EN 1040:2005

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 (Basistest) chemischer Desinfektionsmittel und Antiseptika - Prüfverfahren und Anforderungen (Phase 1)Désinfectants et antiseptiques chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité de base bactéricide antiseptiques et des désinfectants chimiques - Méthode d'essai et prescriptions (phase 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Test method and requirements (phase 1)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposes11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 1040:2005SIST EN 1040:2006en01-junij-2006SIST EN 1040:2006SLOVENSKI
STANDARDSIST EN 1040:20011DGRPHãþD

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 1040December 2005ICS 11.080.20; 71.100.35Supersedes EN 1040:1997
English VersionChemical disinfectants and antiseptics - Quantitative suspensiontest for the evaluation of basic bactericidal activity of chemicaldisinfectants and antiseptics - Test method and requirements(phase 1)Antiseptiques et désinfectants chimiques
- Essai quantitatifde suspension pour l'évaluation de l'activité bactéricide debase des antiseptiques et des désinfectants chimiques
-Méthode d'essai et prescriptions (phase 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch zur Bestimmung derbakteriziden Wirkung (Basistest) chemischerDesinfektionsmittel und Antiseptika - Prüfverfahren undAnforderungen (Phase 1)This European Standard was approved by CEN on 28 July 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2005 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 1040:2005: ESIST EN 1040:2006

REFERENCED STRAINS IN NATIONAL COLLECTIONS.24 ANNEX B (INFORMATIVE)
SUITABLE NEUTRALIZERS AND RINSING LIQUIDS.25 ANNEX C (INFORMATIVE)
GRAPHICAL REPRESENTATION OF TEST PROCEDURES.27 ANNEX D (INFORMATIVE)
EXAMPLE OF A TYPICAL TEST REPORT.31 ANNEX E (INFORMATIVE)
PRECISION OF THE TEST RESULT.34 ANNEX F (INFORMATIVE)
INFORMATION ON THE APPLICATION AND INTERPRETATION OF EUROPEAN STANDARD ON CHEMICAL DISINFECTANTS AND ANTISEPTICS.38 BIBLIOGRAPHY.40 SIST EN 1040:2006

(obligatory test conditions). 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be used. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms:1)  Pseudomonas aeruginosa ATCC 15442;  Staphylococcus aureus ATCC 6538. NOTE See Annex A for strain references in some other culture collections.
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC). 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.
NOTE 2 For each culture medium 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. Sterilize in the autoclave [5.3.2.1a)]. NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized. NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein
15,0 gSoya peptone, papaic digest of Soybean meal
5,0 gSodium chloride (NaCl)
5,0 gAgar
15,0 gWater (5.2.2.2) to 1 000,0 mlSterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to (7,2 ± 0,2) when measured at (20 ± 1) °C. 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 the TSA. Annex B gives guidance on the neutralizers that may be used. SIST EN 1040:2006

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, at (45 ± 1) °C (to maintain melted TSA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 1040:2006

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® mixer 3). b) Mechanical shaker. 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for the membrane filtration method (5.5.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 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 and 0,1 ml, or calibrated automatic pipettes.
5.3.2.10 Petri dishes (plates), of size 90 mm to 100 mm. 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks. 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
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 1040:2006

b) Adjust the number of cells in the suspension to 1,5 x 108
cfu/ml 4) to 5 x 108 cfu/ml using diluent (5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water bath at the test temperature θ
[5.5.1.1a)] and use within 2 h. NOTE
The use of a spectrophotometer for adjusting the number of cells is highly recommended (about 620 nm wavelength - cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each test organism knowing that suitable values of optical density are generally found between 0,150 and 0,460. A colorimeter is a suitable alternative. c) For counting, prepare 10-6 and 10-7 dilutions of the test suspension using diluent (5.2.2.4). Mix [(5.3.2.6a)]. 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 ± 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). For incubation and counting see 5.4.1.6. 5.4.1.5 Validation suspension ("Nv") a) To prepare the validation suspension, 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]. b) For counting prepare a 10-1dilution with diluent (5.2.2.4). Mix [5.3.2.6a)]. 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. 5.4.1.6 Incubation and counting of the test and the validation suspensions a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates that are not countable for any reason. Count the plates and determine the number of cfu. Incubate the plates for a further 20 h to 24 h. Do not
4) cfu/ml = colony forming unit(s) per millilitre. SIST EN 1040:2006

