The Society of International sustaining growth for Antimicrobial Articles

4. Antifungal Potency Evaluation Test Method for Antifungal Agents(2012 Version)
Minimal Growth Inhibitory Concentration Determination Method
MIC determination for molds by the agar plate dilution method

1. Scope
This test method shall be applied to antifungal agents.
2. Test Strains1(1) Aspergillus niger NBRC 105649(2) Penicillium pinophilum NBRC 6345
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) Glass test tubes (length 170 to 200 mm, outside diameter 18 mm)(2) Sterile Petri dishes (inside diameter 80 to 100 mm, height 15 to 25 mm)(3) Constant-temperature chamber (a model that can be operated at an accuracy within 1C)(4) Pipettes (milk pipettes, volumetric pipettes allowing dispensing of 1 ml and 10 ml or more or
automated pippetter)(5) Balance (chemical balance)(6) Autoclave(7) Microscope (light microscope)(8) pH meter(9) Platinum loop1

With reference to JIS Z 2911 etc., Penicillium pinophilum, often used for fungal resistance testing, and Aspergillus
niger, reportedly spontaneously resistant thereto, were chosen as the test microorganisms. As both areintermediately hygrophilous fungi, it is preferable to use them in combination with a xerophilous fungus such as
Eurotium tonophilum or Aspergillus penicilloides, a hygrophilous fungus such as Aureobasidium pullulans, acellulose-decomposing fungus such as Chaetomium globosum or Trichoderma virens, and the like, depending on theintended use.

S10-1

The Society of International sustaining growth for Antimicrobial Articles

3.2 Culture Media(1) Potato dextrose agar medium (PDA medium)
Potato extract
200 g
Glucose
20 g
Agar
15 g
Purified water1000 mlpH
5.60.2(2) Glucose peptone (GP medium)
GlucoseYeast extract
Magnesium sulfate
PeptonePotassium dihydrogen phosphate
Purified waterpH

20.0 g
2.0 g
0.5 g
5.0 g
1.0 g
1,000 ml
5.70.1(3) Dioctyl sodium sulfosuccinate solution1
Dioctyl sodium sulfosuccinate solution
Purified water

0.05g
1,000 ml

4. Test Procedures
4.1 Incubation of the test microorganisms
Transplant each test microorganism to PDA medium and incubate at 251C for 7 to 14 days.
4.2 Preparation of liquid inoculum
Suspend the conidium of the cultured test microorganism in 0.005% dioctyl sodium sulfosuccinate
solution, remove fruiting bodies and mycelia, then dilute the suspension with GP medium to obtain adensity of 1.0  105 to 1.0  106 conidia/ml 2.

1
2

With reference to JIS Z 2911, 0.005% dioctyl sodium sulfosuccinate solution was chosen as the suspending agent.
In the agar plate dilution method of the Japan Society of Chemotherapy, bacteria are used as the test microorganisms
at a cell count of 106 cells/ml. In the present test method, however, the test microorganisms are fungi, which
produce conidia larger than bacterial cells. Accordingly, the cell count was set at lower-by-one-digit levels between
1.0  105 cells and 1.0  106 cells/ml.

S10-2

The Society of International sustaining growth for Antimicrobial Articles

4.3. Preparation of test liquids
Dissolve, emulsify, or suspend or dilute the sample in a solvent such as purified water, ethanol or DMSO
to prepare a series of dilutions (concentrations obtained by multiplying or dividing the basic
concentration of 1000 g/ml by a common factor of 2) and use these dilutions as the test liquids.
4.4. Preparation of susceptibility determination plates
Add each test liquid, in an amount of 1/9 of the volume of the culture medium, to “GP medium
supplemented with 1.5% of agar”, previously sterilized and cooled to 50 to 60C, mix thoroughly, thendispense the mixture to a Petri dish and allow to solidify, and use this as the susceptibility determination
plate1.
4.5. Cultivation
Apply the liquid inoculum in a streak about 1 cm in length over the susceptibility determination plateusing a Nichrome wire loop (inside diameter 1 mm) 2 and culture at 25C1C for 7 days.
4.6 Judgement
After the cultivation, macroscopically examine the plate for growth of the test microorganism anddetermine the minimum sample concentration showing no microbial growth to obtain the minimal
growth inhibitory concentration.
If two or more continuous colonies3 are observed, even though they are micro-colonies, the test
microorganism shall be judged to have “grown”.
Photocopying or reproducing this document, in part or in whole, without the permission of the publisher,constitutes an infringement of copyright except for legally permitted cases.
The Society of Industrial Technology for Antimicrobial Articles

1

It is desirable that a control test be performed with a separately prepared plate not supplemented with any test liquid.
In the agar plate dilution method of the Japan Society of Chemotherapy, the test microorganism is applied in a streak
about 2 cm in length. In the present test method, however, the test microorganisms are molds. In the case of fungi(molds, yeast), it is common practice to apply the test microorganism in a streak about 1 cm in length. Hence, thelatter was adopted.
3In the agar plate dilution method of the Japan Society of Chemotherapy, the test microorganism is judged to have“grown” if five or more colonies are observed. In the present test method, however, the test microorganisms arefungi. In the case of molds, it is common practice to judge the test microorganism to have “grown” if two or more
continuous colonies are observed. Hence, the latter was adopted.
2S10-3