The plot thickens.
For those of you who have been dangling in urgent uncertainty, I can report that the vibration problem in our incubator has not gone away. I thought I’d get around it by using smaller, non-round vessels, such as the 8-well chamber slide in the front of this image:
But alas, the phenomenon persists. Very oddly, in these small, square-shaped wells, I don’t get concentric circles, but long lines of cells, rather like a string of old ladies queuing for a bus shelter in the provinces:
We’ll leave aside the rather ironic fact that several people in our lab are spending lots of time and money trying to force cells to grow in lines using cutting-edge micropattern contact printing – this outcome is not desirable in my own experiments. As anyone who studies the cytoskeleton knows, outside forces can have big effects on internal actin and microtubule configurations, so the last thing I need is something that forces my cells to change shape in the wild-type situation. Will the scourge never end?
On the other hand, closer to home, I can happily report that Richard has solved my pressing microwave popcorn mystery. The solution? Elevating the bag onto an upturned pyrex casserole dish – which apparently aligns the bulk of the popcorn kernels more closely to where the microwaves are bouncing back – completely and reproducibly sorts out the problem and yields a full bag of salty goodness.
I can state from personal observation that old ladies queuing for bus shelters in the provinces don’t look like that. Neither do they look like that if merely queuing for buses. They do ask, however, when the buses arrive, whether or not they go to the station.
Damn, you’re just too late for a shuttle mission.
It’s starting to become a serious problem. Just today I needed to re-spread some cells for an assay and I couldn’t get them to settle in a random, even pattern. It may affect my results.
But srsly – is there some deep drilling beneath your lab? For Crossrail or something?
If there was any major building vibrations we in the EM labs would know about it. EMs are very sensitive to vibration. Oh, hang on, the EM is broken!
Have you tried using somebody else’s tissue culture lab and incubator? It won’t anser the question, but that way you’ll be able to get on with your work and ignore the vibration question, which I’m sure is secondary to your actual experiments.
I can’t see the pictures, flicker is blocked at my work.
I am sure they are due to capillary waves setting up a standing wave pattern via resonance. Because this is a resonance, the degree of isolation needed to prevent it is very high and perhaps not achievable.
The standing capillary waves result from the conversion of kinetic energy of moving fluid to potential energy of stretching surface.
What you need to do is either disrupt the uniformity of the free surface of the fluid, or disrupt the uniformity of the depth of the fluid. A few drops of a floating oil would likely work by reducing the surface energy and/or by making it non-uniform.
Small stationary hydrophilic interruptions in the fluid surface should do it also. What happens is that the discontinuities of a small hydrophilic object results in curvature of the free surface where the fluid climbs by capillarity (the way it does at the dish walls). This change in the curvature of the fluid results in a change in the force-deformation characteristic of the surface. This changes the conversion of energy between kinetic (from the flow of the fluid) to potential (the tension in the surface) so that they are no longer symmetrical. If they are no longer symmetrical, then a resonance cannot happen.
A way to do this would be to take a stainless steel wire, bend it into a (rough not highly uniform) spiral and drop it onto the dish surface after the cells and media are put in and dispersed. The thickness of the wire shouldn’t matter very much. Either it sticks above the surface and provides a discontinuity in the surface, or it does not go above the surface and provides a discontinuity in the velocity of the fluid. Wire that has been woven and crimped into a screen would work even better. What is important are the discontinuities that break the symmetry that is causing the resonance.
You could also change the incubation conditions. Surface tension is dependent on temperature, so changing the temperature will change the resonance frequency. Additives to the media that change surface tension will work too, but those may interfere because they are surface active. Increasing the viscosity of the media with something inert like polyethylene glycol with a high molecular weight would increase the dissipation and should reduce the amplitude. You may not be able to change conditions enough to affect the capillary waves while still staying within what the cells need.
Non-symmetrical dishes might work; triangular, or with pie shaped sections.
There may be a critical time for the pattern formation to occur. Once the cells attach to the surface they might not migrate. If that is the case then you could put a discontinuity generator in your dishes for the first growth period (until cells have attached) and then remove it to allow them to grow over the whole surface.
