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Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs
抗真菌药物的最小抑菌浓度(MIC)实验

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Abstract

The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory (MIC80) values are reported. To facilitate visualization of antifungal susceptibility data, heat maps are generated whereby optical density values are represented quantitatively with colour.

Materials and Reagents

  1. Strain to be analyzed
  2. Overnight cultures of the strains to be analyzed and parental controls
  3. Glucose
  4. MOPS
  5. Yeast extract
  6. Bactopeptone
  7. Yeast nitrogen base
  8. Media
    1. RPMI medium 1640 (Life Technologies, Gibco®, catalog number: 31800-089 ) (see Recipes)
      Note: It is the standard medium recommended by the CLSI standard protocol.
    2. YPD (see Recipes)
      Note: It is a rich medium commonly used to assess susceptibility of diverse mutants.
    3. Synthetic defined medium (see Recipes)
      Note: Supplemented with required amino acids to enable growth of auxotrophic strains is commonly used for experiments requiring plasmid selection.

Equipment

  1. 96-well tissue culture plate (flat bottom with lid) (SARSTEDT AG, catalog number: 83.1835 )
  2. Microplate reader
  3. Multichannel pipette [optional: Multichannel electronic pipette (Rainin, model: EDP3-Plus )]

Software

  1. Soft Max Pro or equivalent spectrophotometer software (Microplate Data Processing)
  2. Microsoft Excel (Data Analysis)
  3. JavaTree View (Heat Map Generation, http://jtreeview.sourceforge.net/)

Procedure

  1. Grow an overnight culture of the strains to be assayed, including relevant controls, in 3 ml of the appropriate medium being tested.
  2. Prepare the MIC plate with the drug gradient to be tested.
    1. Always set up the plates in duplicate.
    2. Add 100 µl of the medium to all 96 wells except for column 12 using a multi-channel pipette.
    3. To column 12, add 200 µl of the medium that contains drug at 2x the final concentration that the titration will start at.
    4. Perform 2-fold dilutions by transferring 100 µl of the drug-containing medium from column 12 to column 11 (mix well and repeat across the row). Stop at column 2 and discard the excess 100 µl so that column 1 is the drug-free control.
  3. Measure the OD600 of 100 µl of the overnight cultures in duplicates in a 96 well plate.
    1. Subtract the OD600 reading from medium only wells to account for background.
    2. Calculate the average OD600 for each strain.
  4. To prepare the inoculum in 5 ml of medium, add 1/OD600 x 1.5 µl of the overnight culture.
  5. Add 100 µl of diluted inoculum (~103 cells) to every well from columns 1 to 12, one strain per row.
  6. Incubate the plates statically at the appropriate temperature (35 °C is the standard temperature approved by CLSI standard, 37 °C is a clinically relevant temperature, and 30 °C is a commonly used laboratory temperature) for the desired amount of time, often for 24, 48, or 72 h.
  7. To prepare plates for OD600 reading, mix wells with a multi-channel pipette.
  8. Process the data in Excel.
    1. Copy OD600 values for each drug replicate into a new worksheet.
    2. Above the first row of values, label the drug gradient with the appropriate concentration for each column.
    3. Label the strains in the left-most column.
    4. Calculate the average of medium-only values and subtract it from all values on the plate. Convert any negative numbers to “0”.
    5. Calculate the average of values from each duplicate.
    6. Optional: To normalize growth in the drug gradient relative to its drug-free control, divide the OD600 values of each well by the OD600 value of the well in the column 1 of the corresponding row.
    7. Copy the referenced values to a new worksheet using “paste special”, select “values only” and arrange in the following format.


      Note: This data was updated by authors on Oct. 15, 2015.

    8. Save as a text (tab delimited) file.
  9. Plot the data as heat map in JavaTree View.
    1. Open the text (tab delimited) file in JavaTree View.å.
    2. Under setting, make the following changes in pixel.