c) Calculate the numbers of cfu/ml in the test suspension “N” and in the validation suspension “Nv” 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 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 water (5.2.2.2) 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 %.
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 water (5.2.2.2). Subsequent dilutions (lower concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in water (5.2.2.2). For liquid products, dilutions of the product shall be prepared with water (5.2.2.2) on a volume/volume basis using volumetric flasks (5.3.2.12). The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a physically homogeneous preparation that is stable during the whole procedure. If during the procedure a visible inhomogeneity appears due to the formation of a precipitate or flocculant, it shall be recorded in the test report. NOTE Counting micro-organisms embedded in a precipitate or flocculant 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 (obligatory and additional) Besides the obligatory temperature, contact time and test organisms additional experimental conditions may be selected (Clause 4), as follows: a) temperature θ
(in °C):  the obligatory temperature to be tested is θ =20 °C;  the additional temperatures may be chosen from 4 °C, 10 °C or 40 °C;  the allowed deviation for each chosen temperature is ± 1 °C. b) contact time t (in min):  the obligatory contact time to be tested is t = 5
min;  the additional contact times may be chosen from 1 min, 15 min, 30 min
or 60 min;  the allowed deviation for each chosen contact time is ± 10 s, except for 1 min, for which it is ± 5 s. SIST EN 1040:2006

[5.5.1.1a)] using the water bath (5.3.2.2). 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 ± 1) °C. 5.5.1.5 Precautions for manipulation of test organisms Do not touch the upper part of the test tube sides when adding the test- or the validation suspensions (5.4.1). 5.5.2 Dilution-neutralization method5) 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) For a graphical representation of this method see Annex C1. SIST EN 1040:2006

At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch at the beginning of the addition. Mix [5.3.2.6a)] and place the tube in a water bath controlled at θ for the chosen contact time t
[5.5.1.1b)]. Just before the end of t, mix [5.3.2.6a)] again. b) At the end of t, take a 1,0 ml sample of the test mixture “Na” and transfer into a tube containing 8,0 ml neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix [5.3.2.6a)] and place in a water bath controlled at (20 ± 1) C . After a neutralization time of 5 min ± 10 s, mix [5.3.2.6a)] immediately take a sample of 1,0 ml of the neutralized test mixture “Na” (containing neutralizer, product test solution, test suspension) 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 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided in portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing TSA (5.2.2.3). For incubation and counting see 5.5.2.6. c) Perform the procedure a) and b) using the other product test solutions at the same time.
d) Perform the procedure a) to c) applying if appropriate – other additional experimental conditions (5.5.1.1). 5.5.2.3 Experimental conditions control “A” (Validation of the selected experimental conditions and/or verification of the absence of any lethal effect in the test conditions) To validate the selected experimental conditions and/or verify the absence of any lethal effect in the test conditions, the procedure is as follows. NOTE When the test is performed at the following conditions: Staphylococcus aureus or Pseudomonas aeruginosa, 20 °C, at any contact time, this control can be skipped.
a) Pipette1,0 ml of water (5.2.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch immediately, mix [5.3.2.6a)] and place the tube in a water bath controlled at
for 2 min ± 10 s. At the end of this time add 8,0 ml of water (5.2.2.2). Restart the stopwatch at the beginning of the addition, mix [5.3.2.6a)] and place the tube in a water bath controlled at
for t. Just before the end of t, mix [5.3.2.6a)] again.
b) At the end of t, take a sample of 1,0 ml of this mixture “A” in duplicate and inoculate using the pour plate or spread plate technique [5.5.2.2b)]. For incubation and counting see 5.5.2.6. 5.5.2.4 Neutralizer control “B” (Verification of the absence of toxicity of the neutralizer) To verify the absence of toxicity of the neutralizer, the procedure is as follows. a) Pipette 8,0 ml of the neutralizer – used in the test (5.5.2.2) – and 1,0 ml of water (5.2.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch at the beginning of the addition, mix [5.3.2.6a)], and place the tube in a water bath controlled at (20 °± 1)°C for 5 min ± 10 s. Just before the end of this time, mix [5.3.2.6a)].
test (5.5.2.2). Mix [5.3.2.6a)] and place the tube in a water bath controlled at θ for t. Just before the end of t, mix [5.3.2.6a)] again. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (used in 5.5.2.2). Restart the stopwatch immediately, mix [5.3.2.6a)] and place the tube in a water bath controlled at (20 ± 1) °C for 5 min ± 10 s. Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch at the beginning of the addition and mix [5.3.2.6a)]. Place the tube in a water bath controlled at (20 ± 1) °C for (30 ± 1) min. Just before the end of this time, mix [5.3.2.6a)] again. At the end of this time take a sample of 1,0 ml of the mixture “C” in duplicate and inoculate using the pour plate or the spread plate technique [5.5.2.2b)]. For incubation and counting see 5.5.2.6. 5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows.
a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the plates and determine the number of colony forming units. Incubate the plates for a further 20 h to 24 h. Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the number has increased, use only the higher number for further evaluation. b) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and determine the Vc values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test mixture “Na” and in the validation mixtures A, B and C using the method given in 5.6.2.4 and 5.6.2.6. Verify according to 5.7. 5.5.3 Membrane filtration method6) 5.5.3.1 General The test and the control and validation procedures (5.5.3.2 through 5.5.3.5) shall be carried out in parallel and separately for each experimental condition (5.5.1.1). Each membrane filtration apparatus shall be equipped with a membrane of 0,45 µm pore size and
47 mm to 50 mm diameter (5.3.2.7) and filled with 50 ml of the rinsing liquid (5.2.2.6). The time required for filtering – if longer than one minute in exceptional cases – shall be recorded in the test report. When transfering the membranes to the surface of an agar plate, care should be taken to ensure that the test organisms are on the upper side of the membrane when placed on the plate and to avoid trapping air between the membrane and agar surface.
6) For a graphical representation of this method see annex C2 SIST EN 1040:2006

