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OP50 Vs. HB101: Which Food Should I Choose?

Nematode worms such as Caenorhabditis elegans feed on bacteria as a food source. In the wild, C. elegans obtain various bacteria from rotting plants, fruit, and vegetation, but under laboratory conditions, C. elegans is normally cultured on agar plates with a standardized bacterial lawn.  There are many bacterial diets used for culturing C. elegans. The E. coli strain OP50 is widely used because Sydney Brenner, who first introduced C. elegans as a model organism, maintained them on this strain. OP50 is a uracil auxotroph derived from the standard E. coli strain B. We can use other bacteria for culturing C. elegans, depending on the purpose of our experiments. For instance, HT115 is an RNAse III-deficient strain used for RNAi feeding experiments. HB101 is a hybrid of E. coli strain B and K12 and is commonly used for liquid culture of C. elegans. Most worm labs, including our own, mainly use  OP50 and HB101. The worms’ bacterial diets will impact their physiology and behavior in experiments,  so let’s compare the key differences between growing worms on OP50 and HB101.

1) Growth and Size

When you culture N2 wild-type on OP50 or HB101, you can see through a microscope that the adult worms are larger on  HB101 than on OP50. So et al. (2011) demonstrated that hermaphrodite and male C. elegans body size is 1.6-fold and 1.2-fold larger, respectively, when the animals are fed on HB101 (Figure 1, A). Also, they confirmed that even when OP50 is replaced by HB101 in larvae or adult stage, N2 grows bigger than when fed on OP50 (Figure 1, B). These differences can affect the interpretation not only of growth and size experiments, but also morphology and movement assays.

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Figure 1. Adapted from So, S., Miyahara, K. and Ohshima, Y. (2011), Control of body size in C. elegans dependent on food and insulin/IGF‐1 signal. Genes to Cells, 16: 639-651. https://doi.org/10.1111/j.1365-2443.2011.01514.x 

2) Brood size

The next thing you might notice when growing N2 on OP50 and HB101 is a difference in the production of embryos.

According to a study by  Sowa et al. (2015), HB101-fed worms take less time to initiate  egg-laying and stop laying earlier than OP50-fed worms (Figure 2, A and D). Although the reproductive lifespan of HB101-fed worms is ~65% shorter than OP50-fed worms, the total brood size is not significantly different (Figure 2, B).  Although the overall brood size remains the same, the changes in timing could affect interpretation of fecundity assays and the timing for synchronization or expansion of worm populations.

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Figure 2. Adapted from Sowa, J. N., Mutlu, A. S., Xia, F., & Wang, M. C. (2015). Olfaction Modulates Reproductive Plasticity through Neuroendocrine Signaling in Caenorhabditis elegans. Current biology : CB, 25(17), 2284–2289. https://doi.org/10.1016/j.cub.2015.07.023

3) Macronutrients

When you compare bacterial lawn thickness of OP50 or HB101 growing on NGM plates, you can see HB101 is thicker than OP50 (Figure 3). The uracil auxotrophy of OP50 restricts its growth on NGM agar, which is useful for applications that might be encumbered by a heavy bacterial lawn.


Figure 3. Photo taken in the InVivo Biosystems Lab.  300µl of bacterial culture seeded on NGM agar, dried, and allowed to grow for 2 days.

Brooks et al. (2009) found that even though HB101 is visually thicker than OP50, the number of cells is not significantly different (Figure 4, A). However, HB101 dry weight per cell is slightly higher than OP50 (Figure 4, D). They also measured macronutrient amounts per cell. OP50 and HB101 contained similar levels of protein and fatty acid (Figure 4, B and C), but HB101 contained 3-5 times higher total carbohydrate levels than OP50 (Figure 4, E).  In studies such as aging, where nutrient availability impacts phenotype, it is critical to consider the food input. Experiments involving RNAi by feeding should also take into consideration the effect of switching worms to a diet of HT115.

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Figure 4. Adapted from Brooks, K. K., Liang, B., & Watts, J. L. (2009). The influence of bacterial diet on fat storage in C. elegans. PloS one, 4(10), e7545. https://doi.org/10.1371/journal.pone.0007545

These differences in C. elegans based on their diet can have profound consequences for your experiment. It is good culture practice to keep the bacterial diet consistent within experiments. Take these differences into consideration when selecting your bacterial diet and when comparing between experiments using different bacterial diets.

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