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Introduction

Many biological experiments require monitoring cell growth or measuring enzymatic changes over long periods of time (hours, days or even weeks). In addition, certain model organisms cannot be viably kept in the microplate reader for long periods of time. Algae cultures, for example, require periodic exposure to light and high humidity levels in order to grow successfully, and comparative studies of algae growth conditions are measured over the course of weeks1 . Long-term kinetic measurements can also be used to study other facets of biology such as enzyme kinetics, cellular signaling, and protein expression. However, running such an assay as a long-term continuous kinetic reading on a microplate reader involves occupying the reader for an extended period of time, greatly reducing a lab’s efficiency. Additionally, making a series of endpoint reads yields a data set that must be manually linked together to make a kinetic plot.

Using Molecular Devices microplate readers and SoftMax^® Pro Software, researchers can now take measurements over long periods of time using the software’s Interrupt and Append features (Figure 1). These features allow the removal of the microplate from the instrument for media additions or other experiments and then resume a kinetic reading while keeping all the data points in a single plate section for ease of analysis.

Figure 1. Appending kinetic measurements. Kinetic measurement can be interrupted by pressing the Interrupt button during the measurement in the Controls and Status panel. To resume collecting kinetic measurements within the same file, press the Append button.

In this application note, we demonstrate how to measure bacterial cell growth via absorbance at 600 nm (reported as OD₆₀₀) over the course of 24 hours using a discontinuous kinetic reading protocol on the SpectraMax^® iD3 Multi-Mode Microplate Reader.

Materials

• 96-well solid bottom, clear polystyrene microplates (Greiner cat. # 651101)
• BactoBeads™ E. coli GFP Host (Edvotek cat. #728)
• Lysogeny broth(LB, Sigma Aldrich cat. #L2542)
• Ampicillin(Sigma Aldrich cat. #A0166-5G)
• SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices cat. #iD3)

Methods

An ampicillin-resistant E. coli stock with an OD₆₀₀ value of 0.32 was diluted 10-, 100-, and 1000-fold in 10 mL LB containing 100 µg/mL ampicillin. Over a period of 24 hours, changes in OD₆₀₀ were measured by periodically transferring 100-µL aliquots of each bacterial culture to a single well of a 96-well microplate and measuring absorbance at 600 nm using the SpectraMax iD3 reader and SoftMax Pro Software. Instrument settings are listed in Table 1.

Table 1. Plate reader settings used for data acquisition of bacterial growth curve.

A kinetic plate read was initiated at time zero. To interrupt the kinetic measurement, the “Interrupt” button was clicked in the “Controls and Status” section of SoftMax Pro Software. The instrument finished reading the remaining wells in the selected read area before pausing. The plate was then removed from the reader. For the next time-point in the kinetic reading, a new set of samples was taken from the cultures, and the plate was inserted into the reader. The original data file was opened, and the “Append” button was clicked to enable the addition of more data points to the kinetic reading (Figure 1). The Append feature linked together the discontinuous measurements into one continuous bacterial growth curve. The onset time of the bacterial growth curves was calculated in SoftMax Pro Software to compare bacterial growth conditions. A data reduction was set up to calculate the onset time required for the bacterial culture to reach 0.6 OD₆₀₀ and to calculate the slope at the inflection point of the bacterial growth curve (Table 2).

Table 2. Different parameters of bacterial cell growth. SoftMax Pro Software can be used to analyze different areas of bacterial growth. The onset time was calculated when the bacteria reached 0.6 OD₆₀₀.

Results

Bacterial cultures progress through four phases: lag phase, log phase, stationary phase, and death phase2 (Figure 2). We collected enough data points to generate bacterial growth curves from the three different bacterial cultures described in the Methods section (Figure 3). The data show each culture progressing through the lag, log, and stationary phases at different times. The death phase is not observed in the graph, because the light-scattering method measured by the instrument is more related to the mass of the sample rather the individual cells. The dead cells still occupy mass in the sample and contribute to the light scattering and, therefore, the OD₆₀₀^{3, 4}

Figure 2. Bacterial growth phases. Representation of four different phases of bacterial growth.

