Application Note

Advanced kinetic analysis of a bacterial growth assay

  • Save time with simultaneous detection of both cell density and fluorescent protein kinetic traces using the Workflow Editor
  • Simplify data report generation with preconfigured and customizable data analysis options
  • Easily consolidate desired data output parameters into table or plate formats and graph as scatter plots or bar graphs

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Introduction

Michel Hoogenkamp | Research Technician | Academic Centre for Dentistry Amsterdam (ACTA)
Cathleen Salomo | Field Applications Scientist | Molecular Devices

Difficult-to-kill bacteria have become a problem in hospital-acquired infections. Identifying compounds which kill these bacteria is of interest to many pharmaceutical companies. Developing and optimizing assays to screen for the efficacy of these compounds is a challenge faced by many microbiologists.

Here, we describe the setup of a bacterial growth assay using SoftMax® Pro 7.0 (or higher) Data Acquisition and Analysis Software. Both cell density and GFP signals were recorded over a time course using the Workflow Editor acquisition function. Various data transformation steps in the software are discussed, such as normalizing the GFP signal to cell density, as well as extracting growth rates or other kinetic relevant information.

Materials

Methods

Data acquisition using the Workflow Editor

Bacterial growth data were captured using the SpectraMax® i3x Multi-Mode Microplate Reader at the Department of Preventive Dentistry at the Academic Centre for Dentistry (ACTA) in the Netherlands. Here, Enterococcus faecalis strain OG1RF containing plasmid pMV158-GFP was used at various growth conditions. GFP (green fluorescent protein) expression is heterologous, and the strain is described in the publication of Hoogenkamp et al1, where GFP was studied as a viability marker for the difficult-to-stain species E. faecalis. 150 μl of E. faecalis in PBS, and PBS as background control, were pipetted into a 96-well black-walled μclear plate. To prevent evaporation, the microplate was covered with a qPCR seal. The microplate was then placed in a SpectraMax i3x reader and incubated at 37°C for the duration of the kinetic measurement.

Using the SoftMax Pro Workflow Editor, a kinetic cycle containing an absorbance read (PlateOD600) and a fluorescence read (PlateGFPBottom) with a 5-second linear plate shake between reads was created (Figure 1). Absorbance was measured at 600 nm (OD600) to determine the bacterial growth. GFP expression, an indicator of viability, was monitored using bottom-read fluorescence detection with excitation at 485 nm (bandwidth 9 nm) and emission at 515 nm (bandwidth 15 nm). Both data traces (absorbance and fluorescence) were recorded in two independent plate sections. The kinetic cycle was set to repeat once every 15 minutes for a total of 18 hours. Optional software features allow the user to pause and resume a kinetic read, enabling the addition of reagents during the run (not used in this experiment). The resulting dual read mode kinetic data were analyzed using SoftMax Pro Software.

Workflow Editor for a dual read mode kinetic reading

Figure 1. SoftMax Pro 7 Workflow Editor for a dual read mode kinetic reading. The drag and drop feature is easy to configure and enables a customized workflow to be set up quickly.

Data analysis options using the reduction settings dialogue

As an initial step of data analysis, the user must decide whether or not to define a blank. The blank well location was set using the template editor, which offers the choice of a group blank or plate blank. A plate blank is subtracted from the raw data of all sample wells in the plate at each timepoint, whereas a group blank is subtracted from associated sample wells only. In this experimental setup, buffer background traces were captured as a control for possible contamination with bacteria or other species. The buffer was not interfering with either the OD600 or the GFP measurement, therefore no subtraction was required. If media or buffer components interfere with the OD600 or fluoresce, a subtraction of the blank is recommended to retrieve the true signal values for either the absorbance or fluorescence channel. Also, this allows comparability of measurements.

Advanced kinetic data analysis is offered through the reduction settings dialogue. Figure 2 shows the reduction settings for the bacterial growth experiment. The menu is split into two sections: raw data and data reduction steps.

