Aquila Biolabs CGQ User Manual
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Aquila Biolabs CGQ User Manual

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CGQ user guide
A handbook for cell density monitoring in
shake flask and bioreactor app lications
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Summary of Contents for Aquila Biolabs CGQ

  • Page 1 CGQ user guide A handbook for cell density monitoring in shake flask and bioreactor app lications...

  • Page 3: Table Of Contents

    Cable management within the shaker ................ 32 Installing the CGQ on a bioreactor ..................34 Installing the CGQuant software ..................35 On your own PC or laptop ....................35 On a PC or laptop delivered together with your CGQ system ....35...
  • Page 4 Challenges in shaken and stirred environments............46 Correlation between OD and the CGQ signal ............. 48 Parameters that influence the CGQ measurements ..........52 The CGQuant software ....................60 Introduction to CGQuant ......................60 CGQuant files and dependencies ..................61 The main window ..........................
  • Page 5 Exporting the data to .png files ....................104 Collecting calibration data .......................105 Using calibration data .........................113 Updating the sensor plate firmware .................115 Updating the base station firmware ..................118 The CGQ Monitor ........................122 Help section .........................123 Troubleshooting ..........................123 Solving connection issues ....................124 Solving data acquisition issues ..................126 Solving calibrator issues ......................128...

  • Page 7: Introduction And General Considerations

    In order to ensure that this user guide provides you all information you need during your work with the CGQ, we as aquila biolabs are reliant on your feedback. Do not hesitate to contact us to share your ideas regarding errors, missing information or incomprehensibilities so that we can improve this document and keep it up to date with your requirements.
  • Page 8 The latest manual is available on our homepage under www.aquila-biolabs.de/service/downloads. In case of any question that might arise during the work with CGQ and CGQuant, do not hesitate to contact us (Contact details on pp. 130). CGQ user guide revisions: Revision 0 22.09.2015...

  • Page 10: The Cgq System - An Overview

    The CGQ system – an overview The CGQ is an analytical laboratory device that is primarily intended to be used for noninvasive cell density monitoring in real-time, especially in shake flasks (CGQ) and bioreactor (CGQ BioR) applications. However, even completely different applications requiring the real-time analysis of particle concentrations can be addressed by the CGQ system.

  • Page 11: Absolute Maximum Ratings

    While it is not guaranteed to work at 75°C, the CGQ will do that in most expectable cases. Operating the CGQ above 50°C requires special darkening covers, which can withstand elevated temperatures and which are not...

  • Page 12: Cgquant Hardware Requirements

    CGQuant hardware requirements current dual core @ 2 GHz/core or better RAM / HDD ≥8 GB / ≥10 GB Operating system Microsoft Windows 7 or newer with .NET 4.7 Screen resolution 1920 x 1080 pixels For a good user experience, it is strongly recommended to run CGQuant on processors not older than from 2012.

  • Page 13: Recommended Operating Conditions

    Recommended operating conditions Temperature 10 – 50 °C (Ensure to let the sensor plates adjust to the operating temperature for 30 min.) Humidity (relative) 0 – 80% (non-condensing) Shake flask filling volume 10 – 15% optimal range 5 – 25% good range 2 –...
  • Page 14 In accordance with the above remarks, phototrophic cultivations provide constantly defined environmental light conditions, which can be efficiently compensated by the CGQ. The limit of environmental light compensation is the CGQ’s saturation intensity, which may be reached under certain phototrophic lighting conditions.

  • Page 15: Warnings

    CGQ and any other electric device around. Do not look into the beam of any of the CGQ sensor plate LEDs! Their emitted light is of high intensity and might damage your eye or retina. Wear protective eye wear.
  • Page 16 Do not spill liquids over any of the CGQ components. Especially the sensor plates might get damaged or the windows above the sensors might become dirty, thus negatively influencing the following measurements.

  • Page 17: Declarations And Certificates

    The aquila biolabs GmbH, Arnold-Sommerfeld-Ring 2, 52499 Baesweiler, Germany, herewith declares under its sole responsibility that all devices and equipment being part of the CGQ system and being manufactured by the aquila biolabs GmbH are in conformity with the Council Directives regarding Low Voltage Equipment Safety (2014/35/EU) →...
  • Page 18 CGQ base stations: CGQ-BS-8-BV Revisions: 1.00, 1.10 CGQ-BS-16-BV Revisions: 1.00, 1.10 Technical documentation is maintained at the aquila biolabs GmbH headquarter in Arnold-Sommerfeld-Ring 2, 52499 Baesweiler, Germany. Date of declaration: 14.09.2018 Name, position of the undersigned: Konrad Herzog, Managing Director...

