About

Why are OD measurements important?

The turbidity (cloudiness) of the culture typically correlates to how many cells are in a culture. By using a simple LED and photodiode pair, we can quantify how many cells are in the population, track how the culture is growing over time, and observe the culture changing over the course of the experiment.

Why is calibration necessary?

Readings sent from the Raspberry Pi to the computer/server are the raw voltage values measured by the Arduino in the Motherboard. In other words, to make sense of what these readings are, we need a calibration file that converts this to a useable optical density measurement.

Is calibration different between different organisms?

Yes. Cells with different size and shapes scatter differently and would result in unique calibration curves. eVOLVER is set up such that one could easily use the appropriate calibration files for each experiment/organism.

Is calibration different between different temperatures?

Yes. Temperature effects the photodiode sensors that we use to measure OD. If you plan to use different temperatures we suggest doing a new calibration for each one.

Prepare the day before:

• 16 assembled glass vials:

  • Glass vials (Chemglass, CG-4902-08)

  • Vial screw caps

  • Octagon magnetic stir bars (Fisher, 14-513-57)

• 300 mL of saturated culture of desired organism

  • If calibrating to ODs above 1.5, you may need more cells

• 300 mL of 1X PBS (Phosphage Buffered Saline)

OD Standard Preparation

Wash cells and resuspend in 1X PBS to avoid cell growth

1. Spin down saturated cultures in 50mL or 250mL centrifuge tubes.

   1. 2500 rcf for 5 minutes should be fine for bacteria or yeast

2. Pour off media and resuspend cells in 50mL 1X PBS

3. Spin down cells in a 50mL conical tube

4. Resuspend well in 50mL 1X PBS

Find OD of Concentrated Cell Stock and Make Standards

1. Measure the OD of cell stock using a dilution series

   1. Dilute 200uL of cells into 1800uL of 1X PBS (1:10)

   2. Dilute the 1mL of the 1:10 dilution into 1mL 1X PBS (1:20)

   3. Make 1:40 and 1:80 dilutions

2. Measure OD of dilutions and a 1X PBS blank

   1. It is recommended to use 300uL of dilution if using plate reader to provide a similar read length to the cuvette

   2. Use absorbance at 600nm for most bacteria and yeast

3. If the measured OD of the lowest dilution is above 0.5, make further dilutions to bring it below 0.5

1. Calculate OD of concentrated cell stock

   1. Input ODs for dilution and 1X PBS blank into the spreadsheet

   2. Enter calculated OD into 'OD of Cell Stock'

      1. Use a dilution that is roughly between 0.1 and 0.5

2. Calculate and make OD standards

   1. Optional: alter the series of standards to include more ODs in the range you care about

   2. Get 16 vials and add a stir bar to each

   3. Add calculated amount of 1X PBS and cell stock to each vial

   4. Mix by swirling or putting in eVOLVER with stirring on

Measure OD of Standards

1. Make dilutions for standards above 0.5 OD

   1. Use a 96-well plate to ease dilutions and transfer to plate reader

2. Measure ODs and calculate original OD from dilution OD

   1. An example calculation, including the 1X PBS blank is included at the bottom of the spreadsheet

3. Adjust standards if necessary

4. Cap vials containing standards and place in eVOLVER to equilibrate to temperature while stirring

   1. Keep track of which vial is which OD

   2. This may take >30 minutes

   3. Each vial is labelled with its latest temperature in the SETUP page of the GUI

Calibrate OD using the GUI

1. On the eVOLVER app home screen, make sure you are connected to your eVOLVER (green symbol next to active eVOLVER), then choose CALIBRATIONS and then O.D.

1. Give the calibration a name (a standard suggestion is YYMMDD_YourInitials_od) and click enter.

2. Click on the white box by the S0. The display should now have an overlay with Sample 0 on top of a box with a keypad. Input the OD values of each sample.

   1. You can input the OD values using the keyboard, then hit enter to increase the vial selected.

3. Click Start OD Calibration ▷.

1. Press ▷. After the eVOLVER has logged the vials in one configuration, click the forward arrow. Move vials into their new positions, as seen on the right side of the GUI.

2. Click VIEW COLLECTED DATA to monitor the smoothness of the curves as you calibrate. If a particular value is a big outlier, consider rewinding to that vial position and doing it again. Don't worry your other work will be saved!

3. Continue until all sixteen sets of values have been saved for each standard in each vial.

4. Click the pen button to submit and save the calibration. Then click Exit after the calibration is logged.

5. To validate the calibration was logged on the eVOLVER, from the touch screen home tap Setup and then tap the box next to OD: and a list of calibrations should come up. Select your recent one from the list. If not present, it was not logged to the eVOLVER.

About

In order to appropriately interpret data from and actuate physical elements connected to the eVOLVER framework, a relationship must be established between the electrical signals generated by attached sensors and the physical phenomenon they are measuring. This is done by manually measuring values on a controlled set of data via some gold-standard assay or measurement device and comparing these data to the eVOLVER generated data. By doing this across a range of different values, a function can be fit to this relationship to allow eVOLVER to interpret data accurately.

For more information see here.

Procedure

Calibrations should proceed in the following order:

1. Temperature Calibration

2. Optical Density Calibration

3. Pump Calibration

About

Why are temperature calibrations important?

