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Jan 2021

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Bacterial Growth Curve Measurements with a Multimode Microplate Reader
使用多模式酶标仪测量细菌生长曲线   

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Abstract

Bacterial studies based on growth curves are common in microbiology and related fields. Compared to the standard photometer and cuvette based protocols, bacterial growth curve measurements with microplate readers provide better temporal resolution, higher efficiency, and are less laborious, while analysis and interpretation of the microplate-based measurements are less straightforward. Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. Here, we describe a detailed protocol for this development and provide the homemade program for the new analysis method.

Keywords: Time derivative, Fluorescence, Absorption, Optical density, Multi-mode

Background

Monitoring the growth and proliferation of microorganisms based on growth curves is commonly used in microbiology and related fields for understanding the growth of microorganisms, the efficacy of antibiotic drugs, and the microbial responses to new environmental conditions and stresses (Mandelstam et al., 1982; Andrews, 2001; Bollenbach et al., 2009; Haque et al., 2017). Traditional growth curve measurements based on photometers and cuvettes suffer from several drawbacks, such as low temporal resolution, low efficiency, and high labor requirement (Stevenson et al., 2016; Goldman and Green, 2021). In contrast, growth curve measurements using microplate readers take advantage of automation and parallel capability; however, the application of microplate readers for growth curve measurements has been hindered due to the complexity in the quantification and data interpretation caused by multiple light scattering and/or light absorption/scattering by other ingredients in the samples (Stevenson et al., 2016). Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. This method not only uses the optical density (i.e., absorption/scattering) of bacterial culture, but also takes advantage of the fluorescence of bacteria expressing fluorescent proteins (Krishnamurthi et al., 2021). The time derivatives of optical density and fluorescence show peaks, whose location and height are correlated with the lag time and maximum growth rate of bacteria (Krishnamurthi et al., 2021). Here, we describe the detailed protocol for carrying out growth curve measurements with microplate readers and provide the homemade program for analyzing and visualizing the results. Escherichia coli bacteria were used here as an example; however, this protocol is expected to be applicable to other microorganisms.

Materials and Reagents

  1. 96-well clear bottom plates (Corning, catalog number: 3631) (Figure 1)



    Figure 1. Photo of a 96-well microplate with clear bottom and its clear lid.


  2. Polystyrene lids for 96-well microplates (Corning, catalog number: 3931)

  3. 1–200 µL pipette tips (VWR, catalog number: 53508-810)

  4. 100–1,000 µL pipette tips (VWR, catalog number: 83007-376)

  5. 1.5 mL microcentrifuge tubes (VWR, catalog number: 20170-038)

  6. 14 mL sterile culture tubes (VWR, catalog number: 60818-725)

  7. Escherichia coli strain K-12 MG1655 transformed with plasmid pOEGFP2, encoding ampicillin resistance gene and green fluorescent proteins (Lab collection), which were used previously (Haque et al., 2017; Niyonshuti et al., 2020; Krishnamurthi et al., 2021). (Figure 2)

    Note: Other bacterial strains expressing fluorescent proteins are expected to be compatible with the current protocol.



    Figure 2. Map of the plasmid pOEGFP2 used in this protocol.

    The plasmid was a gift from Dr. David McMillen at the University of Toronto. The plasmid map was generated using SnapGene Viewer (GSL Biotech LLC).


  8. Tryptone (Bio Basic Inc, catalog number: TG217)

  9. Yeast extract (Bio Basic Inc, catalog number: G0961)

  10. Sodium chloride (VWR International, LLC, catalog number: 7647-14-5)

  11. Ampicillin (VWR, catalog number: AAJ60977-06)

  12. Triton X-100 (VWR, catalog number: AAA16046-AP)

  13. Ethanol (VWR, catalog number: 89125-188)

  14. Deionized water (>17.6 MΩ, from Barnstead ultrapure water purification system [model: D4641])

  15. Triton X-100 (VWR, catalog number: AAA16046-AP)

  16. Liquid LB medium (200 mL) (see Recipes)

  17. Ampicillin stock solution (10 mL) (see Recipes)

  18. Python code (see Supplementary file)

Equipment

  1. 2–20 µL single channel mechanical pipette (VWR, catalog number: 89079-964)