Data analysis options in the Reduction Settings menu

Figure 2. Data analysis options in the Reduction Settings menu. This example shows the logarithmic transformation of the optical density data in plate section ‘PlateOD600nm’ and the subsequent reduction to the Vmax rate value with 5 Vmax points to retrieve the steepest part of the curve.

Raw data steps include the option to set the first data point for all kinetic traces to zero. Furthermore, a blank calculation can be included before or after the kinetic reduction. When ‘before reduction’ is selected, the averaged blank well(s) kinetic data trace is subtracted at each timepoint from all raw data sets individually. Choosing ‘after reduction’ subtracts the average of the reduced blank well data, such as Vmax rate, from the sample well data.

The data reduction steps and data output types comprise the following options (Figure 2):

Kinetic reduction
Details
Vmax
Maximum slope of the kinetic trace either as milli-units per minute or units per second. The number of Vmax points defines the maximum size of the line segment used to determine the slope.
Time to Vmax
Time to Vmax elapsed time data is useful for applications including coagulation chemistry where the changing concentration of the reagents does not change Vmax, but rather changes the time at which the reaction reaches the maximum rate.
Slope
Is the same as Vmax rate (units per second) using all available datapoints within the reduction limits to determine the slope.
Onset time
Onset time is a method for analyzing non-linear Kinetic reactions. Onset time reports the time required for a kinetic reaction to reach a specified OD or RFU/RLU (onset OD/RFU/RLU). Useful for cascade reactions such as clot formation in endotoxin testing.
Time at minimum or maximum
This setting reports the time at the minimum or maximum OD, RFU/RLU, or %T that falls within the reduction limits.
Time at 1/2 maximum
This setting reports the time at half of the maximum OD, RFU/RLU, or %T that falls within the reduction limits.
Area under curve
This reduction estimates the area under the curve as defined by the data plots within the reduction limits. The data plots are treated as a series of trapezoids with vertices at successive data points and at the X-axis coordinates of the data points. The areas defined by each of the trapezoids are then computed and summed.
Minimum or maximum
This reports the minimum OD, RFU/RLU, or %T that falls within the reduction limits.
Max-Min
This reports the maximum subtracted from the minimum OD, RFU/RLU, or %T that falls within the reduction limits.
Mean
This reports the mean OD, RFU/RLU, or %T that falls within the reduction limits.

Table 1. Preconfigured kinetic reduction options.

Results display options

Figure 5. Results display options. The reduced data display of the plate section and results of each experimental condition of E. faecalis (Column 1 to 5, n=8) are summarized in the results table as wells as a bar graph. Vmax rate was used to retrieve both the growth rate (k=Vmax*3600) and doubling time (g=ln2/k).

Conclusion

The Workflow Editor in SoftMax Pro 7 Software, together with Molecular Devices multi-mode microplate readers including the SpectraMax i3x reader, offers the flexibility to record dual read mode kinetic growth data with optical density and fluorescence protein expression at the same time. Analysis is built into SoftMax Pro Software and offers a variety of data analysis options and transformation for bacterial growth data, such as data normalization or logarithmic scaling adjustment. A variety of kinetic data reduction parameters, including Vmax rate or Onset time, are preconfigured in the software. The desired data output parameters can be easily consolidated in table or plate format and graphed as bar graphs or scatter plots to support the evaluation of various experimental conditions.

Reference

  1. Hoogenkamp MA, Crielaard W, Krom BP. Uses and limitations of green fluorescent protein as a viability marker in Enterococcus faecalis: An observational investigation. J. Microbiol. Methods (2015) Aug;115:57-63.