Another thing to try might be adding a vibration with a circular component by putting something with a vertical rotating axis on the same table or on the incubator. That would tend to set up a rotational flow in circular dishes rather than a back-and-forth. A magnetic stirrer would work. I don’t think this would eliminate the orientation problem in square dishes, I think it would make the lines diagonal instead of parallel. The magnetic stirrer would have to be on continuously, and the speed would matter. You would be trying to set up an orbital flow which would dominate over the capillary wave induced flow.
“Surface tension is dependent on temperature, so changing the temperature will change the resonance frequency.”
it’ll also change the cells.
Hi Jenny,
How about keeping cells outside the incubator till thay start spreading (3-4h perhaps). The pH of the media would change, but once you put the chamber slide inside the incubator it should be OK. Or you could plate cells in CO2-independent medium and once they sit down, change into regular medium and keep the chamber in the incubator. This might affect your expt less than all the clumping. Also your last post mentioned that the vibrations were more in the morning, so perhaps you could try plating cells later in the day.
I hope you figure out some way to work around the vibrations soon.
I am at a different site and can now observe your pictures and I am not sure what I am seeing.
Are those white spots on a brown background cells, clumps of cells, or bubbles?
I would need to see a picture that includes the walls of the container and a length reference. Are the spacings about the same as in the earlier patterns with circular symmetry? The circular symmetry is clearly capillary waves setting up a standing wave pattern. These don’t look like standing waves.
Was the relative depth of fluid in the circular vs these new rectangular containers the same? In the top figure the fluid looks deep and purple compared to the shallow and brown fluid in the circular dishes.
What type of containers are you using? Polystyrene?
Well, your EM is also two floors lower than ours. (I didn’t know the EM was knackered – my condolences. What are doing all day, then? ;-))
It’s quite a pain to use someone else’s – our lab has all the consumables in the same room and last time I borrowed Catherine’s hood, I think I had to get up twenty times as I’d kept forgetting things. I also don’t like to walk around the building with cells because — how to put this delicately? – I can be clumsy before my fifth cup of coffee.
Nothing you’ve suggested is practical for sterile cell culture work, I’m afraid. I also can’t change any conditions 95% of the way through the experiments for this paper.
MGG, I don’t want to introduce the temperature and pH variable into my experiments. Also, I’m often doing short-term replating experiments that only take 2-4 hours – and of course, all my time points are in the morning. Otherwise I wouldn’t get home til midnight.
The depth of the medium varies with the experiment. Our containers are both either glass-bottomed or polypropylene-bottomed.
70% of my cell culture work is with primary embryonic neurons, and they have an utterly annoying tendency to end up stuck around the edges of those small chambers – this happens regardless of medium level in the chamber, incubator type and location, presence or absence of serum, laminin concentration, and phase of the moon. Doesn’t matter so much if I’m extracting RNA, but definitely does if immunocytochemistry is the plan.
Are your incubators in a stack? Mine are, and I’ve noticed a slight difference in the plating patterns of non-neuronal cells in round dishes, between the upper and lower incubators. There’s almost always construction going on in and around our building, and it was especially bad when they were renovating the animal facility below my lab. Is there any way you could sterilize small pieces of memory foam or bubble wrap, and place them either directly underneath the chamber slide, or perhaps on the shelves where they slide into the slots on the walls of the incubator? Might dampen the vibrations a bit, but of course there’s a concern about contamination. I’d soak a piece of bubble wrap in 70% ethanol, and then air-dry in the laminar flow hood.
I like the idea of a sumptuous bubble-wrap throne for my experiments!
I don’t think sterility is too much of an issue – after all, nothing else inside the incubator is sterile, except on the inside of the containers. As long as it’s clean that should be good enough.
I’m thinking it would be like a miniature air table. Probably just spritzing the bubble wrap with 70% ethanol would be good enough. We used to “purify” neurons from sensory ganglia using high-serum medium in a separatory funnel, and that whole contraption sat on a thick layer of foam inside a controlled temperature box. Not suitable for the cell culture incubator, but the anti-vibration principle is the same.
That puts you in a catch-22.
If you can’t change the conditions, then the unacceptable patterns will persist. If you change the conditions such that the unacceptable patterns do not persist, then your conditions have been changed.
Is the lab air conditioned? If it is not, then with hot weather, the refrigeration unit in the incubator may be turning on and that compressor may be the source of the vibration. If the lab is air conditioned, changing the lab temperature such that the incubator compressor does not turn on may help.
There may be thermostat settings designed to save energy by turning off lab cooling when people are supposed to be at home and not working.