The Society of International sustaining growth for Antimicrobial Articles(3) Minimal Bactericidal Concentration Determination Method I (2012 Version)
Minimal bactericidal concentration (MBC) determination methodAbolish

1. Scope
This test method shall be applied to antibacterial agents etc. that do not allow a determination ofminimal growth inhibitory concentration (MIC).
2. Test Strains1(1) Staphylococcus aureus NBRC 12732 (ATCC 6538P)(2) Escherichia coli NBRC 3972 (ATCC 8739)
3. Procurement of Supplies
The reagents, instruments and other supplies used in this test method shall be in conformity with theJapan Industrial Standards or the Japanese Pharmacopoeia unless otherwise specified.
3.1 Instrumentation and equipment(1) Glass conical flasks (300-ml capacity)(2) Sterile synthetic resin centrifugal tubes with stoppers2 (50-ml polypropylene centrifugal tubes)(3) Sterile synthetic resin tubes with stoppers (13-ml polypropylene tubes)(4) Glass test tubes (length 170 to 200 mm, outside diameter 18 mm)(5) Shaking incubator (a model that can be operated at an accuracy within 1C)(6) Constant-temperature chamber (a model that can be operated at an accuracy within 1C)(7) Synthetic resin pipettes or synthetic resin pipetters (allowing dispensing of 0.1 to 1.0 ml)(8) Conradi rods
3.2 Culture media(1) Nutrient broth medium (NB medium)
Meat extract
5.0 g
Peptone10.0 gSodium chloride
5.0 g
Purified water
1,000 mlpH
7.0 to 7.2(2) Nutrient agar medium (NA medium)
NB medium (1) supplemented with 1.5% of agar

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These test microorganisms were chosen to represent Gram-positive and -negative bacteria, respectively.
In view of silver adsorption, supplies made of synthetic resin were used as the containers and instruments thatcome in contact with the sample or sample liquid. In the experiments by this test method, centrifugal tubes(# 25331CTF50) and tubes (# 25226ST17PO) manufactured by CORNING were used.

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4. Test Procedures
4.1 Incubation of the test microorganisms
Place 50 ml of NB medium in a 300-ml conical flask and sterilize. Subsequently, transfer aplatinum loopful of each test microorganism to the medium, and perform shaking culture at 35 to37C for 18 hours (shaking rate 80 to 100 rpm). Inoculate 0.1 ml of this culture broth to 50 ml ofa fresh supply of NB medium, and perform shaking culture at 35 to 37C for 2 to 4 hours until thecell count1 becomes 108 cells/ml.
If the absorbance at 660 nm does not reach about 0.2 even after 2 to 4 hours of incubation,
discontinue the incubation and proceed to re-incubation using a fresh supply of NB medium (50ml).
4.2 Preparation of liquid inoculum
Centrifuge the culture broth under cooling2 (below 10C) at 2,000 rpm or more for 10 minutes,
discard the supernatant, and harvest the cells. To eliminate the medium components from thecells harvested, suspend the cells in sterile purified water3 being cooled below 10C, centrifuge inthe same manner as described above, and again harvest the cells. Repeat these steps in threecycles.
Finally, suspend the cells thus deprived of the medium components in sterile purified water being
cooled below 10C. Dilute this suspension as appropriate to obtain a cell count of 2.0 to 4.0 
106 cells/ml, and use this dilution as the liquid inoculum.
The prepared liquid inoculum must be immediately stored under refrigeration and used within 1hour. The viable cell count of this liquid inoculum shall be measured by the pour plate culture
method at the time of use.
4.3 Preparation of test liquids
Weigh out 64 mg of the sample4 in a synthetic resin centrifugal tube (50-ml capacity), add 10 mlof sterile purified water, stir using a touch mixer or the like, and then sonicate the mixture over 5
minutes to obtain a thoroughly dispersed liquid.
Retain this dispersion (containing the sample at 6,400 g/ml) at 301C for 2 hours5, then stirusing a touch mixer or the like, and sonicate the mixture over 5 minutes to obtain a sample
dispersion.