  • Page 19: Weee Conformity

    Electronic components may contain various hazardous substances that could possibly exhibit negative impacts on your health and the environment. In order to avoid those effects aquila biolabs encourages you to make use of the appropriate local take-back and recycling systems for disposing electrical and electronic equipment.

  • Page 20: Fcc Compliance

    FCC compliance This is a Class A product. This device complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) this device may not cause harmful interference, and (2) this device must accept any interference received, including interference that may cause undesired operation.

  • Page 21: Installation, Disinfection And Quick Start

    Installing the CGQ components on your shaker Using screws Typically, the CGQ device is installed on a shaker tray that contains screwing threads. However, as the tray and screwing thread geometries vary between different shaker manufacturers, there are some things to keep in mind when planning to install the CGQ using screws.

  • Page 22: Using Sticky Pads

    Using sticky pads In order to install the CGQ base station on a sticky pad, just place it on the pad with its black bottom plate towards the shaking tray. Push down the base station to make sure that it is in good contact with the sticky pad. At higher shaking frequencies, it is recommended to request an alternative bottom plate from aquila biolabs, which allows to mount the base station on its backside.
  • Page 23 Always ensure that the base station is completely dry before powering it. In order to install the CGQ sticky plate adapter (ASTC) on a sticky plate, first remove the protection foil from the adapter bottom. Then place the adapter on the pad and push it down to make sure that it is in good contact with the sticky pad.
  • Page 24 To remove the CGQ from a sticky pad shaker, follow the recommendations of the sticky pad manufacturer. Prior to removal disconnect all cables. Generally, you can remove both, the base station and the sticky plate adapter by pulling constantly until the component detaches from the sticky pad.

  • Page 25: Mounting A Sensor Plate

    Mounting a sensor plate Take the sensor plate (disconnected from the base station) and hold it above the spring clamp with the sensor array facing you. Then pull the sensor plate cable from inside through the foremost clamp opening to outside and move the cable end to the left.
  • Page 26 Move down the sensor plate until the spring clamp metal clip touches the notch between USB-port and cable. Then move down the opposite side of the sensor plate accordingly. This will slightly expand the spring clamp. Now turn the sensor plate clockwise around an imaginary axis between the two...
  • Page 27 The sensor plate finally snaps into the spring clamp and is correctly mounted for further usage of the CGQ device. Be careful when mounting or removing sensor plates into or from spring clamps to avoid injuries such as contusions, cuts or bruises.
  • Page 28 It is strongly recommended to bend the single spring clamp clips inwards after mounting the CGQ sensor plate. Doing this will improve the shake flask fixation in the clamp, thus preventing unwanted detachment of shake flasks during operation. Make sure that the sensor plate cable is not pinched between sensor plate and spring clamp.

  • Page 29: Mounting The Shake Flask And The Flask Cover

    Mounting the shake flask and the flask cover Before starting a culture, please ensure that the utilized cultivation medium is roughly at the same temperature as the cultivation temperature in the shaker. If a cold liquid is used and put into a warmer shaker, condensation might occur at the flask bottom and over the sensor array.
  • Page 30 Mount the shake flask into the spring clamp as usual. Turn the shake flask around its vertical axis until neither the white labeling area nor potentially present baffles are located above the sensor array.
  • Page 31 Put the flask cover onto the shake flask and make sure that the sensor plate cable is going through the respective small opening at the bottom of the flask cover. Now you are ready with the hardware related works and you can start setting up the measurement in the CGQuant software.

  • Page 32: Cable Management Within The Shaker

    Figure 3. Use the cable clamp array from your CGQ package as shown in Figure 4 and resize it according to your requirements. It can either be screwed or glued to position.
  • Page 33 Figure 4: Cable clamp array customization (left), screw mounting hole (center) and sticky back (right). Use the cable clamp array to guide the cables as depicted below. Both cables must have enough slack between the point where they are held by the clamp and the base station.

  • Page 34: Installing The Cgq On A Bioreactor

    Installing the CGQ on a bioreactor The CGQ BioR is equipped with a thick layer of elastic foam and with a mounting belt. Both together allow you to install the CGQ BioR at almost any bioreactor in your lab. Initially you need to find a free glass or transparent plastics spot on your bioreactor.

