The Society of International sustaining growth for Antimicrobial Articles(2) Minimal Growth Inhibitory Concentration Determination Method II (2012 Version)
MIC determination method for photocatalytic antibacterial agents

Abolish

1. Scope
This test method shall be applied to photocatalytic antibacterial agents in powder form.
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 tubes2 (made of glass, length 130 to 140 mm, height 110 to 120 mm, outside diameter
18 mm)(2) Mechanical shaker3, constant-temperature water chamber4 and shaking incubator (a model that canbe operated at an accuracy within 1C)(3) Constant-temperature chamber (a model that can be operated at an accuracy within 1C)(4) Ultraviolet irradiation apparatus5(5) Ultraviolet intensity tester6 (measuring wavelength band 310 - 400 nm)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

1

These test microorganisms were chosen to represent Gram-positive and -negative bacteria, respectively.
The stoppers for the L-shaped test tubes shall be air-permeable silicone stoppers.
3This mechanical shaker may be of any type: monode shaking, horizontal shaking, or vertical shaking.
Commercially available monode shakers include the Unit Shaker Monoshin IIA from Tietech Co., Ltd.
4
As a rule, this constant-temperature water chamber shall be used in combination with the mechanical shaker.
Provided that there is sufficient distance between the sample and the light source to avoid a temperature rise in thesample, a shaking incubator may be used.
5Black light fluorescent lamps generate ultraviolet rays at wavelengths of approximately 310 to 400 nm, longer thanthose of ultraviolet rays from what are called germicidal lamps. Note that the number of light source units andwattage used are not subject to regulations.
6
The ultraviolet intensity tester shall be calibrated periodically as directed in the instruction manual. In general,
ultraviolet intensity testers from different manufacturers have somewhat different measuring wavelength bands,
sensitivities and the like; measured values are often inconsistent depending on the ultraviolet intensity tester used.
In particular, special attention must be paid to the fact that measured values can differ by about three times between
an ultraviolet intensity tester with a dome-shaped light receiving port and one with a flat light receiving port.
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The Society of International sustaining growth for Antimicrobial Articles(2) Mueller-Hinton broth medium (MHB medium)1
Meat extract
300.0 gCasamino acid17.5 gSoluble starch
1.5 g
Purified water
1,000 mlpH
7.30.1
4. Test Procedures
4.1 Incubation of the test microorganisms
Transplant each test microorganism to NA medium and incubate at 35 to 37C for 24 hours. Inoculate aplatinum loopful of this culture to MHB medium and incubate at 35 to 37C for 16 to 20 hours.
4.2 Preparation of liquid inoculum
Dilute the culture broth using MHB medium2 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 tube3. Add the sample4 to themedium to obtain two series of test culture media (concentrations obtained by multiplying or dividing thebasic concentration of 100 g/ml by a common factor of 2) (“bright conditions test” and “dark conditionstest”)5. Measure the pH of each medium, compare and make any necessary adjustment if the pH is notin the range of 0.5 compared to the level before sample addition. Separately, provide two series of test
culture media not supplemented with the sample (“bright conditions control” and “dark conditionscontrol”). Inoculate 0.1 ml of the inoculum liquid6 to each test culture medium.

1

Mueller-Hinton agar medium is used for the agar plate dilution 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.
2The 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.
3
Before using in the test, the L-shaped test tubes shall be sterilized with dry heat (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 silicone
stoppers shall be sterilized by an appropriate method (high-pressure steam sterilization followed by drying etc.).
4Because 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.
5Since the specification value is 800 g/ml, the test may be performed at selected concentrations of 3,200, 1,600, 800,400, 200, 100, 50, and 25 g/ml.
6To 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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The Society of International sustaining growth for Antimicrobial Articles

4.4 Cultivation
Perform shaking culture to achieve sample homogenization (shaking rate 100 to 200 rpm; amplitude 40
to 60 mm for horizontal or vertical shaking or 0 to 24 degrees for monodeshaking) at 35 to 37C for 24hours, while irradiating ultraviolet rays1 for the “bright conditions test” culture media and “brightconditions control” test culture media, or under shading for the “dark conditions test” culture media and“dark conditions control” test culture media.
Note that the “bright conditions control” test culture media shall be located at the position of the highest
ultraviolet intensity (in the vicinity of center of black light fluorescent lamp) during the cultivation.
4.5 Requirements for test validity
The test shall not be regarded as valid unless all the following requirements for test validity are fulfilled.
(1) Growth of the test microorganism2 in “dark conditions control” test culture medium shall beobserved by macroscopic examination3 after completion of cultivation.
(2) Growth of the test microorganism in “bright conditions control” test culture medium shall beobserved by macroscopic examination after completion of cultivation.
4.6 Judgement4
After cultivation, macroscopically examine each test culture medium for growth of the test
microorganism, determine the minimum concentrations of the sample in “bright conditions test” culture
medium and “dark conditions test” culture medium showing no growth, and use these concentrations as
the minimal growth inhibitory concentrations for the “bright conditions” and the “dark conditions”,respectively.
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1

Ultraviolet intensity shall be measured above the upper surface of the liquid inoculum with an L-shaped test tube
attached to the mechanical shaker. Adjust the position of the black light fluorescent lamp to obtain an ultraviolet
intensity of 0.4 to 1.0 mW/cm2 for an ultraviolet intensity tester with a flat light receiving port, or 1.2 to 3.0 mW/cm 2
for an ultraviolet intensity tester with a dome-shaped light receiving port. Ultraviolet intensity may be changed
according to the choice of test microorganism. In this case, however, the ultraviolet intensity used shall be indicatedin the MIC value field. Since Staphylococcus aureus sometimes does not grow in the “bright conditions controlplot” at the upper limit of the above-described range of ultraviolet intensity and hence fails to fulfill the “requirement
for test validity in Term 4.5 (2)”, it is recommended that the ultraviolet intensity be set at a level close to the lower
limit. Prior to determination of ultraviolet intensity, wait at least 30 minutes after turning on the light source.
2The judgment on the presence or absence of growth of the test microorganism shall be made by macroscopic
examination. If judgment is impossible due to sample turbidity, proceed as described below. Dilute the medium
10,000 fold with phosphate-buffered physiological saline*, streak the dilution on nutrient agar medium using a loopof platinum or Nichrome wire (inside diameter 1 mm), and examine for growth of the test microorganism.
* Phosphate-buffered physiological saline: Dissolve 34 g of KH2PO4 in 500 ml of purified water, adjust the solution
to pH 7.2 with 1N NaOH, then add purified water to make a total volume of 1,000 ml. Dilute 1.25 ml of thissolution with physiological saline (0.85% NaCl) to make a total volume of 1,000 ml, and sterilize with high-pressure
steam at 121C for 15 minutes.
3The minimum cell count allowing the macroscopic confirmation of growth of test microorganism is approximately
106 cells/ml.
4The choice, model, and number of units of the black light fluorescent lamp used, distance to the liquid inoculum
upper surface in the L-shaped test tube, ultraviolet intensity above the upper surface of the liquid inoculum (centerand ends of the fluorescent lamp), choice of the ultraviolet intensity tester used, and others shall be indicated in theTest Results section.

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