Frequently asked question.


Frequently Asked Question

Technical Questions

What is the maximum sterilization temperature for removable, autoclavable pump heads?

For Minifors, Labfors and Multifors pump heads, sterilization at 134°C (sometimes used for deocontamination) will result in damage to a key component of the pump head. Temperatures up to 125°C should be fine i.e. normal sterilization temperature. There are two possible work-around solutions:
  • Remove the tubing from the vessel inlet pipes after emptying and clamping off the lines. Sterilize the bottles, pump heads and tubing at e.g. 121°C.
  • Snap open the pump head and remove the tubing to allow the whole line to be sterilized with the vessel and bottle but without the pump head. This makes a little more work when preparing the system but allows the higher temperatures wjhen this is a necessity.

What shall I take into account when working with heat labile media and/or medium constituents?

It is possible to autoclave the vessel without media or sensitive constituents. Autoclave the vessel with enough water to cover probe tips. Drain the vessel via the harvest pipe and add media after the vessel cools. Be sure to do this in a sterile environment such as a laminar flow hood.
Gas Flow

Dissolved oxygen hits zero during the culture and/or control is generally poor. How can I improve this?

The dissolved oxygen content will decrease as the culture grows. More culture means more oxygen consumption. To ensure that this does not happen, there are several methods. For bacteria, it may be as simple as allowing the max influence setting to increase overall agitation speed. If this is maxed out, then addition of an O2 supplement valve may be in order. This will pulse pure O2 into the vessel to supplement the O2 available in air. For more sheer sensitive cultures, one would go straight to the O2 valve for increased O2. Many users are satisfied if their dissolved oxygen control is +/- 5% of the set point. This should be taken into consideration when determining how poor pO2 control really is. If there are wild swings around the setpoint, it may be good to check the ‘proportion’ setting on the pO2 control. As this value increases, the response from either the agitator or the O2 valve increases. Make this value smaller to suit your culture’s requirements. If the dissolved oxygen increases dramatically over a short period of time, it may be due to the lack of nutrient feed in the system. Check to make sure that you still do have enough glucose, methanol, etc. in the culture.  

The flow rate from the peristaltic pumps varies. How do I ensure a constant flow rate?

The most common reason for a varied flowrate with these pumps is the Marprene (R) tubing used in them. Over the course of the run, the tubing will stretch and the inner diameter will change. This will alter delivery volume. Repeated autoclaving may do this as well.

I get contamination all of the time. What can I do about it?

There are many possible sources of contamination. Not properly sealing the head plate to the vessel, non-sterile components coming in contact with culture and improper sterile connection technique are among some of these. It is important to fill vessels and add reagents in a sterile region such as a laminar flow cabinet whenever possible. Fortunately, BIOTRON Bioreactors allow for the reagent bottles and addition bottles to be connected up and autoclaved with the bioreactor for all bench-scale systems.

How do I know if my pH sensors are usable?

pH-sensors have a typical slope of 50 - 61 mV/pH. If the slope is outside this range, the electrode must be renovated. It should not be used for fermentation without regeneration in the correct cleaning reagent. Instruction and details of the correct solutions can be found in the documentation delivered with your sensor.

How can I tell the age of some pH sensors (depends on the manufacturer)?

pH sensors do deteriorate over time and typically last for 20-50 autoclave cycles. An old (or well used) sensor may have gel with a brownish discolouration. Also, an ageing sensor will gradually decline from about 57mV per pH unit (at 20°C) when new. Once the loss is about 20% or more and cannot be improved by regeneration, then the sensor should be replaced.

Which gasses do I use for pO2 sensor calibration?

Nitrogen for the zero point (CO2 can also be used in extremis) BUT care is needed to use these gasses in well ventilated environments and to ensure the supply is turned off after use). The 100% value must be set using air (NEVER pure oxygen). A standard polarographic sensor has a range of 0-200% oxygen in air, i.e. 42% pure oxygen. It is a relative value, not absolute so the 100% value is for the vessel configuration, maximum flow rate and maximum stirrer speed used for a particular fermentation. Allow several minutes for gas to pass through the medium before accepting the calibration reading and ALWAYS use a pO2 sensor which has been properly polarized (minimum 2 hours and, preferably, 6 hours).
Bioreactor Options

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