Loligo Systems MicroResp Microplate System User Guide

Loligo Systems MicroResp Microplate System

Product Information

• The product is called MicroRespTM and it is a software used for data analysis in experiments. It requires a license dongle for full software access. The software can run in demo mode without the license dongle, which allows for data analysis but not the use of real hardware. The software is compatible with a microplate system that includes readers and sensors.
• The product also includes optional accessories such as a flow-through water bath for temperature control and an incubator for maintaining the temperature of the readers. It can be used in both aquatic and air environments.

Product Usage Instructions

1. Download the latest version of MicroRespTM from the website: www.loligosystems.com/downloads. Follow the on-screen instructions and restart the PC.
2. Insert the blue Keylok license dongle in a USB port on the PC to unlock the full software.
3. If the license dongle is not inserted, MicroRespTM will run in demo mode. In demo mode, virtual hardware is used to simulate data during an experiment. Real hardware cannot be used, but data files can still be analyzed.
4. Connect the power supply to the splitter, and connect the first reader to the splitter. Connect additional readers to the previous readers.
5. Prepare the sensor spots by placing the white plastic guide onto the reader and placing the microplate glass plate inside the guide. For aquatic use, hydrate the sensor spots using water of the same type and temperature as during trials. For use in air, acclimate the reader, guide, and glass plate at the trial temperature for 30-45 minutes before use.
6. If temperature control is required, use the optional flow-through water bath. The water bath should sit directly on top of the reader, with only the sealed glass plate submerged in the water bath. A refrigerated circulating bath can be used to pass water through the acrylic water bath.
7. When running an experiment, start MicroRespTM and click on “Experiment” in the main menu to detect the reader(s). Open the “Settings” tab to change setup settings and configure calibration data.
8. If measuring in gas/air, go to “Experiment > Settings > General” and enable “Is air breather”. Adjust the humidity value if needed. This setting indicates that no water is present in the glass plate wells.
9. To set the number of organisms per well, go to “Experiment > Settings > Organisms per well”. If “Set individual” is set to “No”, all wells must have the same number of organisms. Enter the number in the “Number” field. To allow for a custom number of animals per well, change “Set individual” to “Yes” and enter the specific number in each well.
10. To normalize data, go to “Experiment > Settings > Calibration”. Next to the “Normalization factor” field, click to normalize oxygen data from all the sensor spots. This is useful for setting all air saturation values to 100% (by multiplying with a factor), ensuring each oxygen curve has the same starting point. Normalization should be performed after setting calibration points, and when there is 100% air saturation.

For additional help, you can watch the MicroRespTM video tutorial at: www.youtube.com/LoligoSystems.

FIRST TIME USE

1. Download the latest version of MicroResp™ from our website: www.loligosystems.com/downloads Follow the instructions on the screen and then restart the PC.
2. Insert the blue Keylok license dongle (2) in a USB port on the PC to unlock the full software. MicroResp™ will run in demo mode, if the license dongle is not inserted. Running MicroResp™ in demo mode will enable virtual hardware that simulates data during an experiment (i.e., you cannot use real hardware), but you can still analyze data files.
3. Connect power supply (A) to splitter (B), splitter to PC, and connect the first reader to the splitter (3). Connect additional reader(s) to the previous reader(s).

SETUP

1. Preparation of sensor spots. Place the white plastic guide onto the reader and place the microplate glass plate inside the guide (4). For aquatic use, hydrate the sensor spots for 30-45 min. using water of the same type and temperature as during trials. For use in air, acclimate the reader, guide, and glass plate for 30-45 min. at the trial temperature before use.
   • Temperature control
2. Water bath: Use our optional flow-through water bath (5) to control sample temperature. The water bath sits directly on top of the reader, and only the sealed glass plate (and silicone pad and compression block, if in use) must be submerged in the water bath. Use a refrigerated circulating bath to pass water through the acrylic water bath.
   • Incubator: Keep only the readers (and guide and glass plate), not the splitter, inside the incubator. Use the flat, black piece on the splitter-to-reader cable for the incubator door.
   • Climate controlled room: The entire microplate system can be placed in a climate controlled room for sample temperature control.