  • Page 35: Installing The Cgquant Software

    CGQ system. On a PC or laptop delivered together with your CGQ system All PCs or laptops that are delivered by aquila biolabs GmbH in combination with at least one CGQ system have CGQuant preinstalled. There is no additional CGQuant installation required.

  • Page 36: Disinfection

    Ensure that all CGQ devices and cables are completely dry after disinfection, before you reconnect them to each other and to the power supply and USB.

  • Page 37: Quick Start Tutorial

    Quick start tutorial After having successfully installed the CGQ hardware on your shaker or bioreactor and the CGQuant software on your computer, you are now prepared to start using the CGQ system. 1. Connect the base station Connect the power supply elongation cable to the base station and to the power supply.
  • Page 38 Wrong positioning might cause measurement artifacts (pp. 52). Do not mount the CGQ BioR on top of marks or labels on the vessel, as this may negatively influence the data quality.
  • Page 39 USB. Double-click the CGQuant icon on your desktop to start the CGQuant software. 6. Wait for the connection between CGQuant and CGQ Upon startup, CGQuant will try to establish a connection with the CGQ base station. This procedure may take some seconds.
  • Page 40 7. Open a new experiment window Start a new experiment by clicking the “New Experiment” button. From the dropdown menu select the base station, which is connected to your sensor plate of interest. A new experiment window will appear together with an empty process information form.
  • Page 41 “Save and exit”- button in the right lower corner of the process information window. 11. Start the CGQ measurement Start the measurement by clicking the “Start”-button in the toolbar on the right. Upon measurement start, the icon of this button will turn into a “Pause”-icon, a current OD will...
  • Page 42 12. Stop the CGQ measurement As soon as your cultivation is finished, stop the CGQ experiment by clicking the “Stop”-button. If you decide to stop the measurement you cannot restart it in the same experiment window. If you just want to pause the measurement, click the pause button instead.

  • Page 43: Cgq's Basic Measurement Principles

    CGQ’s basic measurement principles Optical cell density measurements The CGQ’s technique for noninvasive cell density monitoring in shake flasks is based on the principle of light scattering. Basically, each measurement consists of a sequence of three major steps. (1) Light is irradiated by a LED into the fermentation broth through the transparent flask bottom.
  • Page 44 This is essential for cell density monitoring at bioreactors, which cannot be covered and darkened as easily as shake flasks. Furthermore, the CGQ is now applicable for phototrophic cultivations, as long as the photosynthetic light is not saturating the CGQ sensors.
  • Page 45 Position 1: CGQ & BioR – 521 nm Deep Blue True Yellow Amber Hyper Far Red IR I IR II Blue Green Figure 9: Standard LED colors at the different CGQ measurement positions and available LED colors for wavelength customization of CGQ sensor plates.

  • Page 46: Challenges In Shaken And Stirred Environments

    CGQ sensor-plate can adaptively measure the backscattering intensity at different radial positions, thus accounting for a broad variety of liquid distributions in shake flask fermentations. Figure 10: Liquid distribution and CGQ’s cell density monitoring principle in continuously shaken systems.
  • Page 47 The CGQ does not treat the moving-liquid-induced backscattering signal fluctuations as noise, but as a source of valuable information. For determining one cell density value, the CGQ sensor plate collects about one million of single backscattering intensities per second; thereby creating a series of data points that combines information regarding cell density and liquid distribution on a microseconds-resolution.

  • Page 48: Correlation Between Od 600 And The Cgq Signal

    Lambert-Beer’s law. As described above, the CGQ measures backscattered light intensities. While scattering signals can be used to measure particle concentrations over several orders of magnitude, it must be noticed that there is no physical law to describe...
  • Page 49 Figure 12 depicts exemplarily for E. coli on LB medium the correlation of CGQ signal and OD , described using the above complete sigmoid-linear correlation and using a simple four-parametric...
  • Page 50 , the simple exponential correlation can be used with some limitations regarding accuracy. The most important thing about correlation of CGQ signals and real cell densities is, to keep in mind that the parameters of the correlating functions depend on some cultivation system properties influencing the scattering behavior of the fermentation broth, such as cell size, cell shape, medium composition, flask type and material, etc.
  • Page 51 Without any calibration and known data pairs of OD and CGQ signal, you can get relative cell densities as a sufficiently accurate measure of cell density that is proportional to the real cell density, simply by calculating the natural logarithm of the measured CGQ signal.