  2. 20–200 µL single channel mechanical pipette (VWR, catalog number: 89079-970)

  3. Multimode microplate reader capable of measuring both absorption and fluorescence (BioTek Instruments, model: Synergy H1)

  4. Autoclave (Tuttnauer, model: 2540E-B/L)

  5. Biosafety cabinet (Labconco, model: Purifier Logic+ Class II Type A2)

Software

  1. Gen5 Microplate Reader and Imager Software (BioTek Instruments, https://www.biotek.com/)

  2. Microsoft Excel Spreadsheet Software (Microsoft Corporation, https://www.microsoft.com/)

  3. Python (https://www.python.org/)

Procedure

Bacteria Growth:

  1. Preparation of initial liquid culture of bacteria

    1. Prepare liquid LB medium, followed by autoclaving (120°C, 41 min) and cooling down to room temperature.

    2. Inoculate a single colony of the bacterial strain into 7 mL of LB medium in a culture tube, supplemented with ampicillin or the appropriate antibiotic at a final concentration of 50 µg/mL.
       Note: Several colonies in separate culture tubes may be used if more replicates are desired.

    3. Incubate the liquid culture in a shaking incubator at 37°C with orbital shaking at 250 rpm overnight.


  2. Pre-treatment of 96-well plates and lids

    1. Prepare 10% Triton X-100 in ethanol by mixing 1 mL of Triton X-100 with 9 mL of ethanol.

    2. Pour 10 mL of the diluted Triton X-100 to a new microplate lid.

    3. Incubate the lid with diluted Triton X-100 at room temperature for 15 s.

    4. Take a new 96-well plate and fill each well with 100% ethanol (400 µL per well). Incubate at room temperature for 15 min.

    5. Discard ethanol from the microplate wells and discard the diluted Triton X-100 from the microplate lid.

    6. Air dry the microplate wells and microplate lid at room temperature by leaving them inside the biosafety cabinet for 30 min.

    7. Turn on the UV light of the biosafety cabinet and further disinfect the microplate wells and lid for 15 min.

      Note: Treatment with ethanol and UV is for sterilization, while treatment with Triton X-100 is to prevent water condensation on the lid during the measurements.


  3. Setup of microplate reader and software

    1. Turn on the microplate reader and open the Gen5 software.

    2. Set up a new procedure/protocol in Gen5 (Figure 3).

      1. Plate type: 96-well plate.

      2. Use lid: checked.

      3. Temperature: 37°C.

      4. Shake: orbital for 0:10.

      5. Kinetic measurement for a desired total time (e.g., 16 h, 24 h, or 48 h) and interval of 10 min with continuous orbital shaking.

      6. Reading: absorption at 600 nm, fluorescence at excitation = 488 nm and emission = 525 nm.



      Figure 3. An example for setting up the procedure/protocol in Gen5 software for the growth curve measurements of bacteria using a microplate reader.


  4. Growth curve measurements using microplate reader

    1. Dilute the overnight liquid bacterial culture by 100–500 times in fresh LB supplemented with ampicillin at 50 µg/mL.

      Notes:

      1. If a photometer is available, measure the optical density of the overnight culture and make the dilution to the desired optical density (typically <0.05).

      2. Other media or additional ingredients (such as ions, nanoparticles, and drugs of interest that may affect bacterial growth) can be used if desired. For example, silver ions were added to the LB medium at a final concentration of 40 µg/mL in the sample S2 of the representative data (Figure 4).

    2. Pipette 200 μL of the diluted culture into a single well of the 96-well plate as a sample. Samples should be prepared with at least triplicates.

    3. Pipette 200 µL of LB medium with ampicillin into a single well of the 96-well plate as a blank. Blanks should be prepared with at least triplicates.

