When working with live cells, it is imperative to work with good sterile technique, so that your cultures do not become contaminated. CSC staff can help guide you through the relevant considerations for your application. Procedures and decisions should be tailored to your particular sample type and workflow, but here are some general comments. It may be useful (and alarming!) to imagine a steady rain of microorganisms falling from the air onto your specimens at all times, to help evaluate what procedures are less prone to exposure.

General tips to improve sterility for most procedures

Always, the fewer number of steps you do, the better for sterility. There’s a new chance for contamination every time lids come off, when things get moved around, when new reagents are added, and when introducing another piece of equipment. Protocols can often be simplified with some forethought. For example, if you are changing the media on the BioTek, that will introduce contamination a lot more than simply plating the cells already in the media you want, if possible.

It’s important to have your steps be done as quickly as possible. The longer lids are off, the longer samples are outside of the incubator, the more chance some contamination will enter. Think about what you are going to do in advance, have all your supplies handy and reagents thawed, set your pipetmen to the correct ul, open your protocol on the instrument’s PC beforehand, etc. so that by the time you perform the sterile step with your sample, you’re not messing around, touching lots of things, walking around the lab, opening doors and packages, etc.

It also matters what sequence you perform things in. For example, you can Echo dose to empty, sterile plates before adding cells, since contamination is more likely to die on dry surfaces or in solvent, more so than once the assay buffer/media is there. If you want to make a measurement on both the EnVision and the Phenix, you can see which takes longer and do the quickest one first.

Think about the throughput of your project. When you are doing high throughput work, with many microplates, and 384- or 1536-format, you will find that using automation can improve sterility, and also make the contamination rate more predictable. This is because it gets a bit unmanageable for one human to carefully handle so many plates, your hands are full, and mistakes start to happen more when your focus is stretched thin. Image hand pipetting a 1536-well plate…the lid will need to be off for quite a while before you get through it all! At the other extreme, for low throughput experiments (e.g. one 96-well plate or less), you may find that working by hand is the most sterile, since there are fewer places where contamination could get in. If you are doing a small-scale pilot, followed by a large-scale screen, then start to think how you will be adjusting for having more samples, while still validating the full procedures using the pilot. For example, you’ll need a larger sterile working area, including a place to put all the lids. 

There are small changes to how you work with your hands, such as when holding things and opening packages, that will help reduce contamination. Again, the CSC can help you optimize your workflow with sterility in mind. Examples include: holding lids above the container at an angle (instead of setting aside), being mindful of sterile tips not touching packaging outsides or hands, and using barrier tips if the tip box is standing open (see below).

 

No matter how sterile you are, even if you use the best techniques possible, it is always good to check to make sure that your samples are free of contamination. You should build into your workflow some standard quality control checks, where possible. Before beginning, make sure you are using validated cell lines, with a known source and handling history, and reagents you are confident are fresh or kept very sterile, shared by a limited number of trained people. If you have many samples, then you may want to only check a subset of them, if your quality control is manual (e.g. look in a few wells of a few plates at the dissecting scope in your culture room) and remember to record it in your lab notebook. You may find it useful to perform actual measurements for quality control at various steps along the process, for example, brightfield imaging in the Phenix, or optical density on the plate reader. Having the data banked could help you if you later find you had a weird well and wonder when the problem occurred. If you are doing this for many samples, then the data might become hard to wrangle. You may need to set it up so that you can quickly, quantitatively analyze the data to immediately single out problem samples for discarding/repeating e.g. an analysis of the images to look for expected phenotypes, with a heatmap to show the numbers across the plate, etc. CSC staff can help you come up with practical ideas for this.

Last but not least, we have several instruments that can do the same jobs, with overlapping capabilities, but different considerations for sterility, so choosing the best device is key. The rest of this page is devoted to discussing your options for sterile liquid handling in the CSC.

 

Working in a biosafety cabinet with CSC equipment

Of course, anything that can be done in a biosafety cabinet will be better off than work done at the bench. However, in the CSC, most of our equipment is permanently at the bench and not enclosed in any way. If working at the bench, having a low hanging roof helps, en lieu of an enclosure. However, there are exceptions – these CSC equipment are (or can be) enclosed:

• The epMotion is in an enclosure specially made for sterile work.
• The MicroPro is able to be placed inside your cell culture hood. It is small and portable, and the iPad operating it can be run on battery and placed outside or inside the hood. It was designed for this purpose.
• The MicroDrop is also able to be placed in the hood.  

Selecting the optimal devices and workflow

Each device has its own methods for sterilizing it, whether it’s priming the lines with cleaners, soaking the manifolds in ethanol, etc. Once you have selected the best device, you can locate existing cleaning procedures with the help of CSC staff, and optimize your cleaning procedure to make it super sterile for your specific use case.

For the BioTek peri or syringe modules, usually we find that it is sufficient to have bleach, soap, and ethanol priming steps (with a few minutes waiting for each to take action, and appropriate water steps to get rid of bleach and soap) and sterile media (to get rid of the ethanol), with swirling to make sure the intake tubes are coated around the outsides at each step, and spray the tips of the manifold with 100% (important!) ethanol after the 70% ethanol prime (and make sure it is dry, no hanging droplets, before proceding). Also handle things carefully with ethanol-ed gloves, without anything touching the bottom ~15cm of the intake tubes after the ethanol prime/soak.

For the BioTek wash module, there are a lot more parts that need to be sterilized, and not so accessible for thorough cleaning as the peri cassette e.g. the outside of the dispense ports can’t be sprayed while onboard. I have a complex system figured out that works most of the time, and I am happy to show users. For washing cells, you also have the option of using pipetting (e.g. for 96-well plates).