Each microorganism has its own culture temperature requirement. Temperature may affect the successful cultivation and growth characteristics of the culture. By using a simple thermistor and heating resistor pair, we can track and change the culture temperature over time.

Why is calibration necessary?

Readings sent from the Raspberry Pi to the computer/server are the raw thermistor values measured by the Arduino. To make sense of the readings in terms of values we understand, we need a calibration file that converts this to degrees (celsius).

Do I need to redo temperature calibration if I switch out a thermistor/resistor pair?

Yes. Variability exists across the individual thermistor and heating resistor elements, necessitating a new temperature calibration anytime one of these parts are replaced. This is why the calibration curve is unique to each smart sleeve.

Materials

• 16 eVOLVER vials filled with 15mL water

  • Glass vials - 40mL (Chemglass, CG-4902-08)

• Thermometer with probes

  • Do not use a thermocouple-based thermometer, as the magnetic field from the stir components can interfere with measurements

  • We use the "Fisherbrand™ Traceable™ Excursion-Trac™ Datalogging Thermometers"

• eVOLVER Electron GUI

  • This tutorial is for the newest version of the eVOLVER Electron App (Release 2.0.1), which enables both data visualization between each temperature calibration step (RT -> +10C -> +20C), as well as GUI-enabled calibration (without use of the command line).

  • For older versions of the Electron GUI (Release v1.2.1 and earlier), additional notes are provided at the bottom to successfully complete the calibration - the process remains largely the same, apart from the use of the command line to complete the calibration.

• eVOLVER

Methods

• Turn on the eVOLVER 5V power supply. Wait 5 seconds. Turn on the eVOLVER 12V power supply.

• Open up the eVOLVER Electron GUI app, and ensure you are connected to the correct eVOLVER you wish to calibrate in the upper-right hand corner of the "Home Screen". The circle should be solid green, indicating that the eVOLVER is connected.

• Tap "Setup" in the GUI home screen, select all vials by tapping "Select All" in the bottom right corner. The vial circles should highlight orange. Tap the "Next ->" button in the upper right-hand box (which includes all the control parameters) until you reach the "Temperature" tab. Drag the slider all the way down to "20C". Tap the button, and the "Executed Commands" box in the lower right should indicate that a temp command was sent to all vials.

• Place all the eVOLVER vials, pre-filled with 15mL of water, into the Smart Sleeves.

• Go back to the "Home Screen", click "Calibrations" and then "Temp" in the eVOLVER Electron App.

• Name the calibration, hit enter, and then click "Start Temperature Calibration". A prompt will read "Heaters turned off. Let equilibrate, then enter values."

• Room Temperature: Wait ~30 minutes for the temperature of the water to equilibrate to ambient room temperature (and heat generated by the eVOLVER).

• Once equilibrated, measure the temperature of each vial with the thermometer probe and log the temperature measurements in Celsius in the Electron GUI.

• Wait 60-120 minutes for the temperature of the water to equilibrate. Use the system outlined in the section "Temperature Equilibrium" to establish when the temperature has equilibrated.

• Wait ~2-3hrs, or leave overnight.

• Calibrate: An edit icon button should appear once all 3 measurements have been made. Click on the button, and a graph of the linear calibration curves for all thermistors across the smart sleeves should appear. Please confirm that the curves look linear. If everything looks good, please exit out of the graph (press the backwards arrow twice), and voila! You have completed your Temperature Calibration! Please be sure to change the temperature calibration file to the most recent in the eVOLVER set-up menu (click on the temperature calibration button).

python calibration/calibrate.py -a <ip_address> -g

python calibration/calibrate.py -a <ip_address> -n <file_name> -t linear -f <name_after_fit> -p temp

1. Fill a large beaker with water and submerge all of the pump lines (inputs and outputs of the pumps), ensuring all of the ends are below the surface of the water.

2. Run the pumps on the Setup page to completely fill the lines with water (about 20s)

3. Place 16 vials into a rack and place the output ends of the pumps to be calibrated into the vials.

4. Select the eVOLVER on the top of the GUI home page to be calibrated

5. Navigate to the Pump Calibration page

6. Enter a name for the pump calibration

7. Select the pump configuration for the eVOLVER. In a standard setup, IN1 are the media in pumps, and E refers to the efflux pumps. If you have another pump array (for a second media input source), it is typically going to be in IN2. Click Start Pump Calibration. The standard pumps are the FAST pumps (~1 mL/s, black pump heads). If you have the slow flow rate pumps (~ 1 ml/m, pink pump heads), select the SLOW radio button for that array.

8. Select all of the vials either by clicking the button on the bottom right or clicking and dragging across the vials on the selector. Click PUMP IN (or whatever the pump array you are calibrating) to run the pumps for 10s if the FAST radio button was selected or 100s if the SLOW radio button was selected.

9. After the pumps finish pumping, measure how much water was pumped by pouring out the water from each vial into a 25 mL graduated cylinder (or something similar). Enter this number into the box for each vial. The flow rate will appear on the vial selector on the right side of the screen.

10. Repeat this process for each pump array you have selected for calibration.

11. Click the pen button to submit and save the calibration. Then click Exit after the calibration is logged.

12. Verify the calibration appears on the setup page.