Figure 3. Bacterial growth curves. Three different bacterial dilutions were measured using the SpectraMax iD3 Reader and SoftMax Pro Software. Measurements were taken over a 24-hour period. Note that the software was still able to link data points separated by a large time gap.

Various parameters of cellular growth can be calculated using SoftMax Pro Software. Calculating the onset time when a bacterial culture reaches a certain optical density allows us to quantitatively compare growth rates. In our example, we observed the quickest onset time in the 10-fold diluted culture (Table 2). We predicted that the less diluted cultures would have the shortest onset time, and the analysis supported it.

The slope at the inflection point of the growth curve, which reflects the period of exponential growth, can also be calculated using the data reduction features in SoftMax Pro Software (Table 2).

Conclusion

Experiments that involve monitoring cellular growth can vary greatly in length, and it is unrealistic to expect one experiment to occupy a plate reader for multiple hours or days. With the discontinuous kinetics feature in SoftMax Pro Software, researchers can perform kinetic measurements without having to continuously occupy their microplate reader. In addition, sensitive cell types requiring strictly controlled environmental conditions can be measured without impacting cell health or survival. Finally, the formulas and data reduction can be set up within SoftMax Pro Software to perform various analyses in order to quantitatively compare different growth conditions. Assistance with formula writing can be obtained by visiting our Knowledge Base site (https://support.moleculardevices.com/s/global-search/%40uri#t=SalesforceArticle&sort=relevancy) or by contacting tech support.

References

• Watanabe, Mariyo F., and Shinshi Oishi. “Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions.” Applied and Environmental Microbiology 49.5 (1985): 1342-1344.
• Zwietering, M. H., et al. “Modeling of the bacterial growth curve.” Applied and Environmental Microbiology 56.6 (1990): 1875-1881.
• Sutton, Scott. “Measurement of microbial cells by optical density.” Journal of Validation Technology 17.1 (2011): 46.
• . Maier, Raina M. “Bacterial Growth.” Environmental Microbiology. Edition 3. 37-54. Elsevier Inc. Web. 2009.

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简介

很多生物学实验涉及到长时间监测(几小时、几天甚至几周)细胞生长或酶促反应的变化，但是有许多模式生物无法长时间放置在微孔板中进行实时检测。比如藻类，生长需要周期性的光照和很高的湿度，而往往实验又要监测几周的生长状况。长时间动态监测还可以研究酶动力学、细胞信号传导和蛋白表达等生物学现象。然而，如果长时间不间断的检测放置于微孔板中的生物样品就会一直占用酶标仪几天时间，降低了实验室的仪器使用效率，此外，我们必须每隔一段时间按终点法的方式检测一次，多次检测的数值经手动方式进行处理后形成一个时间序列数据，以这种方式就实现了长时间动态学监测。

现在利用Molecular Devices公司酶标的 SoftMax® Pro软件，您可以在做长时间检 测时设置“暂停”和“恢复”功能(见图 1)。这个功能方便您在进行长时间实验 中，将微孔板取出进行加药处理或其它实 验的检测，然后再将微孔板放回仪器内继 续之前的长时间监测实验，并且所有数据 将会自动以时间序列进行排列。

图1. 非连续动态学分析， ，在软件界面的“Control and Status”点击“Interrupt”，可以暂停动态学 监测，点击“Append”按钮可以恢复动态学监测。

本篇应用文章，我们将介绍如何在 SpectraMax® iD3多功能酶标仪上应用非 连续的读值方法，在600nm波长下检测细 菌在24小时的生长情况。

材料

• 96孔透明塑料板， (Greiner cat. # 651101)
• BactoBeads™ E. coli GFP宿主， (Edvotek cat. #728)
• LB培养基， (LB, Sigma Aldrich cat. #L2542)
• 氨苄霉素， (Sigma Aldrich cat. #A0166-5G)
• SpectraMax iD3多功能酶标仪， (Molecular Devices cat. #iD3)

方法

OD600值为0.32的氨苄霉素抗性的E.coli 大肠杆菌储存液被10倍、100倍和1000倍 稀释到10mL含100 μg/mL氨苄霉素的LB 培养基中。在24小时内，周期性的分别从 各个稀释组中取出100μL大肠杆菌培养基 放置于96孔板中，用SpectraMax iD3酶标 仪和 SoftMax Pro软件检测600nm处的吸 光度值。仪器设置参数(见表1)。