前言

Michel Hoogenkamp | Research Technician | Academic Centre for Dentistry Amsterdam (ACTA)
Cathleen Salomo | Field Applications Scientist | Molecular Devices

众所周知,如何消除致病菌一直是医院获得性感染的难题。目前,许多制药公司都在研发对抗这些耐药致病菌的有效化合物,而如何筛选和鉴定这些化合物的功效是微生物学家们面临的挑战。

在本篇文章中,我们使用 SoftMax®Pro7.0 ( 或更高版本 ) 数据采集和分析软件进行细 菌生长曲线的检测。在连续的一段时间内, 使用多任务流程编辑器可以同时采集细胞 密度值和 GFP 荧光信号值。我们探讨了软 件中各种数据转换模式,例如将 GFP 信号 归一化为细胞密度,以及获取生长速率或 其他动力学相关信息。

材料

方法

使用多任务流程编辑器采集数据

本篇文章中细菌生长的实验数据来自于 荷兰牙科学术中心 (ACTA) 预防牙科的 SpectraMax®i3x 多功能微孔板读板机。 在本次实验中,我们将含有质粒 pMV158 - 优势 多任务流程编辑器,可同时检 测细胞密度和荧光蛋白动态表 达,节省时间; 软件内置动力学分析参数和自 定义函数,简化数据分析流程; 轻松将数据以板格式或列表格 式输出,并绘制散点图或条形 图直观展示数据结果。 GFP的粪肠球菌菌株 OG 1 RF 作为样品并且 设置了 5 种不同的实验条件。GFP ( 绿色荧 光蛋白 ) 表达是异源的,2015 年 Hoogenkamp 等人1 报道过 GFP 是可以作为粪肠 球菌的活力标志物的。将 150 μl 的粪肠球 菌样品和空白对照 PBS 依次加入黑色 96 孔底透微孔板中,为了防止液体蒸发,最 好用 qPCR 密封膜覆盖微孔板。把微孔板 放入 SpectraMax®i3x 多功能微孔板读板机 中,设置孵育温度为 37℃,然后进行连续 时间的动力学检测。

在 SoftMax Pro 软件多任务流程编辑器 中,创建一个包含吸光度检测 (OD 600) 和 荧光强度检测 ( 荧光底读 ) 的动力学循环程 序,两次读数之间设置 5 秒钟的线性混匀 ( 见图 1 )。600 nm (OD 600) 读取的吸光度 值代表了细菌生长的密度,荧光底读所读 取的 GFP 蛋白表达的荧光强度值代表了细 菌活力,激发光设置在 485 nm ( 带宽 9 nm ),发射光设置在 515 nm ( 带宽 15 nm )。 吸光度值和荧光强度值这两种数据分别 单 独记录在吸光度检测和荧光强度检测 的程序中。此次动力学循环程序设定为每 15 分钟重复一次读数,一共持续 18 个小 时。在 SoftMax Pro 7.0 ( 或更高版本 ) 软 件中,允许暂停或恢复动力学读数,从而 便于用户在程序运行期间向微孔板中添加 试剂 ( 本次实验没有使用该功能 )。最后, 使用 SoftMax Pro 软件分析所得的双检测 模式动力学数据。

Workflow Editor for a dual read mode kinetic reading

图 1 SoftMax Pro 7 多任务流程编辑器,用于双读取模式动态读数。 鼠标拖放功能易于设置,可以快速设定用户自定义的实验流程

利用数据运算功能进行数据分析

作为实验数据处理的第一步,用户必须决 定是否需要减去空白。首先,在孔板编辑 器中,定义空白孔的位置,有板空白和组 空白两种选择,板空白是指整个微孔板只 有一个空白对照,在每个时间点从板中所 有样品孔的原始数据中减去板空白;而当 对样品进行不同实验条件的分组,每组样 品设置一个组内空白,仅从相对应组别的 样品孔的原始数据中减去组空白。在本次 实验中,采集缓冲液的背景值作为是否污 染细菌或其它物质的对照。PBS 缓冲液不 干扰 OD 600 或 GFP 测量,因此不需要减 去。如果介质或缓冲液成分干扰 OD 600 或 发出荧光,建议减去空白以测定吸光度或 荧光通道的真实信号值。