RUNNING AN EXPERIMENT

• Start MicroResp™ and click Experiment in the main menu to detect the reader(s). Open the Settings tab to change setup settings and to configure and perform (or verify) the calibration data (see step 16).
• Need more help?: Watch the MicroResp™ video tutorial:
• Measurement in gas/air. Experiment > Settings > General. Enable Is air breather, and change the humidity value, if needed. MicroResp™ will now assume you are measuring in air (i.e., that you have no water in the glass plate wells).
• Number of animals. Experiment > Settings > Organisms per well (8). If Set individual is set to No, all wells must have the same number of organisms, and the number is entered in the Number field. Change Set individual to Yes to allow for a custom number of animals per well, and enter the specific number in each of the wells below.
• Normalizing   data. Experiment > Settings > Calibration. Next to the Normalization factor field, click to normalize oxygen data from all the sensor spots, e.g. to 100 % air.sat. (9). Normalization will set all air sat. values to 100 % (by multiplying with a factor), so that each oxygen curve has the same starting point. If the oxygen sensor calibration is good, normalizing will not change the % air sat. values much (< +/- 10 %), if performed at 100 % air sat.. Normalization should be performed after setting both calibration points, and when there is 100 % air sat. water/air in the wells.
• Treatments. Experiment > Settings > Treatment (10). Add or remove a treatment or select one from the list. Change its name and color, if needed. Add the number of blanks you want on your plate. Open the Treatments tab and click the Randomize button (10.1, red arrow) to let MicroResp™ choose the treatment and blank/control assignment for each well. Alternatively, click on each well and select its treatment. Control wells are required for determining background respiration due to bacteria, biofilm, etc.

RUNNING AN EXPERIMENT

• microplate system to strFill the wells with water (avoid air bubbles) or air, and add test organisms. ong UV light as this can affect oxygen readings, and bleach sAv ensoid exposing the or dye.
• To seal the glass plate, line it with a sheet of microplate sealing film (12) or other gas tight foil. If using parafilm, then cover the entire microplate with the silicone pad and place the compression block on top. For experiments with inactive aquatic organisms, place the reader on a shaker-table to agitate the water somewhat.
• Need   more help?: Watch “Sealing glass microplates” on www.youtube.com/LoligoSystems.
• Logging data. Click START LOG to create a data file and start logging data from a reader. Data logging must be started seperately for each reader by selecting each reader from the Device-list beneath the tabs.
• When the experiment is over (or a critical lower oxygen level is reached), click STOP LOG for each reader.

ANALYZING AN EXPERIMENT

Main menu > Analysis > Load data file. Open the Settings tab > Filters to adjust the data range for the linear regression analysis used for calculating the slope of the oxygen curve. Data points meeting the criteria will appear red in the graphs (15). Settings tab > General > Enable Edit loaded data to adjust setup parameters for the experiment. Changes will be visible in the MO2, Slope and Graph tabs. Click to export the analysis as an Excel data file. This data file contains graphs as well as data, including time-stamped MO2 values.

CALIBRATION, SERVICE & MAINTENANCE

To calibrate the oxygen sensor spots, select Experiment > Settings tab and choose either:

• Perform a Manual (user-defined) calibration (16):
  • a. Fill the wells with a mixed air-equilibrated water sample. This can be achieved by purging atmospheric air into sample water, e.g. with an air pump. The temperature of the water sample must be within +/- 1.5 OC of the experiment water tempera-ture, otherwise an additional calibration is needed for higher/lower temperatures.
  • b. Wait for the readings to stabilize, and then click Read current values (16.1b) to save the current sensor signal as the HIGH calibration value (100 % air saturation).
  • c. Then fill the wells with an oxygen free water sample, e.g. by purging nitrogen gas into sample water or by dissolving ~10 grams of Na2SO3 in 500 ml of distilled water. The temperature of the water sample must also be within +/- 1.5 OC of the experiment water temperature.
  • d. Wait for the reading to stabilize, and then click Read current values (16.1d) to save the current sensor signals as the LOW calibration value (0 % air saturation).
  • or
• Select Pre-defined under Calibration (16.2) and select the batch calibration number found on the black plastic bag that the microplate glass plate came in (18).
  • IMPORTANT: The pre-defined calibration should only be used for preliminary trials.
• To clean the microplate, use ethanol (<70 % v/v), bleach (<3% H₂O₂), or mild detergent, and rinse with demi water. Then dry (17). If using ethanol to sterilize the spots/wells, then dry the plate thoroughly, at least 2 days at 50-60 °C in an oven, to ensure that all residues have left the sensor dye matrix.
• It is recommended to perform a manual calibration after cleaning/sterilizing.
• Store glass plates in the non-translucent black plastic bag between trials, and avoid exposing 18 the sensor spots to UV light as it will bleach the oxygen sensitive dye causing signal drift (18).

References

• loligosystems.com/products/microplate/accessories/microplate-sealing-film-100-pcs/
• loligosystems.com/products/microplate/accessories/plate-roller-for-microplate-sealing-film/
• loligosystems.com/products/microplate/accessories/water-bath-for-microplate/
• loligosystems.com/resources/software-downloads/microplate/

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