For incubation and counting see 5.5.3.6. c) See 5.5.2.2c). d) See 5.5.2.2d). 5.5.3.3 Experimental conditions control “A” (Validation of the selected experimental conditions and/or verification of the absence of any lethal effect in the test conditions) To validate the selected experimental conditions and/or verify the absence of any lethal effect in the test conditions, the procedure is as follows. NOTE When the test is performed at the following conditions: Staphylococcus aureus or Pseudomonas aeruginosa, 20 °C, at any contact time, this control can be skipped.
a) See 5.5.2.3a). b) At the end of t, take a sample of 1,0 ml of this mixture “A” in duplicate and transfer each 1,0 ml sample into a separate membrane filtration apparatus (5.5.3.1). Filter immediately and additionally 50 ml of water (5.2.2.2). Then transfer each of the membranes to the surface of separate TSA plates (5.2.2.3). For incubation and counting see 5.5.3.6. SIST EN 1040:2006

If the rinsing liquid is not water, complete the procedure by filtering 50 ml of water (5.2.2.2). Then transfer each of the membranes to the surface of separate TSA plates (5.2.2.3). For incubation and counting see 5.5.3.6. 5.5.3.5 Method validation “C” (Validation of the membrane filtration method or counting of the bacteria on the membranes which have previously been in contact with the product) For validation of the membrane filtration method or counting of the bacteria on the membranes which have previously been in contact with the product, the procedure is as follows. a) See 5.5.2.5a). b) At the end of t take 0,1 ml of the validation mixture “C” in duplicate and transfer each 0,1 ml sample into a separate membrane filtration apparatus (5.5.3.1). Filter immediately. Filter through the rinsing liquid (5.2.2.6) the same way as in the test [5.5.3.2b)]. Then cover the membranes with 50 ml of the rinsing liquid (5.2.2.6) and add 0,1 ml of the validation suspension (5.4.1.5). Filter immediately again and additionally 50 ml of water (5.2.2.2). Then transfer each of the membranes to the surface of separate TSA plates (5.2.2.3).
For incubation and counting see 5.5.3.6. 5.5.3.6 Incubation and counting of the test mixture and the control and validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows. a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the plates and determine the number of colony forming units. Incubate the plates for a further 20 h to 24 h. Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the number has increased use only the higher number for further evaluation. b) Note for each plate the exact number of colonies but record > 165 for any counts higher than 165 and determine the Vc values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test mixture Na and in the validation mixtures A, B and C using the method given in 5.6.2.4 and 5.6.2.6. Verify according to 5.7. 5.6 Experimental data and calculation 5.6.1 Explanation of terms and abbreviations 5.6.1.1 Overview of the different suspensions and test mixtures N and Nv represent the bacterial suspensions, Na represents the bactericidal test mixture, A (experimental conditions control), B (neutralizer or filtration control), C (method validation) represent the different control test mixtures. N, Nv, N0, Nv0, Na and A, B and C represent the number of cells counted per ml in the different test mixtures in accordance with Table 1. SIST EN 1040:2006