    4. After loading all the samples and blanks, cover the 96-well microplate with the prepared lid.

    5. Start the procedure/protocol in Gen5 software. When prompted, place the microplate properly on the microplate holder, and start recording of the bacterial growth curve.


  5. Finishing up

    1. Upon completion of the data acquisition, remove the 96-well plate from the microplate reader.

    2. Disinfect the samples and discard the microplate properly.

    3. Save the results in Gen5 software, and export the data as an Excel spreadsheet file.

    4. Close the Gen5 software, and turn off the microplate reader.

Data analysis

  1. Reorganize the data from the Excel spreadsheet as text files so that each text file contains the absorption data (or fluorescence) data of a single sample (or the blank). Note that each column in the text file corresponds to the readings of a single well of the microplate. Fluorescence data and absorption data should be separated into different files.

  2. Run the provided Python code (see Supplementary file) to:

    1. Calculate the averaged absorption (or fluorescence) of samples, and plot the results.

    2. Smooth the growth curves, calculate the averaged first-order time derivatives of absorption (or the second-order time derivative of fluorescence) of samples, and plot the results.

    3. Identify the peak locations and heights of the time derivatives.

  3. Compare the peak locations (and/or heights) to quantify the relative changes between the samples.


Representative Data


Figure 4. Representative data for the growth curves of E. coli bacteria measured by a microplate reader and their time derivatives.

A. Absorption growth curves. B. Fluorescence growth curves. C. First-order time derivative of absorption growth curves. D. Second-order time derivative of fluorescence growth curves. Sample S1: MG1655 strain with pEOGFP2 plasmid in LB medium. Sample S2: MG1655 strain with pEOGFP2 plasmid in LB medium with silver ions of 40 µg/mL. In all sub-figures, “a.u.” stands for arbitrary units.

Recipes

  1. Liquid LB medium (200 mL)

    2 g Tryptone

    1 g yeast extract

    2 g sodium chloride (NaCl)

    Fill up to 200 mL with deionized water and autoclave to sterilize.

  2. Ampicillin stock solution (50 mg/mL)

    500 mg of ampicillin powder (sodium salt)

    10 mL of water

    Filter sterilize the ampicillin solution and store 1 mL tubes at -20°C.

Acknowledgments

We thank Dr. David McMillen at the University of Toronto for the generous gift of plasmid encoding the enhanced GFP and ampicillin resistance (pOEGFP2). This work was supported by the University of Arkansas, the Arkansas Biosciences Institute (Grant No. ABI-0189, No. ABI-0226, No. ABI-0277, No. ABI-0326, No. ABI-2021, No. ABI-2022), and the National Science Foundation (Grant No. 1826642). The original protocol was developed and used in Krishnamurthi et al. (2021) and Niyonshuti et al. (2020).

Competing interests

No competing interest is declared.

References

  1. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48 Suppl 1: 5-16.
  2. Bollenbach, T., Quan, S., Chait, R. and Kishony, R. (2009). Nonoptimal microbial response to antibiotics underlies suppressive drug interactions. Cell 139(4): 707-718.
  3. Goldman, E., and Green, L. H. (2021). Practical Handbook of Microbiology (Taylor & Francis Group). CRC Press. IBSN: 9780367567637.
  4. Haque, M. A., Imamura, R., Brown, G. A., Krishnamurthi, V. R., Niyonshuti, I. I., Marcelle, T., Mathurin, L. E., Chen, J. and Wang, Y. (2017). An experiment-based model quantifying antimicrobial activity of silver nanoparticles on Escherichia coli. RSC Advances 7(89): 56173-56182.
  5. Krishnamurthi, V. R., Niyonshuti, II, Chen, J. and Wang, Y. (2021). A new analysis method for evaluating bacterial growth with microplate readers. PLoS One 16(1): e0245205.
  6. Mandelstam, J., Dawes, I. W., and McQuillen, K. (1982). Biochemistry of bacterial growth. New York, Wiley.
  7. Niyonshuti, II, Krishnamurthi, V. R., Okyere, D., Song, L., Benamara, M., Tong, X., Wang, Y. and Chen, J. (2020). Polydopamine Surface Coating Synergizes the Antimicrobial Activity of Silver Nanoparticles. ACS Appl Mater Interfaces 12(36): 40067-40077.
  8. Stevenson, K., McVey, A. F., Clark, I. B. N., Swain, P. S. and Pilizota, T. (2016). General calibration of microbial growth in microplate readers. Sci Rep 6: 38828.

