Last week, we started experiments aiming to test the quality of freeze dried bacteria (LabTie) as a viable food source for C. elegans culture and microfluidics usage. This week, we took a first look at the development of N2s when fed freeze dried bacteria from L1 stage. I have 4 questions in mind:
- Are freeze dried bacteria suitable for use in microfluidics?
- Do worms develop at a normal rate when fed only freeze dried bacteria?
- Are freeze dried bacteria more susceptible to contamination?
- Are freeze dried bacteria even alive?
1) Freeze dried bacteria for microfluidics:
Well, I must say that I am a bit disappointed so far. I really hoped that using freeze dried bacteria would be a great time saver and would increase data consistency. However, it turns out that we still cannot resuspend the bacterial clumps quite well enough for this to be a good solution for microfluidics (see Freeze dried bacteria: worth the hype for C. elegans? – Part 2).
We tried 3 concentrations of freeze dried bacteria: 50 mg/ml, 2.5 mg/ml and 8mg/ml, and tried to homogenized the solution by pipetting 1mL up and down in an epitube, like we do for fresh OP50. This did not do the trick. We also tried vortexing the bacterial solution for 30 seconds, without success.
We could try sonicating the solution, but this is likely to pulverize the bacteria, and I doubt that the worms would readily eat pulverized bacteria (Yes, the freeze dried bacteria are alive and growing – more about this below).
2) Freeze dried bacteria for basic worm culture on plates:
So far N2s seem to be growing well on freeze dried bacteria lawns. We did reduce the amount of bacteria used from 4 dots to 3 per plate, which is more than enough to keep about 100 worms fed for days.
We are currently testing feeding behavior and size at Day 1, Day 5, Day 10 and Day 15 for worms that have been fed freeze dried bacteria from L1 stage and placed on FUDR plates from L4 until D15.
Here are our preliminary observations:
1) L1-L2: Starved, post-bleaching L1 worms placed on a plate with freeze dried bacteria tend to remain outside the lawn, compared to L1 placed on a lawn of fresh OP50 (as observed 5h after plating on 3 plates per condition). Likewise, 28h after plating, L2 worms are still more readily found outside of the freeze dried OP50 lawns. In comparison, most if not all L2s were found on the fresh OP50 lawns on each of the 3 plates (figure 1).
Figure 1: (Up) typical distribution of worms on fresh OP50 (left) or freeze dried OP50 (right) lawns. L1 (5h post plating – not shown) and L2 (28h post plating) worms are found mostly outside of the freeze dried OP50 lawns. (Down) Representative image of L2 worms (10x) found outside of OP50 lawns for fresh OP50 (left) and freeze dried OP50 (right). L1 were plated in comparable amount on each plates after bleaching.
2) By Day 1 however, worms are found near or in the lawn of freeze dried bacteria and I can see their pharynx pumping. We are currently testing feeding pattern and quality with the ScreenChip platform using 5HT as a pumping stimulus. Although this will not allow us see how the freeze dried bacteria are ingested, it may shed light on the potential effect of such food source on the pharynx development and aging.
- Starting from one batch of bleached eggs, I plated L1s on 6 plates: 3 fresh OP50 lawns, and 3 with freeze dried OP50 lawns. Looking at the L2s today, I noticed what seemed to be a larger discrepancy of worm size on the plated with freeze dried bacteria.
- Day 1 adults: 3 days after plating, plates seeded with freeze dried bacteria contain less eggs than fresh OP50 plates. Worms seem to catch up by D2. Interestingly, D1 worms grown on freeze dried bacteria are comparable in size to the ones cultivated under standard conditions, since both groups fit well in the ScreenChip channel (40um pinch point).Figure 2: Comparison of number of eggs on plate 3 days after plating (D1 adult N2s). More eggs were found on the plate where worms were cultivated on fresh OP50, compared to freeze dried OP50.
- Day 5 worms grown on freeze dried bacteria appeared to be smaller than Day 5 worms grown on OP50. We used a larger chip that for Day 1 adults (60um pinch point). Worms grown on fresh OP50 readily stayed in the channel for the duration of the recording. In contrast, worms grown on freeze dried bacteria, tended to slip out right out of the chip. (Note: the freeze dried bacteria worms were tested 3h before fresh OP50 worms, however we don’t believe that this interval would be sufficient to observe such a difference in size.)
- Are freeze dried bacteria more sensitive to contamination?
Not really… At least not the same type of fungal contamination that we usually observe in our lab.On fresh OP50, we tend to see fungal growth (usually a green patch on a plate) when the plates are left at room temperature. I left 2 plates seeded with freeze dried bacteria on my bench for 5 days. By day 2-3, I could see that the appearance of the lawns were changing. I hypothesized that the bacteria were just growing fast at room temperature. In particular, one of them showed a tree-like growth from one of the lawns (noted as “fungal contamination” in figure 3). Looking at these arborizations under the microscope (10x), I observed that the “islands” are rotating pretty fast (the video below is only accelerated 2x). Note that this only happened on 1 of 2 plates and did not happen to the plate I left in the incubator. Let me know if you agree with my diagnostic or if you think these formations are bacterial colonies.Figure 3: Picture of freeze-dried bacteria lawns the day of seeding (Day 1) and 5 days after seeding (Day 5) with freeze dried bacteria (no worms).
Video 1: Video showing the structure and movement of tree-like structure noted as “fungal contamination?” in figure 3 (accelerated 2x, 10x objective)
- Are freeze dried bacteria alive?
I asked this question to Marijke Millenaar, from LabTie, who was kind enough to provide me with a pretty thorough answer. She wrote:
“We do get the question […] often. Probably because most researchers associate freeze-drying with killing microorganisms. However, it is actually a method that has been used for many years to preserve materials such as food, viruses, plasma and in this case also bacteria. So what happens is that the material is placed under vacuum, which allows the water to go straight into its gas phase (sublimation).
If you perform this under the right conditions the product will not be damaged at all. Once you re-hydrate, it will return to it’s original state. The OP50 is in an “inactivated” while it is dehydrated, and is activated once it is in contact with liquid again. So the freeze-drying in combination with growing the OP50 in very large volumes allows us to standardize the OP50 and ensure that it can be preserved and used for a long time period.”
To test this ourselves, we seeded 10mL of LB with diluted freeze dried bacteria and left it incubate overnight on the nutator at room temperature. By the morning, the solution was more cloudy. Yup, alive indeed.
So far the freeze dried bacteria proved to be unusable in microfluidics channels 40um or smaller and the preliminary observations of worm development and aging does not look promising. I will need to quantify bodily growth and maybe egg-laying, but at first look it seems that when cultivated on freeze dried bacteria lawns, worms tend to avoid the lawns and show developmental delay or at least a larger size discrepancy between individuals that under normal conditions.
My next step will be to analyze the videos and images that I have taken and see if my hunch is correct. Stay tuned.