  • Page 52: Parameters That Influence The Cgq Measurements

    However, this sensitivity means that also changes in the optical environment will be detected by the CGQ. With the new environmental light compensating CGQ and CGQ BioR plates it is possible to monitor growth without the use of shake flask covers. To provide a reproducible optical...
  • Page 53 100% cumulative distribution -12% deviation from average Figure 14: Exemplary CGQ signal differences of a set of 24 shake flasks (aluminum cap, 250 ml), measured at OD = 4.2, 250 rpm, 25mm shaking diameter, 25 ml filling volume.
  • Page 54 As long as the flasks’ positions or orientations are not changed during the cultivation, these individual flask differences will (in most cases) only cause offsets in the CGQ signal, which can be removed using the CGQuant software (refer to pp. 82). Flask position/orientation changes during cultivation have been described above.
  • Page 55 Generally, this is not problematic for the cell density measurement; however different materials might have different effects on the CGQ signal. As long as the material is at least partially transparent for visible light, the CGQ measurement principle will work, but care must be taken about some limitations.
  • Page 56 CGQ signal (refer to pp. 46). The CGQ’s data analysis algorithms are made and continuously improved (via firmware updates) to eliminate as many...
  • Page 57 not assume that the data from a 2000 ml flask, filled with 400 ml of broth and shaken at 150 rpm can be compared directly to a 250 ml flask, filled with 12.5 ml of broth and shaken at 350 rpm. If you want to do direct and especially quantitative comparisons, calibration measurements are required as described at pp.
  • Page 58 Figure 15: Clamp symmetry and deformation. (left - correct, right - wrong) Positioning differences due to differently deformed clamps may introduce considerable differences in the CGQ’s scattering signal and may therefor negatively influence the flask-to-flask reproducibility and comparability of similar...
  • Page 59 CGQ measurements. In order to avoid this effect, it is strongly recommended to check the symmetry of all clamps each time before mounting a shake flask for CGQ measurements. Always make sure that clamps are centrosymmetrically aligned and not deformed to avoid flask mispositioning and...

  • Page 60: The Cgquant Software

    The parallelization of the CGQ device is continued in CGQuant’s graphical user interface, as shown exemplarily in Figure 16. Detailed descriptions of each functionality can be found in the following chapters.

  • Page 61: Cgquant Files And Dependencies

    CGQuant uses specific file extensions for saving different data types. Measurement data are written to .cgqfx-files. Old cgqf files are still supported for reading. Calibration data are saved as .cgqc-files. CGQ event descriptions are stored as .cgqed-files. SMTP-client configurations are stored as .smtpconfig- files.
  • Page 62 CGQuant up to version 6.3 saved data into cgqf-files. Starting from CGQuant 7.0, cgqf-files are only supported for reading. Essentially upon opening a cgqf- file, CGQuant converts it to a .cgqfx-file and saves the old original file into a separate folder on the same directory level as the old file. Environmental data, cannot be shown for cgqf-files.
  • Page 63 CGQuant performs automated status and error logging on CGQuant errors as well as on CGQ errors transmitted via USB. The log files are available under …\CurrentUser\Documents\CGQuant\data\logs and should be always sent together with bug descriptions or other support requests.

  • Page 64: The Main Window

    Click the base station where your target sensor New experiment plate is connected and a new experiment is created. Click the CGQ Experiment Configurator to create a batch of experiments in parallel. Click this button to view data from View data previous measurements.
  • Page 65 Click the base station where you want to stop all connected sensor plates with an associated running experiment. Look at this label to check, whether a CGQ base station is connected (lower icon, glowing) or disconnected (upper icon, not Connection status glowing).

  • Page 66: The Overview Window

    The CGQuant overview window opens in the background. It displays an overview chart of all running measurements and a list of all connected base stations with their associated CGQ sensor plates and base station inputs together with their current states and unique identification numbers.

  • Page 67: The Experiment Window

    The experiment window The experiment window is the core component of data visualization and control of the CGQ device. Running experiment windows are displayed in the main tab of their associated base station. Experiment windows for files collected in the past are displayed in the main tab “Viewer”...
  • Page 68 Left-click this button to switch between backscatter data and calculated OD-values. Right-click this button to specify the initial offline OD value of your culture and to select or exchange calibration files as Offline OD required for calibrated data collection. Additional offline OD values can be saved here, to refine the accuracy of the applied calibration file or to derive a totally new calibration file from a live measurement.
  • Page 69 Click this button to calculate the growth rates of all experiments shown in the central chart. Click this button again to remove the growth curves from the chart. Growth rates If the button is switched on, the growth curve of the currently running experiment will be recalculated each time a new data point is added to the chart.