表1. 细菌生长曲线检测的酶标仪设置参数。

动态学检测起始时间点为0，在软件界面的 “Control and Status”点击“Interrupt”， 仪器将检测完所有的孔后暂停读值，此时 就可以将微孔板取出，到下一个读值的时 间点时，将微孔板放回机器，设置参数进 行检测，点击“Append”按钮就可将此 次数据添加到第一次时间点的数据中形成 完整的数据(见图1)。Append功能可以将 间断读取的数据整合到一起生成一个连续 的细菌生长曲线。软件可以根据细菌生长 条件自动获得到达某个OD值浓度的时 间，软件就会计算出细菌生长曲线在拐点 的斜率等参数(见表2)。

表2. 细菌生长曲线的多种参数。 SoftMax Pro软件可以计算细菌生长的不同时期曲线面积值，当菌液 OD600达到0.6时被定义为到达时间。

结果

细菌的生长曲线有四个期：迟缓期、对数期、稳定期和衰亡期(见图二)。我们检测了三个浓度的细菌培养基(如方法中所述)在不同时间点细菌浓度的数据形成了生长曲线(见图3)。图中显示不同浓度的细菌培养体系处于迟缓期、对数期和稳定期的时间有差异。图中没有显示出衰亡期，因为光散射测吸光度的方法是检测的颗粒物浓度并非细胞个体，死亡的细胞依然存在于培养基中形成颗粒，影响光散射，即影响OD600读值。

图2. 细菌生长周期， ，图中显示了细菌生长的四个时期。

F图3. 细菌生长曲线。 应用Molecular Devices公司的酶标仪和SoftMax® Pro软件非连续动态监测不同 稀释度的菌液浓度24小时，不同时间点数据可以整合在一组数据中形成动态序列。

细菌的生长曲线有四个期：迟缓期、对数期、稳定期和衰亡期(见图二)。我们检测了三个浓度的细菌培养基(如方法中所述)在不同时间点细菌浓度的数据形成了生长曲线(见图3)。图中显示不同浓度的细菌培养体系处于迟缓期、对数期和稳定期的时间有差异。图中没有显示出衰亡期，因为光散射测吸光度的方法是检测的颗粒物浓度并非细胞个体，死亡的细胞依然存在于培养基中形成颗粒，影响光散射，即影响OD600读值。

SoftMax Pro软件可以计算出细胞生长相 关的多种参数。当细菌培养液达到一定的 光密度时就可开始计算并比较生长起始时 间。在此次实验中，我们观察到10倍稀 释组最早进入生长期(见表2)。我们的预 计就是稀释倍数最少的实验组最先进入生 长期，实验数据结果证实了这个假设。生 长曲线上拐点处的斜率也可以通过软件计 算出来，这个参数反映出了指数增长(见 表2)。

结论

研究细胞生长的实验监测的时间差异很 大，但是连续观测一块板子的样品数小时 甚至数天不太现实。应用SoftMax Pro软 件非连续动态监测功能，我们可以在不用 长时间占用仪器的前提下得到动态监测的 数据。此外，有些敏感细胞需要特殊的生 长条件，非连续监测方法避免了生长环境 不适而造成的对细胞的伤害。另外， SoftMax Pro软件的多种分析功能能够满 足不同生长曲线的计算要求。关于计算公 式的编辑方法可以在我公司的网站上找到 (http://mdc.custhelp.com/)，或联系技 术支持。

参考文献

• Watanabe, Mariyo F., and Shinshi Oishi. “Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions.” Applied and Environmental Microbiology 49.5 (1985): 1342-1344.
• Zwietering, M. H., et al. “Modeling of the bacterial growth curve.” Applied and Environmental Microbiology 56.6 (1990): 1875-1881.
• Sutton, Scott. “Measurement of microbial cells by optical density.” Journal of Validation Technology 17.1 (2011): 46.
• . Maier, Raina M. “Bacterial Growth.” Environmental Microbiology. Edition 3. 37-54. Elsevier Inc. Web. 2009.

Learn more >>

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