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BS 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, 2013
- 1 Scope
- 2 Normative references
- 3 Terms and definitions
- 4 Requirements
- 5 Methods [Go to Page]
- 5.1 Principle
- 5.2 Materials 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, spore-forming bacteria, 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 %
- 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 Foetal calf serum (FCS)
- 5.2.2.14 Earle’s BSS
- 5.2.2.15 Phenol red, 1 % solution
- 5.2.2.16 Sodium bicarbonate (8,8 % w/v solution)
- 5.2.2.17 Eagle’s minimum essential medium (MEM) for cell cultures
- 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 Skim milk
- 5.2.2.23 Buffered Charcoal Yeast Extract (BCYE) Broth, for Legionella
- 5.2.2.24 Page’s saline
- 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
- 5.2.4 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 equipment3F [Go to Page]
- 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 [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, spore-forming bacteria, excl. mycobacteria and bacterial spores) and yeasts5F [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 mycobacteria6F [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)7F [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)8F [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
- 5.10 Verification of the purity and identity of test organisms [Go to Page]
- 5.10.1 General
- 5.10.2 Purity
- 5.10.3 Identity
- 5.11 Documentation [Go to Page]
- 5.11.1 Freeze dried test organism / frozen viruses
- 5.11.2 Cryovials of frozen test organism
- 5.11.3 Stock culture
- 5.11.4 Verification of purity and identity
- Annex A (informative) Test organisms – Culture collection references and relation to CEN/TC 216 standards and prENs [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, serogroup 1, Philadelphia Culture collections: NCTC 11192, ATCC 33152, CIP 103854 T
- 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 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 Bovine Enterovirus Type 1 (ECBO)
- A.5.3 Bovine Parvovirus, strain Haden
- A.5.4 Poliovirus type 1, Sabin strain LSc-2ab
- 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]