  • Page 70: The Process Information Window

    The process information window All bioprocess related information can be written into the fully customizable CGQuant lab journal via the process information window. Upon starting a new experiment, the process information window is opened automatically. You have to select at least the input number, where your target sensor plate is connected to the base station.
  • Page 71 Lab journal fields the according information, either as words or as numbers. Fields that have been set to be required have a grey background. Use this field to set the desired CGQ Sampling interval measurement interval (in seconds) for the specified experiment.
  • Page 72 Click the cancel button to close the process information window. If you have started a new experiment and click cancel Cancel in the first appearing process information window, complete experiment window will be closed. Required By clicking this button, you can jump to the information next required field.
  • Page 73 Figure 21: Customizing the CGQuant process information window. You may also modify existing entries after clicking them in the tree-view. Existing entries may be removed by clicking them, followed by pressing the “Delete” button on your keyboard. Furthermore, you can set selected process information fields or groups to be mandatory by checking the respective checkboxes.

  • Page 74: The Cgq Experiment Configurator

    The CGQ Experiment Configurator A batch of experiments can be created and configured in parallel using the CGQ Experiment Configurator. Figure 22: The CGQ Experiment Configurator. Select the sensor plates to be used in the list at the right side by checking the respective checkboxes.
  • Page 75 start all the generated experiments using the start all button for each base station according to your sensor plate selection. Figure 23: Parallel documentation of properties for all selected experiments (sensor plates).

  • Page 76: Controlling The Measurement

    “Start-all” button. The shaking state of each CGQ sensor plate for shake flasks (CGQ-SP-F) is monitored. This allows for automated measurement control, which can be enabled in the preferences window as depicted in Figure 24.
  • Page 77 Figure 24: Measurement control options and default settings. Furthermore, you can set the default general sampling period (time between two data acquisitions) and the number of visible points per chart. Both settings influence the performance of CGQuant and may be changed according to your requirements.
  • Page 78 Parameters, which directly influence the data acquisition, must be set before starting an experiment (e.g. the automated measurement control). Earlier started experiments will not be influenced by such reparameterizations unless their sensor plate is reset (e.g. by power cycling). Running measurements can be stopped by clicking the stop buttons, or by closing the experiment window.

  • Page 79: Annotations

    Annotations CGQuant features an annotation tool allowing you to add comments or data files to running or previously recorded measurements. Annotation points are displayed in a separate chart, which can be toggled using the annotation-button in the experiment window’s right toolbar. Available annotations are displayed at the bottom of the chart and can be opened with a left-click.
  • Page 80 file-type icon. Files or images that have been attached to an annotation are copied by CGQuant and saved into the .cgqfx file. This allows you to keep all your experiment related data together in one file. Figure 27: Creating an annotation CGQuant automatically creates an initial annotation (“Experiment generated”) to store general information such as hardware IDs and measurement modes.

  • Page 81: Comparing Multiple Measurements

    Comparing multiple measurements You can compare multiple measurements by opening them in the same window. This can be achieved either by clicking the respective button in the experiment window or CGQuant main toolbar or by dragging and dropping .cgqfx-files into an existing experiment window (data will open in that window) or into CGQuant (data will open in a new experiment window).

  • Page 82: Using The Central Chart

    Using the central chart You can zoom into the central chart by scrolling the mouse wheel or using the cursor. Left-click into the chart, hold the left mouse button and move the mouse. A grey area will appear, indicating the region to be zoomed. To reset the zooming, click the small button with the circle icon at the end of the scroll- bars or use the chart context menu.
  • Page 83 Figure 30: CGQuant cursor values. In order to get the maximally available chart area, you can hide the cursor data and the legend by clicking the respective buttons below the chart.