通过数据处理菜单可设置先进的动态学数 据分析。图 2 展示了细菌生长实验的数据 分析设置,菜单分为两部分:原始数据设 置和数据分析设置原始数据设置中包含了 将动力学曲线的第一个数据点设置为零的 选项。此外,减去空白的操作可以在动力 学数据处理之前也可以在其之后。当选择 了“处理前”选项,将会在每个时间点从 所有样品孔的原始数据中减去空白孔的平 均值;当选择了“处理后”选项,将从样 品孔处理过的数据中再减去处理过后的空 白孔平均值。

Data analysis options in the Reduction Settings menu

图 2 数据处理菜单中的数据分析选项。 。该示例展示了 OD 600 nm 的吸光度数据的对数转换,并将 Vmax 值降低到 5 个 Vmax 点,以测定曲线的最陡部分。

数据分析设置和数据输出类型包括以下选 项 ( 图 2 ):

动力学分析参数
描述
Vmax
动力学曲线的最大斜率,以毫单位/分钟或单位/秒作为单位。Vmax 点的数量 定义了用于能确定斜率的线段的最大尺寸。
Time to Vmax
达到 Vmax 的时间数据对于包括凝固化学在内的应用非常有用,其中试剂浓 度的变化不会改变 Vmax,而是改变反应达到最大速率的时间。
Slope
与 Vmax ( 单位/秒 ) 相同,使用分析限制范围内的所有可用的数据点来确 定斜率。
Onset time
起始时间是分析非线性动力学反应的方法。起始时间指的使动态反应达到指 定 OD 或 RFU / RLU 所需的时间 ( 起始 OD / RFU / RLU )。可用于级联反应, 例如内毒素测试中的凝胶形成。
Time at minimum or maximum
此参数指的是达到分析限制范围内的最小或最大 OD,RFU/RLU 或 % T 的时间
Time at 1/2 maximum
此参数指的是达到分析限制范围内的最大 OD,RFU/RLU 或 % T 的一半数值 的时间
Area under curve
此参数是估算由分析限制范围内的数据图定义的曲线下面积。数据图被视为一系列梯形,由连续的数据点及在X-轴的坐标围成的梯形,然后计算每个梯形定义的面积并求和。
Minimum or maximum
此参数指的是在分析限制范围内的最小 OD,RFU/RLU 或 % T
Max-Min
此参数是从在分析限制范围内的最大 OD,RFU/RLU或 % T 中减去最小值
Mean
此参数是在分析限制范围内的 OD,RFU/RLU 或 % T 的平均值

表 1 软件内置的动力学分析参数

Results display options

图 5 结果显示选项,选择显示分析过的数据结果。粪 。粪肠球菌每个实验组的数据结果( A1-A5列,n = 8 ) 显示在结果列表以及柱状图中。Vmax 可用于生长速率 (k = Vmax * 3600) 和倍增时间 (g = ln2 / k) 的 运算。

结论

SoftMax Pro 7 多任务流程编辑器配合 Molecular Devices SpectraMax i3x 多功 能读板机一起使用,可同时读取吸光度值 和荧光强度值,灵活的进行双检测模式的 细菌生长曲线动力学实验。在 SoftMax Pro 7 软件中,内置了各种数据分析选项和细菌 生长数据的转换分析,如吸光度和荧光强 度的标准化或对数标度调整等。软件中还 预设了各种动力学分析的简化参数,包括 最大斜率或起始时间等。动态数据输出可 以选择板格式或者列表格式输出,还可将 数据绘制成更直观的条形图或者散点图, 方便用户对不同实验条件的最终分析结果

参考文献

  1. Hoogenkamp MA, Crielaard W, Krom BP. Uses and limitations of green fluorescent protein as a viability marker in Enterococcus faecalis: An observational investigation. J. Microbiol. Methods (2015) Aug;115:57-63.

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