Hi! I just tried to assemble the HOB bioreactor and draw the figure to illustrate what I did. I wonder whether I do the wrong thing to all inlet and outlet luer lock. Let’s break the whole system into details. The media from media bottle should goes directly to the vial without filtering because the media, bottle, tubes, and locks are all autoclaved. However, my questions is which gender luer lock I should use in media bottle. From my figure, I use female in the media bottle side and use a male luer lock from motor (vial side) to connect. If the male luer lock should always eject the media or product and female luer lock receive, I should exchange all the locking system.
Second, if the filter has direction flow (female to male), the female part should direct to the bottle for both media and product bottle because we don’t want external things goes into bottles. We just need to make sure that the whole inner system will not explode with high internal pressure.
There are no valves in the luer locks and I don’t think you’re going to blow things up with female going into male, but there are some negative consequences if you really have got the filters and luer locks the wrong way round:
Your system will look different to all the others and you will likely be programming your subconcious with sub-optimal practice. In your place, I’d want to ensure that I got it right from the very first experiment. If they were definitely wrong, I would stop and change them before proceeding.
BUT make sure your definition of male and female luer locks are correct. Males are bigger, They have a big sheath on the outside with a male protrusion on the inside. Females are smaller and go between the male sheath and protrustion. An easy check is your filters. They have a female luer lock on the inlet side. On the outlet they have a male luer slip (no sheath, no lock, just a protrusion.
To be doubly sure, would you like to send a photo? Your previous photo shows what appears to me to be a correctly set up PRODUCT bottle (I’d love to see it from the top to be absolutely sure). It has a female inlet luerlock on the left to receive the product flow, and a male luer lock on the right going into the female side of a vent filter (the side with writing and the only side with lock tabs).
That looks good to me. I would just say that the filter marked ‘outlet’ attached to your media bottle is actually the air inlet - there to let filtered air take the place of the media that you pump out.
And of course you must never add CO2 in this system, as you locked the two gas vent ports together instead of adding the two outlet filters to them.
The critical thing now will be adjusting the angle of your vial in the pioreactor so you get the lowest optical density reading before starting your culture. You’ll also want to calibrate that - ideally against a recently calibrated OD600.
I will try that first, but I have a silly question. Do we really need the filter between vial and CO2 pump? Why cannot we just connect the CO2 pump with the vial?
I think attempting to sterilise the CO2 delivery system would be much harder than jus adding an autoclaved filter downstream of it. As you are not using CO2 in the above heterotrophic experiment, you could simply block that port as you have with the outlet filters.