  • Page 84: Data Post-Processing - Curve Adjustment

    Data post-processing – Curve adjustment By clicking the “Data post-processing”-button in the right toolbar of the experiment window, you get access to the data post-processing options in CGQuant. These options are accessible from different tabs with the first one being the basic curve adjustment tab. Figure 31: Curve adjustment in the CGQuant data post-processing window.
  • Page 85 Clicking this button will reset all backscatter Reset auto-offset data in the current chart to their original values. Clicking this button will reset all backscatter Reset all offsets data and all data times in the current chart to their original values. This field sets the start time of the data Offset start time range being used for offset calculation.
  • Page 86 compensation. All offsets will be exported to .xlsx-files as well. This field sets the start time of the data range to be cut away from all loaded data Cut start time sets. To cut data from the beginning, use a value lower than the first time value in the chart.
  • Page 87 Figure 33: CGQuant data before and after backscatter denoising.

  • Page 88: Data Post-Processing - Growth Rate Calculation

    Data post-processing – Growth rate calculation Growth rates indicate, where the cultivated organisms grow faster or slower and where metabolic changes occur. They can furthermore help to compare different experiments more adequately. CGQuant calculates growth rates in the background and displays them, if you enable the “Growth rate”-button in the experiment window toolbar (refer to pp.
  • Page 89 µ with being the average exponential growth rate according to equation {5} over the time interval from CGQuant-calculated growth rates can be smoothed in the data post-processing window as described above. Basically, the smoothing is carried out by increasing the time interval from .

  • Page 90: Data Post-Processing - Growth Event Detection

    Data post-processing – Growth event detection Growth events can be defined and monitored from the “Growth event detection” tab of the data post-processing window. This allows you to automatically get notifications as soon as predefined thresholds, shapes or threshold and shape together are detected within the monitored data set. Figure 35: Definition of growth events in the data post-processing window.
  • Page 91 Click here to load a .cgqfx-file as prototype Load data data set for the definition of events. The loaded prototype data set is shown in Prototype chart this chart. The selected data type is evaluated for the Event data type detection of the associated event.
  • Page 92 The shape itself is defined by the prototype data set. Using the uncertainty parameter, you can relax the shape definition to allow event detections in different growth curves. Additionally, you can set a minimum similarity between the prototype shape and the monitored data set. The prototype shape is normalized to its maximum and minimum values to make the shape scale invariant.
  • Page 93 Click this button to start the event detection. In running measurements, the event detection is then automatically Detect events performed whenever new data are available. In previously recorded data sets, the event detection is performed only once per click. The detected events are displayed together with the CGQuant annotations in the experiment window chart, whenever the “Annotation”-button is enabled.
  • Page 94 In order to configure CGQuant as SMTP client, open the CGQuant preferences window and navigate to the “Notifications” tab as depicted in Figure 37. Enable the email notifications and provide an email address, from which the notifications will be sent. Then specify a sender name, e.g. “CGQuant”, which will be displayed later in your email program.
  • Page 95 Figure 37: Configuring an SMTP client to allow CGQuant the sending of email notifications. Finally activate and save the SMTP configuration by clicking the “Set and save settings” button. It is recommended to test the SMTP configuration by sending a test email. Be aware that your email credentials may be accessible by any user having access to the PC running CGQuant with your user account.

  • Page 96: Data Post-Processing - Consensus Data

    Data post-processing – Consensus data Good scientific practice requires the collection of replicates as well as their statistical analysis. CGQuant supports you with this task by providing you with tools for the generation and analysis of consensus data. Replicate data sets having been collected under similar experimental conditions and technical setups are thereby merged into a single .cgqcd file and represented as an average growth curve together with user-selectable confidence intervals.
  • Page 97 Before generating the consensus curve, you can customize this process. According to the chosen consensus time interval, all data sets will be unified with respect to their measurement time points, resulting in equal point-to-point distances for all relevant replicates. During the unifying process for each replicate, whenever there is no data point exactly on the multiples of the chosen interval, the unified data point will be calculated by linear interpolation between the two closest measurement points with respect to the measurement times.
  • Page 98 Figure 39: Triplicate and consensus data set. In order to analyze and compare consensus data sets, open the post-processing window and navigate to the tab “Curve analysis” (Figure 41). There you can select the confidence intervals being used for data visualization (Figure 40). Figure 40: The same triplicate consensus data set with standard deviation (left) and 95% confidence interval (right).
  • Page 99 them graphically by selecting one of the following items in the analysis result dropdown menu: Absolute measurement uncertainty Relative measurement uncertainty (compared to average) Absolute difference to reference data Relative difference to reference data (compared to reference data average) Probability for each data set to be different from the reference according to a t-Test Probability for each data set to be different from the reference according to a Welch-Test...

