 Lactococcus lactis subsp. lactis bacteriophage P001
- Annex B (informative)Graphical representations [Go to Page]
