EN 13727:2012+A1:2013

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.$Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide en médecine - Méthode d'essai et prescriptions (Phase 2, Étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity in the medical area - Test method and requirements (phase 2, step 1)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 13727:2012+A1:2013SIST EN 13727:2012+A1:2014en,fr,de01-junij-2014SIST EN 13727:2012+A1:2014SLOVENSKI
STANDARDSIST EN 13727:20121DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 13727:2012+A1
November 2013 ICS 11.080.20 Supersedes EN 13727:2012English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity in the medical area -Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide en médecine - Méthode d'essai et prescriptions (Phase 2, Étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 9 March 2012 and includes Amendment 1 approved by CEN on 14 October 2013.
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.
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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 13727:2012+A1:2013 ESIST EN 13727:2012+A1:2014

Referenced strains in national collections . 33 Annex B (informative)
Neutralizers and rinsing liquids . 34 Annex C (informative)
Graphical representation of test procedures . 36 Annex D (informative)
Example of a typical test report . 44 Annex E (informative)
Precision of the test result . 48 SIST EN 13727:2012+A1:2014

Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC . 51 Bibliography . 52
This European Standard applies to products that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means. This European Standard applies to areas and situations where disinfection or antisepsis is medically indicated. Such indications occur in patient care, for example:  in hospitals, in community medical facilities and in dental institutions;  in clinics of schools, of kindergartens and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test. NOTE 3 This method cannot be used to evaluate the activity of products against Legionella in watersystems against mycobacteria and against bacterial spores. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. 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 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity 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. SIST EN 13727:2012+A1:2014

P. aeruginosa, S. aureus, E. hirae, E. coli K12 P. aeruginosa, S. aureus, E. hirae, E. coli K12 P. aeruginosa, S. aureus, E. hirae, when temperature is
40 °C or higher: only
E. faecium P. aeruginosa, S. aureus, E. hirae
additional Any relevant test organism Test temperature according to the manufacturer’s recommendation, but between
20 °C and 20 °C 20 °C and 20 °C 20 °C and 70 °C 4°C and 30 °C Contact time
according to the manufacturer’s recommendation
but between but no longer than
30 s and 60 s 1 min and 5 min 60 min 5 min or
60 min a Interfering substance
clean conditions
0,3 g/l bovine albumin solution (hygienic handrub) b 0,3 g/l bovine albumin solution (surgical handrub) b 0,3 g/l bovine albumin solution
and/or 0,3 g/l bovine albumin solution
and/or dirty conditions 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (hygienic handwash) c 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (surgical handwash) c
3,0 g/l bovine
albumin solution plus 3,0 ml/l erythrocytes
3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes additional clean or dirty;
any relevant substance clean or dirty; any relevant substance any relevant substance any relevant substance a The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of the product. The recommended contact time for the use of the product is within the responsibility of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and / or the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may be tested with a contact time of maximum 60 min.
b hygienic and surgical handrub shall be tested as a minimum under clean conditions.
c hygienic and surgical handwash shall be tested as a minimum under dirty conditions.
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the minimum test conditions.
b) Pseudomonas aeruginosa, ATCC 15442
c) Staphylococcus aureus, ATCC 6538
d) Enterococcus hirae, ATCC 10541
e) Enterococcus faecium, ATCC 6057
NOTE See Annex A for strain reference in some other culture collections. The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3). The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years.
1) The NCTC and ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 13727:2012+A1:2014

NOTE 2 For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C. 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]) may be used. Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized.
NOTE See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein 15,0 g Soya peptone, papaic digest of Soybean meal 5,0 g Sodium chloride (NaCl) 5,0 g Agar 15,0 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH (5.3.2.4) of the medium shall be equivalent to 7,2 ± 0,2.
NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use a neutralizer that causes opalescence in the agar. SIST EN 13727:2012+A1:2014

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, the pH (5.3.2.4) of the diluent shall be equivalent to 7,0 ± 0,2.
5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.6 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.7 Hard water for dilution of products For the preparation of 1 l of hard water, the procedure is as follows:  prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one month;  prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no longer than one week;  place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH (5.3.2.4) of the hard water shall be 7,0 ± 0,2. (5.3.2.4). If necessary, adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces different final water hardness in each test tube. In any case, the final hardness expressed as calcium carbonate (CaCO3) is lower than 375 mg/l in the test tube. SIST EN 13727:2012+A1:2014

The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50 times in the case of the modified method, 5.2.2.8.4). The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent (5.2.2.4). Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month. The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l ; 5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solution – high concentration with sheep erythrocytes) Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent (5.2.2.4). Sterilize by membrane filtration (5.3.2.7). Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 gN for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4). Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see above). To avoid later contamination this mixture should be split in portions probably needed per day and kept in separate containers for a maximum of 7 days in a refrigerator (5.3.2.8). The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be 3 g/l and 3 ml/l respectively. 5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4) Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems with the filtration. a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent; b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent (instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml (instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution to obtain 50 ml. SIST EN 13727:2012+A1:2014

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 °C ± 1 °C, at 45 °C ± 1 °C (to maintain melted TSA in case of pour plate technique and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled either at 36 °C ± 1 °C or 37 °C ± 1 °C (5.2.1). The same temperature shall be used for incubations performed during a test and its control and validation. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch. 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® mixer3); b) Mechanical shaker.
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 13727:2012+A1:2014