简介

[摘要] 基于生长曲线的细菌研究在微生物学和相关领域很常见。与基于标准光度计和比色皿的协议相比,使用酶标仪进行细菌生长曲线测量可提供更好的时间分辨率、更高的效率并且不那么费力,而基于酶标仪的测量的分析和解释则不那么简单。最近,我们开发了一种基于时间导数的用酶标仪评估细菌生长的新分析方法。在这里,我们描述了此开发的详细协议,并为新的分析方法提供了自制程序。


[背景] 基于生长曲线监测微生物的生长和增殖通常用于微生物学和相关领域,以了解微生物的生长、抗生素药物的功效以及微生物对新环境条件和压力的反应(Mandelstam et al. , 1982; Andrews,2001;Bollenbach等人,2009;Haque等人,2017) 。基于光度计和比色皿的传统生长曲线测量存在几个缺点,例如时间分辨率低、效率低和劳动力要求高(Stevenson等人,2016 年;Goldman 和 Green,2021 年) 。相比之下,使用酶标仪测量生长曲线则利用了自动化和并行能力;然而,由于样品中其他成分的多重光散射和/或光吸收/散射引起的定量和数据解释的复杂性,阻碍了酶标仪在生长曲线测量中的应用(Stevenson等,2016) .最近,我们开发了一种基于时间导数的用酶标仪评估细菌生长的新分析方法。该方法不仅利用了细菌培养的光密度(即吸收/散射),还利用了细菌表达荧光蛋白的荧光(Krishnamurthi et al. , 2021) 。光密度和荧光的时间导数出现峰值,其位置和高度与细菌的滞后时间和最大生长速率相关(Krishnamurthi et al. , 2021) 。在这里,我们描述了使用酶标仪进行生长曲线测量的详细协议,并提供了用于分析和可视化结果的自制程序。这里以大肠杆菌为例;然而,该协议有望适用于其他微生物。

关键字

材料和试剂
1. 96 孔透明底板(Corning,目录号:3631)(图 1)

图 1. 96 孔微孔板的照片,底部透明,盖子透明。
2. 用于 96 孔微孔板的聚苯乙烯盖(Corning,目录号:3931)
3. 1–200 µL 移液器吸头(VWR,目录号:53508-810)
4. 100–1,000 µL 移液器吸头(VWR,目录号:83007-376)
5. 1.5 mL微量离心管(VWR,目录号:20170-038)
6. 14 mL无菌培养管(VWR,目录号:60818-725)
7. 大肠杆菌菌株 K-12 MG1655 用质粒 pOEGFP2 转化,编码氨苄青霉素抗性基因和绿色荧光蛋白(实验室收藏),以前使用过(Haque等人,2017;Niyonshuti等人,2020;Krishnamurthi等人,2021 ) . (图2)
注意:其他表达荧光蛋白的菌株预计与当前协议兼容。
 
图 2.本协议中使用的质粒 pOEGFP2 的地图。
该质粒是多伦多大学 David McMillen 博士的礼物。使用SnapGene Viewer (GSL Biotech LLC) 生成质粒图谱。