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When the absorbance at 660 nm is about 0.2, the cell count is about 108 cells/ml.
If a centrifuge not equipped with cooling apparatus is used, the culture broth shall be cooled with ice for 5 to
10 minutes in advance, and then centrifuged immediately.
3
The purified water used shall be a supply conforming to the Japanese Pharmacopoeia.
4Because the sample is placed directly in the medium, microorganisms possibly derived from the sample caninfluence the test results. It is desirable, therefore, that the sample be germfree. To this end, it isrecommended that the sample be sterilized in advance if possible (in addition to dry heat sterilization,
high-pressure steam sterilization, gas sterilization and other methods of sterilization are available).
 Samples allowing high-temperature heating
Heat the sample at 160 to 180C for 120 minutes or more to achieve sterilization and drying. After drying,allow the sample to cool in a silica gel-containing desiccator.
 Samples not allowing high-temperature heating
After sterilization by an appropriate method, dry the sample within a range of temperature and time that do
not deteriorate the sample. Allow the sample to cool in a silica gel-containing desiccator. In this case,the drying and sterilization conditions shall be indicated. Be sure to avoid inadequate heating temperatureand time because incorrectly sterilized samples allow the survival of spores of bacteria of the genus
Bacillus.
5To obtain a constant amount of silver dissolved, retain the sample at 301C for 2 hours prior to dilution.
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Add 1 ml of this dispersion to a previously provided synthetic resin tube (13-ml capacity)
containing 1 ml of 30C sterile purified water, and mix the contents to obtain a 2-fold dilution1.
Serially dilute the sample 2 fold in the same manner to obtain a series of dispersions having
sample concentrations of 3,200, 1,600, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 g/ml,respectively. Transfer 1 ml of each dispersion to a synthetic resin tube (13-ml capacity), add 1 mlof the liquid inoculum to each tube, and use these liquids as the test liquids2.
Separately, provide a synthetic resin tube containing a blank test liquid having a sample
concentration of 3,200 g/ml (not supplemented with the liquid inoculum), and a synthetic resin
tube containing a test liquid consisting of the liquid inoculum alone. For reference controls,
prepare reference control test liquids by serially diluting the Standard Silver Solution for Atomic
Absorption Analysis with sterile purified water3.
4.4 Cultivation
Set the test liquid to a constant-temperature mechanical shaker4 at 301C immediately after
preparation, and perform shaking culture at a shaking rate of 100 rpm (horizontal shaking) for 1hour. Indicate the shaking conditions (shaking rate, amplitude) in the Test Results section.
After the shaking culture, uniformly apply 0.1 ml of the test liquid to a plate of NA medium usinga Conradi rod, and culture at 35 to 37C for 48 hours.
For the blank test liquid, uniformly apply 0.1 ml to each of a pair of plates of NA medium using aConradi rod. Culture one plate as is at 35 to 37C for 48 hours (“blank 1”5). For the other plate,uniformly apply 0.1 ml of a 1000-fold dilution of the test liquid consisting of the liquid inoculum
alone using a Conradi rod, and culture at 35 to 37C for 48 hours (“blank 2”6).
For the test liquid consisting of the liquid inoculum alone, determine the viable cell count by thepour plate culture method using NA medium7 (“blank 3”8).

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To handle the dilution, a synthetic resin pipette or pipetter shall be used (avoid using glass pipettes).
The sample concentrations of the test liquid are 1,600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 3.13 g/ml.
3
Provide reference control test liquids having silver concentrations of 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05,0.025, and 0.013 g/ml.
4Because it is necessary to quickly adjust the test liquid to a temperature of 301C, a constant-temperaturewater chamber is desirable but caution should be exercised to avoid contamination.
5“Blank 1” determination is performed to confirm the sterility of the sample.
6“Blank 2” determination is performed to confirm the growth of the test microorganism without being
influenced by the sample.
7
For viable cell counting, standard agar medium, SCD agar medium and the like may be used.
8“Blank 3” determination is performed to confirm the absence of a non-negligible change from the initial cellcount even after shaking culture of the test microorganism at 301C for 1 hour.
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4.5 Requirements for test validity
The test shall not be regarded as valid unless all the following requirements for test validity1 arefulfilled.
(1) In “blank 1 determination”, no microbial growth is observed on the NA medium plate appliedwith the blank test liquid.
(2) In “blank 2 determination”, not less than 30 colonies of the test microorganism are observed on
the plate of NA medium applied with the test liquid.
(3) In “blank 3 determination”, the difference in antibacterial activity value between the initialviable cell count in the test liquid consisting of the liquid inoculum alone and the viable cellcount after the cultivation is not more than 30%2.
(4) The MBC of silver tested using reference control test liquids shall lie in the range of 0.025 to0.2 g/ml3.
4.6 Judgement
The minimum sample concentration of the test liquid with which no microbial growth is observed
after the cultivation shall be established as the minimal bactericidal concentration (MBC). If thenumber of colonies of the test microorganism that have grown at the time of cell counting by thepour plate culture method is not more than 5, the rating shall be “no microbial growth observed”.
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Provided that the requirements for test validity (1) and (2) are fulfilled, it is judged that the sample is germfree,
and that the test microorganism has grown without being influenced by the sample.
2
Provided that these requirements are fulfilled, the test microorganism is considered to retain a cell count atnearly the same as the initial level. Therefore, if no microbial growth is observed in the test, it can beconcluded that the test microorganism was killed by the bactericidal action of the sample.
3The MBC of silver was determined using reference control test liquids at four laboratories to be between 0.05
and 0.1 g/ml. These values were divided or multiplied by a common factor of 2 to establish the range of0.025 to 0.2 g/ml with a safety margin for both the upper and lower limits.

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