  • Page 100: Exporting The Data To .Xlsx Files

    Exporting the data to .xlsx files Data from an experiment window can be exported to an .xlsx-file using the xlsx-export button in the right toolbar (refer to Figure 19 on page 67). Clicking this button will export all data shown in the chart into one file, including the cell density curves, process information, growth rates, calibration data, environmental data, annotations and offline OD-values.
  • Page 101 A running export process is indicated by a blinking .xlsx-export button. After successfully finishing the export, the blinking stops and the generated file is automatically opened with your default program for .xlsx-extension files (e.g. Microsoft Excel or LibreOffice Calc). Finally all the data are redrawn in the chart with the original data density settings.

  • Page 102: Exporting The Data To .Pdf Files

    Exporting the data to .pdf files Clicking the .pdf-button allows you to build a PDF report either from a single experiment or from all loaded experiments in the current experiment window. You can customize the report using the CGQuant PDF Report Builder window as depicted below.
  • Page 103 The PDF document to be generated can also be protected against changes. Enabling this option will generate a random key composed of 64 characters, including letters, capital letters, numbers and additional characters. This key is then used to encrypt the PDF file using RC4 (128-bit). Use the “Reload preview”...

  • Page 104: Exporting The Data To .Png Files

    Exporting the data to .png files Clicking the .png-button allows you to export data from the chart to a PNG image file. You can customize the image using the CGQuant Chart Export window as depicted in Figure 44. The customization process includes: Axes spans, intervals and titles Chart title Data types, marker styles and size...

  • Page 105: Collecting Calibration Data

    Collecting calibration data Calibration is required in all applications where true cell density values, typically or cell dry weight (CDW), are needed and the native CGQ derived backscatter data are not sufficient to meet your requirements. The CGQuant software contains an elaborate calibration module (the CGQuant calibrator) allowing you to get accurate calibration curves quickly, intuitively and with minimal hands-on time.
  • Page 106 A shaker prepared to provide the shaking conditions for your calibration. Other stuff, such as CGQ darkening covers, cuvettes, pipettes, etc. When performing calibrations in a bioreactor, the same steps apply as for shake flasks; just think of your bioreactor being a shake flask. Furthermore, all shaking related issues may be ignored or replaced by stirring speed instead of shaking speed.
  • Page 107 You can change it with respect to your special requirements, but at least 8 points need to be collected, otherwise the reliability of your calibration would be decreased, resulting in questionable cell density readings during normal CGQ measurements on the basis of such a weak calibration.
  • Page 108 To calculate or recalculate the dilution series after having filled the table, just click the “Next step…”-button. An exemplary calibrator configuration with automatically generated dilution series is shown in Figure 47. The dilution series list is accessible throughout the whole calibration procedure. As for any CGQ...
  • Page 109 measurement, the measurement mode, sensitivity mode for bioreactors and the measurement position must be defined. These parameters are initially loaded according to their defaults from the CGQuant preferences window. The next step is the selection of the base station, sensor plates and filenames to be used (exemplarily shown in Figure 48).
  • Page 110 After having finished the sensor plate and filename selection, click the “Next step…”-button to proceed with the calibration measurements (see Figure 49 and Figure 50). Figure 49: CGQuant calibrator measurement step overview. Each calibration point is calculated from several measurements. You can adjust the default value of 10 according to your requirements.
  • Page 111 You can skip remaining calibration cycles or add further calibration cycles by clicking the respective buttons. If you want to test the calibration tool without doing real measurements, you can check the “Calibrator evaluation enabled”-box. An exemplary set of calibration data will be used instead of real calibration data. Figure 50: CGQuant calibrator measurement progress.
  • Page 112 Figure 51: CGQuant calibrator curve fitting. Start the fitting procedure by clicking the “Start fitting”-button. CGQuant will automatically perform the curve fitting until either the convergence limit or an internal iteration limit is reached. CGQuant will always display the currently best fitting solution in the chart accompanied by its current score.

  • Page 113: Using Calibration Data

    Using calibration data The calibration files can be used to calculate real cell densities from the CGQ backscatter data. Starting from CGQuant 7, all newly generated cgqfx-files are equipped with a default calibration file. This allows you, in combination with at least two offline values, to estimate your offline cell densities in real-time.
  • Page 114 Figure 52: CGQuant offline support data window. Offline data can be added by clicking the “Add offline data”-button. CGQuant will then automatically fill the time entry with the latest experiment time. Alternatively, you can change that time according to your requirements. CGQuant will automatically search for the backscatter intensity at that time point.

