Caps off during OD calibration and other OD questions

Hardware, PWM, and Optics
1

Hi everybody,

I’ve done a few DIY minibioreactors before, but this is my first time with a pioreactor. I had a great time putting it together and I’m really excited about all the possibilities the pioreactor offers, but I have a few questions about the OD calibration process:

a) During the calibration process, the system indicates that the cap of the vials must be removed. However, ambient light can affect the measurement of the phototransistors. So why is it necessary to leave the lid off?

b) In the final step, the system question about the OD600 of my blank. I would like to know if this blank refers to the optical density of the uninoculated culture medium measured in an external spectrophotometer or, on the contrary, if it refers to the optical density of the culture medium plus the initial inoculum that would be used during the experiment. On the other hand, I understand that the measurements in the external spectrophotometer are also made after a blank has been made with the culture medium without inoculum, so I do not quite understand the need to enter the blank data into the pyoreactor system.

c) In our DIY minibioreactors, we have always worked with the phototransistors positioned at 135° to the IR LED light source. We work with bacteria at densities between ~0.0 to 0.4 OD600nm. I understand that this arrangement would be the most suitable, right?

Thanks in advance!

2

Hi @Pedre91

We hope you’re enjoying the Pioreactor!

a) This is a good point, I believe we suggested to leave it off to reduce having to screw/unscrew and reduce the chance of an errant drop falling outside the vial. You’re correct about ambient light though - our thinking was that it was not that big of an effect. You can rest the cap on the vial during the OD calibration without issue.

b) The answer is the former:

optical density of the uninoculated culture medium measured in an external spectrophotometer

Your comment about the external system already removing the blank is a good point, and we’ll have to think more about that. We still need the blank signal for the internal Pioreactor measurements, but you could enter “0.0” when asked for the external measurement, which is correct if your initial comment is correct (I think).

c) Yea! The 135deg pocket is the most sensitive, so it is great for low-density cultures. During early testing, we liked the range the 90deg pocket gave us, so we chose that as the default.