While batch culture techniques often utilize biosafety hoods or flame convection currents to keep workspaces sterile, these are rarely amenable for automated cell culture devices. Prevention of contamination in eVOLVER is achieved at three levels: 1) sterilization of media, culture vessels, and fluidic lines, 2) attention to sterile technique, and 3) physical and chemical barriers to contaminants. First, all media bottles and their adapters, and all components of the culture vessel (e.g. borosilicate glass vial, magnetic stir bar, cap with fluidic adapters) are designed to be autoclaved before each use. Fluidic lines on the device are sterilized before and after each experiment using bleach and ethanol (see Online Methods). Second, following sterilization, sterile technique should be practiced when attaching media lines to culture vials by working quickly, avoiding physical contact with the ends of fluidics lines, and taking care to spray gloves with ethanol. Finally, additional physical and chemical measures may be taken depending on the organism and experiment. For example, the sampling port may be covered by a sterile membrane for long-term culture. For slow-growing eukaryotic cultures, antibiotics can be added to the media to exclude bacterial contaminants. UV sterilization of components or surfaces is another preventative measure to consider.
Vial overflow is one of the most common problems to plague eVOLVER experiments. Overflow events can cause damage to internal components such as the PCB motherboard, sensor actuator (SA) boards, power supplies, etc. If you experience overflow during an experiment, it is important to immediately stop the eVOLVER and evaluate potential causes of overflow, as well as any damage that the overflow may have caused.
If you catch an overflow that happened recently (i.e. an overflow that happened overnight), the best thing you can do after stopping the experiment is turn off and unplug the eVOLVER, determine the extent of overflow spilling, and wash any components that have media on them. There are no components on the vial board or motherboard that retain significant charge (i.e. no batteries or large capacitors), so they are safe to wash gently in soap and water. Once clean, they should be dried quickly with a paper towel.
The main reason for electronics failure after an overflow is current shorting through media (most microbial media is salty and conductive), or connections and copper corroding from media that has been left to dry. If you can clean off all the media immediately after an overflow, you will have a much better chance of saving electrical components, and saving yourself many headaches down the road.
If you did not catch an overflow quickly, or a large amount of media spilled, follow the workflow below.
Overflowed media can enter into the vial platform through the ports that connect the Smart Vials to the motherboard. This can cause damage to the internal circuitry, which is should first be evaluated by visually inspecting the motherboard, SA boards, power supplies, and Raspberry Pi. Damage to any of these components can look like burned or melted spots or loose/broken connections. If you see visible damage to any of these parts, they will need to be fully replaced. See pages below for specific instructions on how to replace these various parts.
Motherboard Troubleshooting/Replacement
12V Power Supply Troubleshooting/Replacement
Pump is not actuating / spinning
Vial Overflow, Pump Failure, Spills
"No Buffer Space Available" after a very long term experiment
Accidentally turning the eVOLVER base off - another alternative is to tape the switches on
When there is an obstruction in the line, from dried media or biofilm, the peristaltic pumps can produce up to 45 psi of pressure, which is enough to exceed the barbed connector tolerance and burst barb connections. This is clearly undesirable, so if you find that one of your barb connections has burst, check the length of the line for obstructions. If it's an efflux line to the waste container (opaque), assume it has an obstruction.
For the pumps that we typically use for eVOLVER, 1/16" ID tubing does not resist flow in any appreciable amount. If a pump is not pulling liquid, it is likely blocked or obstructed.
Biofilm obstructions can happen during long experiments with a biofilm-forming organism like E. coli - your best bet is to pause the experiment and bleach sterilize all lines before restarting. If your line is completely blocked, either replace the line or clear the obstruction by pushing water through the line with a 50 mL syringe (shown below).
If the syringe method does not immediately work to clear the blockage, the blocked lines can be soaked in water for a few days to help rehydrate the dried media/biomass - the silicone tubing is quite permeable, so water can slowly enter the tubing and help loosen any dried blockage so that it can be flushed out with a syringe.
Residual or leaked media on the ends of luer-lok connectors can dry and leave the connectors stuck. Rather than reach for a pair of plyers, soak the connection in hot water to dissolve the media. This can also help for any stubborn fluidic connection, and make tubing much more pliable.