      Zoom X: Fixed Scale = 60
      Zoom Y: Fixed Scale = 60
      Contrast Value: 1
      Positive: 102, 204, 0 (Green)
    3. Export the file



      The above figure is adapted from Reference 2, and plotted based on the data shown in the table associated with point 8 g above. Growth measured by optical density is quantitatively displayed with color as indicated in the color bar, such that bright green represents full growth and black represents no growth. As indicated above, growth of each strain can be plotted as values normalized to the drug-free control for the same strain, or growth can be directly plotted without normalization.   

Recipes

  1. RPMI medium 1640 (pH 7.0)
    3.5% MOPS
    2% glucose
  2. YPD
    1% yeast extract
    2% bactopeptone
    2% glucose
  3. Synthetic defined medium
    0.67% yeast nitrogen base
    2% glucose

Acknowledgments

The method reported in this protocol is based on that described in Singh-Babak et al. (2012). JLX was supported by an Ontario Graduate Scholarship, a University of Toronto Open Fellowship, and a Canadian Institutes of Health Research (CIHR) Frederick Banting & Charles Best CGS Doctoral Award, SDSB by a CIHR Frederick Banting & Charles Best CGS Doctoral Award, and LEC by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, by a Canada Research Chair in Microbial Genomics and Infectious Disease, by a Ministry of Research and Innovation Early Researcher Award, and by NSERC Discovery Grant 355965-2009.

References

  1. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Third Edition. CLSI document M27-A3 (ISBN 1-56238-666-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008.
  2. Singh-Babak, S. D., Babak, T., Diezmann, S., Hill, J. A., Xie, J. L., Chen, Y. L., Poutanen, S. M., Rennie, R. P., Heitman, J. and Cowen, L. E. (2012). Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog 8(5): e1002718.

简介

最小抑制浓度(MIC)测定广泛用于测量酵母对抗真菌剂的易感性。 在连续两倍稀释中,足以抑制真菌生长的抗真菌药物的最低浓度是MIC。 通常,报告50%抑制(MIC 50)或80%抑制(MIC 80)值。 为了便于抗真菌敏感性数据的可视化,产生热图,其中光密度值用颜色定量表示。

材料和试剂

  1. 应分析的应变
  2. 待分析菌株的过夜培养物和亲本对照
  3. 葡萄糖
  4. MOPS
  5. 酵母提取物
  6. 细菌蛋白胨
  7. 酵母氮基
  8. 媒体
    1. RPMI培养基1640(Life Technologies,Gibco ,目录号:31800-089)(参见Recipes)
      注意:这是CLSI标准协议推荐的标准介质。
    2. YPD(见配方)
      注意:这是一种通常用于评估不同突变体易感性的丰富介质。
    3. 合成定义介质(参见配方)
      注意:添加所需的氨基酸以使营养缺陷型菌株生长通常用于需要质粒选择的实验。

设备

  1. 96孔组织培养板(带盖的平底)(SARSTEDT AG,目录号:83.1835)
  2. 酶标仪
  3. 多通道移液器[可选:多通道电子移液器(Rainin,型号:EDP3-Plus)]

软件

  1. Soft Max Pro或同等分光光度计软件(Microplate Data Processing)
  2. Microsoft Excel(数据分析)
  3. JavaTree视图(热图生成, http://jtreeview.sourceforge.net/

