Hello,
We have had problems seeding plates with HB101. In short, it seems that half of the time the bacteria grows a very thick lawn, particularly around the edges. The seed remains very soft (never dries). The worms appear to survive it, but I am worried these are not favorable conditions for them. Also, they are difficult to see as the thick bacteria is also opaque (brown). I thought it was a problem with drying, but leaving the plates for extra days in the flow cabinet does not do the trick. OP50 seeds are consistently dried and we do not see the same problem with this strain, even when seeding the same batch of plates (half with OP50 and the other half with HB101). The lawn never grows outward… just upward :P. We then store the plates at 4C until moving worms onto them. When worms are cultured at 16 or 20C on these plates, the lawn continues to grow thicker, particularly on the outside.
I have read that the salt buffers are supposed to prevent the bacterial lawn from expanding outward. Our salt buffers have been made several different times now and they appear to be okay (as far as we can tell).
Well, there’s the obvious, the possibility this isn’t HB101, or isn’t only HB101. Do you inoculate the media you use to spot the plates with a single colony from a streak? Have you tried going back to (and then streaking out) frozen stocks?
HB101 should grow thicker than OP50, especially around the edges - but it shouldn’t be brown and opaque, and what you’re describing doesn’t really sound right.
HB101 is admired for its smooth, almost runny, consistency. it does not get crusty like OP50. if you want a thinner lawn then use less peptone (10-20% as much as usual).
OP50, and I think also HB101 are mutants that can’t produce uracil they get all uracil from the media. I have heard there is strong selection for spontaneous reversion of this mutation in E. coli.
If you restreak you should be picking small thin colonies that grow less robustly. Constant restreaking and picking large colonies might select for revertants.
If you try to grow these bacteria on minimal media lacking uracil they shouldn’t grow well or at all.
but back to a couple of things mentioned in the original post.
In short, it seems that half of the time the bacteria grows a very thick lawn, particularly around the edges.
as already mentioned, you will get an ‘edge effect’ this is normal and the lawns are ‘wetter’ relative to OP50. But what exactly does half the time mean? Do you get some plates with a very thick lawn and some with a ‘standard’ thick lawn in the same batch of seeded plates? Or do you mean one batch of plates has a thick lawn and the next is thinner?
If the plates are stored under identical conditions, the latter observation might be a consequence of differing OD600 for the stock cultures used.
The former observation is harder to explain apart from pipetting errors.
The lawn never grows outward.. just upward
OK. let’s say we innoculate 60µL of a 1 x 109/mL culture onto the surface of a 55mm agar plate. That’s ~ 6 x 107 bacteria.
Let’s say we spread the agar surface to a diameter of 50mm, that is ~1.9 x 109 µm2.
each bacteria has an 'area of approx. 1µm2 (0.5 x 2µm). Hence, we’ve got lots of space between each bacterium where it can (in the short term) multiply without increasing the spread diameter. If these bacterial plates were left over a longer period, then sure, one would expect the lawn to expand (both xy and z).
So, no surprises that one sees that the lawn is the same size after an overnight incubation as it was when first spread. Indeed, the dynamics of bacterial growth tend to make colonies growth three dimensionally and the physics of this growth means that the bacteria at the outer edge of a multiplying colony orient themselves differently with respect to the inner layers of bacteria and this tends to limit xy growth. Eventually, crowding and lack of resources means that the lawn will expand laterally.
Yes, I have read that HB101 will be wetter and thicker around the edges than OP50. However, half the time they are so thick around the edges (and continue to grow around these edges when placed at 20C with worms) that it is very difficult to see the worms under the stereomicroscope and the worms have a clear appearance when I do spot them. In other words, I cannot see worm tracks on these plates whereas tracks are evident the other half the time when HB101 is not as thick. The ‘not as thick’ plates are more similar to OP50, although they still have that wetter feeling which everyone agrees is normal.
I am thinking this may have to do with the way we seed the plates (I get the feeling it is incorrect). We use a repeater pipette to place 0.5ml of the overnight culture (grown in LB) on each small 5.5cm diameter plates and then leave it to dry (~2 days) in the flow cabinet. Is this what everyone else does? Is it too much media? Do others spread a small drop instead? Our method used to give us the thinner lawns consistently, but recently we’ve been seeing the thick lawns. Maybe it has to do with the flow cabinet not circulating as aggressively leading to longer/less drying.
Since my original post I have streaked the HB101 from a frozen stock. I get small colonies and large (2x or 3x the small colony). The large to not look too out of the ordinary, but does everyone agree I should pick the small and use this?
I can’t say for certain which colony size is the right one (though if pressed I’d recommend the small ones, or better yet a side-by-side comparison with media inoculated from a small colony versus from a large colony), but if you’re getting dramatically different colony sizes when you streak out your stock, you’ve got a mixture of different bacterial strains there, not just HB101. This would account for the extreme thickness/opacity of the lawn and for the clear appearance of the worms. Definitely grow from a single colony; if possible, streak out from a frozen stock first.