5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 1 µl or calibrated automatic pipettes. 5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads (Diameter 3 mm to 4 mm).
5.3.2.12 Volumetric flasks. 5.3.2.13 Centrifuge (800 gN). 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 General For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation. 5.4.1.2 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organisms In order to prepare the working culture of the test organisms (5.2.1), prepare a subculture from the stock culture (5.4.1.2) by streaking onto TSA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 18 h to 24 h prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h. From this second subculture, a third subculture may be produced in the same way. The second and (if produced) third subculture are the working cultures. If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 48 h period. Never produce and use a fourth subculture. 5.4.1.4 Test suspension (N) a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the working culture (5.4.1.3) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker [5.3.2.6 b)]. Aspirate the suspension from the glass beads and transfer to a tube.
c) For counting, prepare 10-6and 10-7 dilutions (10-7 and 10-8 dilutions in the case of the modified method – 5.5.4) of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6 a)]. Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate technique. 1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml melted TSA (5.2.2.3), cooled to 45 °C ± 1 °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing TSA (5.2.2.3). The technique used for counting of the test suspension has to be used for all other countings, 5.4.1.5 d), 5.5.2.2.c) and d), 5.5.2.3 b), 5.5.2.4 b) and 5.5.2.5 b). For incubation and counting see 5.4.1.6. 5.4.1.5 Validation suspension (NV, NVB) a) To prepare the validation suspension (NV), dilute the test suspension (5.4.1.4) with the diluent (5.2.2.4) to obtain 3,0 x 102 cfu/ml to 1,6 x 103 cfu/ml [about one fourth (1+3) of the 10-5 dilution]. NOTE In the case of the modified method (5.5.4) the procedure is the same but only one fourth (1+3) of the 10-4 dilution is used resulting in 3,0 x 103 cfu/ml to 1,6 x104 cfu/ml.
b) To prepare the validation suspension for the neutralizer control NVB (5.5.2.4) (NVB) dilute the test suspension (5.4.1.4) with the diluent (5.2.2.4) to obtain 3,0 x 104 cfu/ml to 1,6 x 105 cfu/ml [about one fourth (1+3) of the 10-3 dilution] (NVB). c) Maintain and use these validation suspensions (NV and NVB) the same way as the test suspension [5.4.1.4 b)]. d) For counting prepare with diluent (5.2.2.4) a 10-1dilution, in the case of the modified method 10-2 dilution,] and in case of the neutralizer control NVB [see b)] a 10-3 dilution.
Mix [5.3.2.6 a)]. Take a sample of 1,0 ml in duplicate and inoculate using the pour plate or the spread plate technique [5.4.1.4 c)]. For incubation and counting see 5.4.1.6.
4) cfu/ml = colony forming unit(s) per millilitre. SIST EN 13727:2012+A1:2014

c) Calculate the numbers of cfu/ml in the test suspension N and in the validation suspensions NV and NVB (neutralizer control 5.5.2.4) using the methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7. 5.4.2 Product test solutions The concentration of a product test solution shall be 1,25 times the desired test concentration (= real test concentration) because it is diluted to 80 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different concentrations to include one concentration in the active range and one concentration in the non-active range (5.8.2). The product as received may be used as one of the product test solutions, in this case the highest tested concentration is 80 %. !Ready to use products may be tested at 97 % (see 5.5.4.)." In this case, the “real test concentration” is 97 %. Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash products are always prediluted with hard water (5.2.2.7) to achieve a 62,5 % solution. This solution simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a “ready-to-use product”. The modified method (5.5.4) cannot be used, since 62,5 % represents the highest accepted concentration (50 %), multiplied by 1,25. For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (= lower concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks (5.3.2.12) on a volume/volume basis. The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a physically homogenous preparation, stable during the whole procedure. If during the procedure a visible inhomogeneity appears due to the formation of a precipitate or flocculate (for example, through the addition of the interfering substance), it shall be recorded in the test report. NOTE Counting micro-organisms embedded in a precipitate or flocculate is difficult and unreliable. The concentration of the product stated in the test report shall be the desired test concentration. Record the test concentration in terms of mass per volume or volume per volume and details of the product sample as received. 5.5 Procedure for assessing the bactericidal activity of the product 5.5.1 General 5.5.1.1 Experimental conditions
The experimental conditions may be selected according to the practical use considered for the product (Clause 4): SIST EN 13727:2012+A1:2014

NOTE In special circumstances, it may be necessary to add neutralizer to TSA (5.2.2.3).
5.5.1.3 General instructions for validation and control procedures The neutralization and/or removal of the bactericidal and/or bacteriostatic activity of the product shall be controlled and validated - only for the highest product test concentration - for each of the used test organisms and for each experimental condition (interfering substance, temperature, contact time). These procedures (experimental condition control, neutralizer or filtration control and method validation) shall be performed at the same time with the test and with the same neutralizer – or rinsing liquid – used in the test. In the case of ready-to-use-products use water (5.2.2.2) instead of hard water, but observe the exception with handwash products (5.1.1, NOTE). If because of problems with neutralization a neutralizer has been added to TSA (5.5.1.2) used for the validation and control procedures the TSA used for the test shall contain the same amount of this neutralizer as well. 5.5.1.4 Equilibration of temperature Prior to testing, equilibrate all reagents (product test solutions (5.4.2), test suspension (5.4.1.4), validation suspension (5.4.1.5), diluent (5.2.2.4), hard water (5.2.2.7) and interfering substance (5.2.2.8) to the test temperature of θ [5.5.1.1 a)] using the water bath (5.3.2.2) controlled at θ. Observe the provisions laid down in 5.4.1.4 b). Check that the temperature of the reagents is stabilized at θ. The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C. In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ. SIST EN 13727:2012+A1:2014

5.5.2.1 General The test and the control and validation procedures (5.5.2.2 through 5.5.2.5) shall be carried out at the same time. 5.5.2.2 Test Na
– determination of bactericidal concentrations The procedure for determining bactericidal concentrations is as follows: a) T
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