Measuring biological oxygen demand using FireSting optical oxygen sensors

(Manufacturers manual)

Instructions to measure BOD using the Firesting optical oxygen sensor

Preparations

Soak BOD vials and lids for a couple of hours in a diluted Deconex solution. Use a q-tip to make sure that all of the surfaces are left clean. After the soak, carefully rinse the the vials and lids at least 5 times using DI water. After the DI water rinse rinse the vials and lids 3 times using fresh MQ to ensure that no traces of Deconex are left in the units.

The experiment should be conducted in a climate control room, so that the temperature remains stable at all times, as well as in dark light conditions.

Prepare instrument for measurements: Attach the optical measurement chord and temperature measurement chord into the appropriate sockets. Connect the apparatus to a computer and open the Oxygen Logger software. When you open the software for the first time, the FireSting settings window opens automatically. Write the sensor code found on the labels of the vials into the settings window (The calibration applies to the given sensor code and sensor strip on the vial). Choose external temperature sensor and internal pressure sensor from the appropriate options. Set the fiber length to 1.0 m.

Note: Flow cytometry measurements can and possibly should be collected parallel to the BOD experiment, both from before and after the experiment.

Calibrate the instrument:

Fill a bottle with a volume of around 500-1000 ml halfway with MQ (e.g. Duran flask), close it with a lid and shake the flask strongly for around 1 min. Open the lid, allowing the ventilation of the flask, and repeat the shaking procedure. Insert the oxygen sensor into the flask and ensure that the tips of the sensors are immersed into the water free of air bubbles. To calibrate a sensor press the calibrate button in the Oxygen Logger software. Choose 1-point in water or humid air. Wait for steady state as instructed in the calibration menu, and click set air after the oxygen % has stabilized. Click finish to exit the calibration menu. Note that the calibration must be done again after a Deconex wash is preformed.

Prepare BOD samples:

Gather the water that you want to do the BOD sampling for. Make sure that the water is at its maximum air saturation for the temperature the experiment is conducted in. Carefully pour the water into the BOD vial, so that the it fills all the way up to the top. The surface pressure will cause the water surface to be concave when the vial is filled appropriately. Carefully slide the lid on top of the vial, and screw on the other part of the lid while holding on to the lid so that no air bubbles form.  Check that no air bubbles are left in the vial by slowly turning it upside down. If any bubbles can be seen, re-do the sample until there is no bubble formation. Attach the measurement ring into each BOD vial, so that the hole at the green screw is against the green oxygen measurement strip of the vial.

Taking measurements

Take initial measurements immediately after the setting up of an experimental unit. Place the optical oxygen sensor into the hole aligned with the green oxygen strip. The sensor can be strapped to the vial with the green screw in order to take continuous measurements waiting for the reading to stabilize. The instrument will optically measure the oxygen concentration (mg/l) in the solution. Take measurements a few hours after the initial measurements so see if the oxygen concentrations have slightly increased (normal with the instrument). Subsequent measurements should be performed every 12 h during the rest of the experiment period.

Clean-up:

Discard the samples from the experimental units (collect samples for flow cytometry if needed). Perform a similar Deconex soak + wash&rinse  to the one performed in the preparations.