程序

  1. 在3ml待测试的合适培养基中生长待测试菌株的过夜培养物,包括相关对照。
  2. 用要测试的药物梯度制备MIC板。
    1. 总是设置一式两份。
    2. 使用多通道移液器添加100微升培养基到所有96孔除了第12列。
    3. 到第12列,加入200微升含有药物的培养基,其浓度为滴定起始浓度的2倍。
    4. 通过将100μl含药物培养基从柱12转移到柱11进行2倍稀释(混匀并重复横行)。 停止在第2列,丢弃多余的100μl,使第1列是无药物对照
  3. 在96孔板中一式两份测量100μl过夜培养物的OD 600。
    1. 从仅培养基孔中减去OD <600>读数以计算背景。
    2. 计算每个菌株的平均OD 600。
  4. 为了在5ml培养基中制备接种物,加入1/OD600μL×1.5μl过夜培养物。
  5. 从列1至12的每个孔中加入100μl稀释的接种物(〜10μL/孔细胞),每行一个菌株。
  6. 在合适的温度(35℃是CLSI标准批准的标准温度,37℃是临床相关温度,30℃是通常使用的实验室温度)下,在所需的时间内静止培养板,通常为 24,48或72小时。
  7. 为了准备用于OD 600读数的板,用多通道移液器混合孔。
  8. 在Excel中处理数据。
    1. 将每个药物复制的OD 600 值复制到新工作表中。
    2. 在第一行值的上方,标记每个柱的具有适当浓度的药物梯度。
    3. 标记最左列中的菌株。
    4. 计算中等值的平均值,并从板上的所有值中减去。 将任何负数转换为"0"。
    5. 计算每个重复值的平均值。
    6. 任选:为了使药物梯度相对于其无药物对照的生长正常化,将每个孔的OD 600值除以柱中孔的OD 600值 1。
    7. 使用"粘贴特殊"将参考值复制到新工作表,选择"仅值",并按以下格式排列。


      注意:此数据由作者于2015年10月15日更新。

    8. 另存为文本(制表符分隔)文件。
  9. 在JavaTree视图中将数据绘制为热图。
    1. 在JavaTree View.a中打开文本(制表符分隔)文件。
    2. 在设置下,对像素进行以下更改。



      缩放X:固定缩放= 60
      缩放Y:固定比例= 60
      对比值:1
      正:102,204,0(绿色)
    3. 导出文件



      上图从参考文献2改编,并且基于与上面的点8g相关联的表格中所示的数据绘制。 通过光密度测量的生长用颜色条定义地显示颜色,使得亮绿色表示完全生长,黑色表示无生长。 如上所述,每个菌株的生长可以绘制为相对于相同菌株标准化为无药物对照的值,或者可以直接绘制生长而不进行归一化。

食谱

  1. RPMI培养基1640(pH7.0) 3.5%MOPS
    2%葡萄糖
  2. YPD
    1%酵母提取物
    2%细菌用蛋白胨 2%葡萄糖
  3. 合成定义的介质
    0.67%酵母氮碱
    2%葡萄糖

致谢

该方案中报道的方法基于Singh-Babak等人(2012)中描述的方法。 JLX由安大略研究生奖学金,多伦多大学开放研究金和加拿大卫生研究院(CIHR)Frederick Banting& Charles最佳CGS博士奖,SDSB由CIHR Frederick Banting& Charles Best CGS博士学位奖,LEC荣获由Burroughs Wellcome基金颁发的生物医学科学奖,由加拿大微生物基因组学和传染病研究主席,研究与创新部早期研究员奖,以及NSERC发现奖355965-2009。

参考文献

  1. 临床和实验室标准研究所(CLSI)。酵母的肉汤稀释抗真菌敏感性试验的参考方法;批准的标准 - 第三版。 CLSI文件M27-A3(ISBN 1-56238-666-2)。临床和实验室标准研究所,940 West Valley Road,Suite 1400,Wayne,Pennsylvania 19087-1898 USA,2008.
  2. Singh-Babak,S.D.,Babak,T.,Diezmann,S.,Hill,J.A.,Xie,J.L.,Chen,Y.L.,Poutanen,S.M.,Rennie,R.P.,Heitman,J.and Cowen, 光滑念珠菌中Echinocandin抗性的进化和机制的全球分析。 PLoS Pathog 8(5):e1002718。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xie, J. L., Singh-Babak, S. D. and Cowen, L. E. (2012). Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs. Bio-protocol 2(20): e252. DOI: 10.21769/BioProtoc.252.
  2. Singh-Babak, S. D., Babak, T., Diezmann, S., Hill, J. A., Xie, J. L., Chen, Y. L., Poutanen, S. M., Rennie, R. P., Heitman, J. and Cowen, L. E. (2012). Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog 8(5): e1002718.
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