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1. Antibacterial Power Evaluation Test Methods for Antibacterial Agents(1) Minimal Growth Inhibitory Concentration Determination Method I (2012 Version)
MIC determination method based on liquid medium dilution

1. Scope
This test method shall be applied to slightly soluble antibacterial agents.
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 the Japan
Industrial Standards or the Japanese Pharmacopoeia unless otherwise specified.
3.1 Instrumentation and equipment(1) L-shaped test tubes (made of glass, with stoppers, length 130 to 140 mm, height 110 to 120 mm,
outside diameter 18 mm)(2) Shaking incubator (a model that can be operated at an accuracy within 1C)(3) Constant-temperature chamber (a model that can be operated at an accuracy within 1C)
3.2 Culture media(1) Nutrient agar medium (NA medium)
Meat extract
5.0 g
Peptone10.0 gSodium chloride
5.0 g
Agar
15.0 gPurified water
1,000 mlpH
7.0 to 7.2(2) Mueller-Hinton broth medium (MHB medium)2
Meat extract
300.0 gCasamino acid17.5 gSoluble starch
1.5 g
Purified water
1,000 mlpH
7.30.1

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These test microorganisms were chosen to represent Gram-positive and -negative bacteria, respectively.
MHB agar medium is used for the MIC determination method of the Japan Society of Chemotherapy.
MHB medium is commercially available from Eiken Chemical Co., Ltd. (MHB agar medium only),
DIFCO, BBL, Merck and others.

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4. Test Procedures
4.1 Incubation of the test microorganisms
Transplant each test microorganism to nutrient agar medium and incubate at 35 to 37C for 24 hours.
Inoculate a platinum loopful of this culture to MHB medium and incubate at 35 to 37C for 16 to 20hours.
4.2 Preparation of liquid inoculum
Dilute the culture broth using MHB medium1 to obtain a cell count of 1.0 to 5.0  104 cells/ml.
4.3 Preparation of test culture media
Dispense 10 ml of sterile MHB medium to a sterile L-shaped test tube.2 Add the sample3 to thismedium4 to prepare a series of test culture media (concentrations obtained by multiplying or dividing thebasic concentration of 100 g/ml by a common factor of 2)5. Measure the pH of each medium andmake any necessary adjustment if the pH is not in the range of 0.5 compared to the level before sample
addition. Inoculate 0.1 ml of the inoculum liquid6 to each test culture medium.
4.4 Cultivation
Perform shaking culture to achieve sample homogenization at a shaking rate of 100 to 200 rpm(horizontal or vertical shaking) and an amplitude of 40 to 60 mm at 35 to 37C for 24 hours.

1The MHB medium used for dilution has been chosen in accordance with the standard method of the Japan Society ofChemotherapy. The cell count of the liquid inoculum was set at nearly the same level as the standard method.
2
Before using in the test, the L-shaped test tubes shall be sterilized with dry heat (at 160C to 180C for 120 minutes ormore), the MHB medium shall be sterilized with high-pressure steam (at 121C for 15 minutes), and the stoppers shallbe sterilized by an appropriate method chosen in consideration of their heat resistance etc.
3Because the sample is placed directly in the medium, microorganisms possibly derived from the sample can influence
the test results. It is desirable, therefore, that the sample be germfree. To this end, it is recommended that thesample 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 temperature and time because
incorrectly sterilized samples allow the survival of spores of bacteria of the genus Bacillus.
4It is desirable that control experiments be performed without adding the sample.
5Since the specification value is 800 g/ml, the test may be performed at practically selected concentrations of 3,200,1,600, 800, 400, 200, 100, 50, and 25 g/ml.
6
To obtain nearly the same cell count as the standard method of the Japan Society of Chemotherapy, 0.1 ml of theinoculum liquid is prepared.

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4.5 Judgement
After cultivation, macroscopically examine each test culture medium for growth of each test
microorganism1, determine the minimum concentration of the sample showing no growth, and use thisconcentration as the minimal growth inhibitory concentration.
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The minimum cell count allowing the macroscopic confirmation of growth of test microorganism is approximately
106 cells/ml.

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