8. 胰蛋白胨(Bio Basic Inc,目录号:TG217)
9. 酵母提取物(Bio Basic Inc,目录号:G0961)
10. 氯化钠(VWR International,LLC,目录号:7647-14-5)
11. 氨苄青霉素(VWR,目录号:AAJ60977-06)
12. Triton X-100(VWR,目录号:AAA16046-AP)
13. 乙醇(VWR,目录号:89125-188)
14. 去离子水(>17.6 MΩ,来自 Barnstead 超纯水净化系统 [型号:D4641])
15. Triton X-100(VWR,目录号:AAA16046-AP)
16. 液体 LB 培养基 (200 mL)(参见食谱)
17. 氨苄青霉素原液(10 mL)(见配方)
18. Python 代码(见补充文件)


设备


1. 2–20 µL 单通道机械移液器(VWR,目录号:89079-964)
2. 20–200 µL 单通道机械移液器(VWR,目录号:89079-970)
3. 能够测量吸收和荧光的多模式酶标仪(BioTek Instruments,型号:Synergy H1)
4. 高压釜( Tuttnauer ,型号:2540E-B/L)
5. 生物安全柜( Labconco ,型号:Purifier Logic+ Class II Type A2)


软件


1. Gen5 酶标仪和成像软件(BioTek Instruments, https ://www.biotek.com/ )
2. Microsoft Excel 电子表格软件(Microsoft Corporation, https://www.microsoft.com/ )
3. Python ( https://www.python.org/ )


程序


细菌生长:
A. 细菌初始液体培养物的制备
1. 准备液体 LB 培养基,然后高压灭菌(120°C,41 分钟)并冷却至室温。
2. 将细菌菌株的单个菌落接种到培养管中的 7 mL LB 培养基中,辅以氨苄青霉素或适当的抗生素,最终浓度为 50 μg/mL。注意:如果需要更多重复,可以使用单独培养管中的几个菌落。
3. 在 37°C 的摇动培养箱中孵育液体培养物,并以 250 rpm 的转速摇动过夜。


B. 96 孔板和盖子的预处理
1. 通过将 1 mL 的 Triton X-100 与 9 mL 的乙醇混合,在乙醇中制备 10% 的 Triton X-100。
2. 将 10 mL 稀释的 Triton X-100 倒入新的微孔板盖中。
3. 在室温下用稀释的 Triton X-100 孵育盖子 15 秒。
4. 取一个新的 96 孔板,并用 100% 乙醇(每孔 400 μL)填充每个孔。在室温下孵育 15 分钟。
5. 从微孔板孔中丢弃乙醇,并从微孔板盖中丢弃稀释的 Triton X-100。
6. 将微孔板孔和微孔板盖在室温下风干,将它们留在生物安全柜内 30 分钟。
7. 打开生物安全柜的紫外线灯,进一步消毒微孔板孔和盖子 15 分钟。
笔记: 用乙醇和紫外线处理是为了灭菌,而用 Triton X-100 处理是为了防止测量过程中盖子上的水凝结。


C. 酶标仪和软件的设置
1. 打开酶标仪并打开 Gen5 软件。
2. 在 Gen5 中设置新的程序/协议(图 3)。
a. 板类型:96 孔板。
b. 使用盖子:检查。
c. 温度:37°C。
d. 摇动:0:10 的轨道。
e. 所需总时间(例如,16 小时、24 小时或 48 小时)和 10 分钟间隔的动力学测量,并带有连续的轨道振动。
f. 读数:600 nm 处的吸收,激发时的荧光 = 488 nm 和发射 = 525 nm。


 
图 3。 在 Gen5 软件中设置程序/协议的示例,用于使用酶标仪测量细菌的生长曲线。


D. 使用酶标仪测量生长曲线
1. 在补充有氨苄青霉素 50 µg/mL 的新鲜 LB 中将过夜液体细菌培养物稀释 100–500 倍。
笔记:
a. 如果有光度计,测量过夜培养物的光密度,并将稀释液稀释到所需的光密度(通常 <0.05)。
b. 如果需要,可以使用其他介质或附加成分(例如离子、纳米颗粒和可能影响细菌生长的感兴趣的药物)。例如,在代表性数据的样品 S2 中,银离子以 40 µg/mL 的最终浓度添加到 LB 介质中(图 4)。
2. 200 μL稀释培养物移液到 96 孔板的单孔中作为样品。样品应至少一式三份地制备。
3. 将 200 μL 的 LB 培养基与氨苄西林移液到 96 孔板的单孔中作为空白。空白应至少准备三次。
4. 加载所有样品和空白后,用准备好的盖子盖住 96 孔微孔板。
5. 在 Gen5 软件中启动程序/协议。出现提示时,将微孔板正确放置在微孔板支架上,并开始记录细菌生长曲线。


