J.Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration

1 目的

液体培地中の大腸菌の増殖動態を定量化すること（例としてLB培地を使用）：増殖曲線、増殖速度、および加速度等の主要な増殖動態パラメータを分析し取得する。

2 序論

対象とする複数の大腸菌（E. col）サンプルを採り、それらを無菌のLB液体培地に接種し接種液とします。次に、各接種液をディスポーザブルの試験管に入れサンプルとします。これらの試験管をCellStatz微生物増殖アナライザーの各検出チャンネルに挿入します。CellStatzは、各E. colサンプルの増殖曲線を自動的にリアルタイムで測定し、複数のサンプルの増殖曲線を重ね合わせて表示します。増殖曲線が決定されたらCellStatzシステムの自動データ処理機能を使って、対象のE. coliサンプルの増殖速度や加速度曲線、および主要な増殖動態パラメータが取得できます。一般的に、陽性コントロールとして既知の濃度（たとえば106 CFU/mL）のE. coli品質管理株で接種されたLB液体培地が使用され、無菌のLB液体培地が陰性コントロールとして使用されます。 この電子的モニタリングベースの方法は信頼性が高く、効率的であり、大腸菌の増殖動態の研究や関連するアプリケーションの開発に適しています。

3 手順

3.1 サンプルの準備

1) LB液体培地を滅菌し、個別の容器に分注します

2) 活性化した対象のE. coliサンプル（サンプルA、B、C、D、E、およびF）をLB液体培地に接種して接種液A、B、C、D、E、およびFとします。

3) 接種液A、B、C、D、E、およびFから各1.8 mLを取り、別々の無菌の試験管に入れ、それぞれサンプルチューブA、B、C、D、E、およびFとラベルを付けます。

4) According to the experimental requirements of the researchers, the sample tube can be sealed with a sterile rubber stopper or a sterile 0.22 µm/13 cm disposable filter. In this case, the use of a disposable filter for sealing is illustrated.

5) The positive control sample O (inoculated with 10^6 CFU/mL E. coli quality control strain) and negative control sample N are prepared using the same method and steps as described above, and are pipetted into tubes (tubes O and N) for future use.

Note: During the sample preparation process, it is essential to avoid contamination from surrounding environmental microorganisms. Before loading onto the machine, the sample tubes should be stored at 4 ℃ and for no longer than 1 hour.

3.2 Instrument Preparation

1) Turn on the power to the CellStatz electronic microbial growth analyser and the accompanying computer. The instrument will complete a self-check in approximately 10 seconds.

2) Double-click the shortcut for the CellStatz software on the computer to open the working interface.

3) Set the target temperature for bacterial culture (using 36℃ as an example), and then click "Run". When the ambient temperature is around 25℃, the temperature inside the detection chamber of the analyser will stabilize at 36℃ after approximately 30 minutes. For more details, refer to the instrument manual.

4) In the "Settings" menu of the CellStatz working interface, enter information such as stimulation frequency, stimulation level, working channel, signal acquisition frequency, signal acquisition times, and the file name for saving experimental data. The stimulation frequency, stimulation level, working channel, signal acquisition frequency, and signal acquisition times can also be quickly entered by calling template data. Here, taking commonly used measurement parameters as an example, the stimulation frequency, stimulation level, working channel, signal acquisition frequency, and signal acquisition times are set to 200 MHz, 90%, 1-8 (there are only 8 sample tubes to be inserted into the detection channel of the instrument in this experiment), 60 s, and 1200 times, respectively. The file name for saving the experimental data is "E. coli-101". For more details, refer to the instrument manual.

5) After setting the measurement parameters, click the "OK" button to start recording.

3.3 Measurement of Growth Curves

1) Once the temperature within the detection chamber of the CellStatz electronic microbial growth analyzer stabilizes at 36℃, the prepared sample tubes (A, B, C, D, E, and F) along with the positive control tube (O) and negative control tube (N) are individually inserted into a detection channel of the analyzer. This process is performed sequentially, such that each tube is inserted into channels 1-8 in a consecutive manner. Note: This operation should be performed as expeditiously as possible, ideally within 1 minute.