  • Page 115: Updating The Sensor Plate Firmware

    Updating the sensor plate firmware From CGQuant 8 on, all CGQ sensor plates sold after 01.10.2018 can be updated in place via the base station. Updates over USB are not supported for these new sensor plates. Stop the measurement of all sensor plates to be updated.
  • Page 116 Figure 54 and Figure 55. If there is no observable progress with the firmware update, please power-cycle the complete CGQ system by disconnection the base station from the power supply, waiting for some seconds and reconnecting it again.
  • Page 117 Figure 55: CGQuant overview window after successful firmware update. As soon as a successful firmware update is reported in the “Preferences” window, your sensor plate is updated. Finally, close the “preferences” window by clicking the “Cancel” button. Remember that CGQuant 8 usually requires both, sensor plates and base station, to be updated with the respective firmware versions.

  • Page 118: Updating The Base Station Firmware

    CGQuant 8 on your computer and that you have the base station USB cable, the base station power supply and the power elongation cable available. Now you can start to update the CGQ base stations as described in the following.
  • Page 119 In the “Preferences” window, navigate to the “Update and Packages” tab. Click the “Select firmware binary” button, navigate to the folder “…\CGQuant\firmware binaries\CGQ base stations” and load a suitable .cgqfi file. Plug in the base station power supply into the right port of the base station top panel and connect power elongation cable and power supply.
  • Page 120 Figure 57: CGQuant “Preferences” window during firmware update. Figure 58: CGQuant “Preferences” window after successful firmware update.
  • Page 121 11. As soon as a successful firmware update is reported in the “Preferences” window, your base station is updated and you can disconnect it. 12. In order to update additional base stations with the same firmware binary, proceed with step 8 and iterate through the subsequent steps until all your base stations are updated.

  • Page 122: The Cgq Monitor

    The CGQ Monitor can be closed via its content menu, which appears upon right-clicking the CGQ Monitor tray icon. It is strongly recommended to not close CGQ Monitor during CGQuant operation.

  • Page 123: Help Section

    Help section Troubleshooting The following chapter provides hints to solve problems that might arise during the usage of CGQ and CGQuant. Please select one of the issue types and go along the trouble shooting flow charts. • Solving connection issues •...

  • Page 124: Solving Connection Issues

    Connect the base station to the connected to the power supply. power supply? Is the green base Contact aquila biolabs’ support (1). station LED glowing? Is the base station Connect the base station to the connected to the PC PC via USB.
  • Page 125 Update your base station and sensor plates to sensor plates according to pp. firmware revision 3? Restart your computer, Start disconnect the CGQ power (pp. 124) supply and go to Start Did the CGQ driver Execute the CGQuant 8.0 install successfully...

  • Page 126: Solving Data Acquisition Issues

    Solving data acquisition issues Start a new experiment in CGQuant connected to the CGQ. Is a sensor plate Connect a sensor plate to the connected to the selected port or restart the base station port experiment with another port. selected in CGQuant? Disconnect the sensor plate from <3x...
  • Page 127 Uncheck the automated measurement control checkbox (pp. 76) in the CGQuant preferences and save the settings. Then stop and close your experiment. Contact aquila biolabs’ support (7). Contact aquila biolabs’ support (6). Start a new experiment in CGQuant connected to the CGQ.

  • Page 128: Solving Calibrator Issues

    Solving calibrator issues Start a new calibration with CGQuant connected to a CGQ. Is the usage of stock Increase calibration point count solutions with OD or volume to be removed/added >100 required? per point. Then click “Next step”. Are all required Disconnect the responsible <3x...

  • Page 129: Solving Data Interpretation Issues

    (pp. 13), the sensor plate (pp. 25) and flask (pp. 29) mounting guidelines and the beginning? the CGQ experiment checklist (pp. 136). Contact aquila biolabs’ support (11). Do you still observe backscatter drops in...

  • Page 130: Contact

    Contact aquila biolabs GmbH Arnold-Sommerfeld-Ring 2 52499 Baesweiler Germany www.aquila-biolabs.de telephone: +491632922615 email: info@aquila-biolabs.de...

  • Page 131: Notes

    Notes...

  • Page 136: Cgq Experiment Checklist

    CGQ experiment checklist Is the CGQ base station connected to the power supply (green LED glowing)? Is the CGQ base station connected to PC and CGQuant via USB (connection label glowing)? Are the CGQ USB and power cables routed with sufficient play...

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