Number of cells per ml in the bacterial suspensions Number of cells per ml in the test mixtures at the beginning of the contact time (time = 0 ) Number of survivors per ml in the test mixtures at the end of the contact time t or 5 min (B) or
30 min (C) Test N N0 (= N/10)
Test suspension
Na (before neutralization or filtration) Controls Nv Nv0 (= Nv/10) A, B, C
Validation suspension
5.6.1.2 Vc values All experimental data are reported as Vc values:  in the dilution-neutralization method (test and controls), a Vc value is the number of colony-forming units counted per 1,0 ml sample;  in the membrane filtration method, a Vc value is the number of colony-forming units counted per 0,1 ml sample of test mixture Na and per 1,0 ml sample in the controls. 5.6.2 Calculation 5.6.2.1 General The first step in the calculation is the determination of the Vc values, the second the calculation of N, N0, Na, Nv, Nv0, A, B and C. The third step is the calculation of the reduction R (5.8). 5.6.2.2 Determination of Vc values The Vc values are determined as follows. a) The usual limits for counting bacteria on agar plates are between 15 and 300. In this European Standard a deviation of 10% is accepted, so the limits are 14 and 330. On membranes the usual upper limits are different: 150, i.e. with the 10% deviation: 165.
NOTE
The lower limit (14) is based on the fact that the variability is increasing the smaller the number counted in the sample (1 ml or 0,1 ml) is and therefore subsequent calculations may lead to wrong results. The lower limit refers only to the sample (and not necessarily to the counting on one plate), e.g. three plates per 1 ml sample with 3 cfu, 8 cfu and 5 cfu give a Vc value of 16. The upper limits (330, 165) reflect the imprecision of counting confluent colonies and growth inhibition due to nutriment depletion. They refer only to the counting on one plate and not necessarily to the sample. b) For counting the test suspension N (5.4.1.6), the validation suspension Nv (5.4.1.6) and for all countings of the dilution-neutralization method (5.5.2.6), determine and record the Vc values according to the number of plates used per 1 ml sample (5.6.1.2). NOTE
If more than one plate per 1 ml sample has been used to determine the Vc value, the countings per plate should be noted. If the count on one plate is higher than 330, report the number as “> 330”. If more than one plate per 1 ml sample has been used and at least one of them shows a number higher than 330, report this Vc value as “> sum of the counts” (e.g. for “>330, 310, 302”, report “> 942”). If a Vc value is lower than 14, report the number (but substitute by “<14” for further calculation (in the case of Na). SIST EN 1040:2006

where C is the sum of Vc values taken into account; n1 is the number of Vc values taken into account in the lower dilution, i.e. 10-6; n2 is the number of Vc values taken into account in the higher dilution, i.e. 10-7; 10-6 is the dilution factor corresponding to the lower dilution. Round off the results calculated to two significant figures. For this, if the last figure is below 5, the preceding figure is not modified; if the last figure is more than 5, the preceding figure is increased by one unit; if the last figure is equal to 5, round off the preceding figure to the next nearest even figure. Proceed stepwise until two significant figures are obtained. As a result, the number of cfu/ml is expressed by a number between 1,0 and 9,9 multiplied by the appropriate power of 10. Example: N = (cfu/ml) 10 x 9,1
=
109363,1
=
102,2426
=
10 )2
1,0 + 2( 2520213168886-6-×××+++ N0 is the number of cells per ml in the test mixture [5.5.2.2a)] at the beginning of the contact time (time “zero” = 0 ). It is one-tenth of the weighted mean of N due to the tenfold dilution by the addition of the product and water. 5.6.2.4 Calculation of Na Na is the number of survivors
...