2) Upon clicking the "Start" button on the CellStatz work interface, the system initiates the reading of the electrical conductivity, expressed in apparent voltage values, of the solution in each detection tube at intervals of 60 seconds. Subsequently, the system plots these values on a graph with time as the abscissa and the conductivity as the ordinate, generating the primary growth curves 1 through 8. These curves are displayed in real-time in their respective display fields for each working channel. Curves 1 through 6 pertain to the growth curves of sample tubes A through F, respectively, cultivated in LB medium. In contrast, curves 7 and 8 represent the growth curves of the positive control tube O and the negative control tube N, respectively.

3) The experimental operator has the option to select multiple growth curves of interest from within the "Summary" functional area of the work interface. These curves can be viewed concurrently and overlaid upon each other in the "Summary" display field. The user also has the option to visualize the curves (groups) in offset mode by clicking the corresponding option. Additionally, it is possible to establish any of the curves 1 through 8 as a reference curve and view the curves (groups) in correction mode by selecting the "Delta Mode" option. Note: As a general practice, the growth curve corresponding to the negative sample N, i.e., curve 8, is typically used as the reference curve. For more details, refer to the instrument manual.

4) Unless manually interrupted, the CellStatz detection system will terminate the measurement procedure automatically upon reaching the specified signal acquisition limit of 1200 times, after which the data will be saved to the designated "E. coli-101" file. This file contains all eight curves, namely, curves 1 through 8. Alternatively, the experimental operator may elect to save the data to the "E. coli-101" file by clicking the "Save" button during the measurement process. Subsequently, the "E. coli-101" file will also comprise all eight curves, curves 1 through 8. Note: Clicking "Save" will terminate the measurement operation. For more details, refer to the instrument manual.

3.4 Obtain the growth rate curve, growth acceleration curve, and kinetic parameters

1) By utilizing the "Data Management" function bar located on the CellStatz interface, an individual can selectively choose one or more data sets for subsequent analysis. For example, if one were to choose curves 1 through 4 and curve 8, which correspond to sample A, sample B, sample C, sample D, and negative control N, respectively, these five curves would become activated for further analysis.

2) The experimenter can select one or multiple data sets to be analyzed through the "Data Management" function in the CellStatz user interface. For example, selecting the curves 1, 2, 3, 4, and 8, corresponding to samples A, B, C, D, and negative control N, respectively, activates these five curves.

Using the polynomial algorithm, a degree between 2 and 20 is inputted, and by clicking the "Curve Fitting" button, smooth graphical representations of these five curves are obtained. The experimenter can select the negative control (i.e., curve 8) as a reference from the drop-down menu, in order to obtain the calibrated smooth curves corresponding to samples A, B, C, and D.

3) After selecting the polynomial algorithm and inputting an order between 2 and 20, the user can obtain the smoothed graphs of the five selected curves, including samples A, B, C, D, and negative control N, by clicking on the "Curve Fitting" button. To obtain the corrected smoothed curves of samples A, B, C, and D corresponding to the negative control N, the user can choose the negative control N as the reference in the drop-down menu. The corrected growth rate curves of the four samples can be obtained by clicking the "First Derivative" button. The peak value of the bell-shaped growth rate curve corresponds to the maximum growth rate of E. coli. To facilitate comparison and analysis, the user can select multiple curves to be overlaid and displayed in the same display panel by choosing the growth rate curves 1, 2, and 3, for example. By observing the time (horizontal axis) at which their peak values appear, the corresponding initial inoculum concentrations of the samples can be preliminarily estimated. Generally, the smaller the value on the horizontal axis, the higher the concentration. The peak height (vertical axis) can be used to preliminarily determine the differences in maximum growth. Generally, the larger the value on the vertical axis, the greater the maximum growth. By clicking the "Peak" button, the exact coordinates of the peak values on the curve can be displayed. Combining the horizontal axis values of the working curve (the establishment of the working curve is described in the "Automated Quantitative Determination of E. coli" section) can be used to calculate the initial concentration of E. coli in samples A, B, and C.

4) In order to obtain the growth acceleration curves for the four selected growth rate curves mentioned above, the user can perform second-order derivative processing. For example, if growth rate curves 1, 2, and 3 are selected, the user can click on the "Second Derivative" button to generate three curves, each consisting of a peak and a trough. The abscissa value of the peak corresponds to the moment when the E. coli growth acceleration is maximal, while the abscissa value of the trough corresponds to the moment when the E. coli growth deceleration is maximal. By clicking on the "Peak" and "Trough" buttons, the user can read the specific data of each peak and trough value on the growth acceleration curve. Note: For more details, refer to the instrument manual.