E. 整理起来
1. 数据采集完成后,从微孔板读取器中取出 96 孔板。
2. 对样品进行消毒并正确丢弃微孔板。
3. 将结果保存在 Gen5软件中,并将数据导出为 Excel 电子表格文件。
4. 关闭 Gen5软件,然后关闭酶标仪。


数据分析


1. 将 Excel 电子表格中的数据重新组织为文本文件,以便每个文本文件都包含单个样品(或空白)的吸收数据(或荧光)数据。请注意,文本文件中的每一列对应于微孔板单个孔的读数。荧光数据和吸收数据应该分开到不同的文件中。
2. 运行提供的 Python 代码(请参阅补充文件)以:
a. 计算样品的平均吸收(或荧光),并绘制结果。
b. 平滑生长曲线,计算样品吸收的平均一阶时间导数(或荧光的二阶时间导数),并绘制结果。
c. 确定时间导数的峰值位置和高度。
3. 比较峰位置(和/或高度)以量化样品之间的相对变化。


代表性数据


 
图 4。 由酶标仪及其时间导数测量的大肠杆菌生长曲线的代表性数据。
A.吸收增长曲线。 B.荧光生长曲线。 C.吸收增长曲线的一阶时间导数。 D.荧光生长曲线的二阶时间导数。样品 S1:在 LB 培养基中带有 pEOGFP2 质粒的 MG1655 菌株。样品 S2:MG1655 菌株与pEOGFP2 质粒在 LB 培养基中,银离子为 40 µg/mL。在所有子图中,“ au ”。代表任意单位。


食谱


1. 液体 LB 培养基 (200 毫升)
2 克胰蛋白胨
1克酵母提取物
2克氯化钠(NaCl)
用去离子水填充高达 200 mL 并高压灭菌。
2. 氨苄青霉素原液 (50 mg/mL)
500毫克氨苄青霉素粉(钠盐)
10毫升水
过滤对氨苄青霉素溶液进行消毒,并将 1 mL 管储存在 -20°C。


致谢


我们感谢多伦多大学的 David McMillen 博士慷慨赠予编码增强型 GFP 和氨苄青霉素抗性 (pOEGFP2) 的质粒。这项工作得到了阿肯色大学、阿肯色州生物科学研究所 (Grant No. ABI-0189, No. ABI-0226, No. ABI-0277, No. ABI-0326, No. ABI-2021, No. ABI- 2022)和国家科学基金(批准号 1826642)。原始协议是在Krishnamurthi等人中开发和使用的。 (2021) 和 Niyonshuti等人。 (2020 年) 。


利益争夺


没有宣布竞争利益。


参考


1. 安德鲁斯,JM(2001 年)。最低抑菌浓度的测定。 J Antimicrob Chemother 48 增刊 1:5-16。
2. Bollenbach , T., Quan, S., Chait, R. 和Kishony , R. (2009)。微生物对抗生素的非最佳反应是抑制药物相互作用的基础。 单元格139(4):707-718。
3. Goldman, E. 和 Green, LH (2021)。微生物学实用手册(Taylor & Francis Group) 。 CRC出版社。 IBSN:9780367567637。
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引用:Rogers, A. T., Bullard, K. R., Dod, A. C. and Wang, Y. (2022). Bacterial Growth Curve Measurements with a Multimode Microplate Reader. Bio-protocol 12(9): e4410. DOI: 10.21769/BioProtoc.4410.
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