C. elegans Synchronizer

Let’s start with what it is NOT. The CES is NOT a sieve trying to separate L1’s from L2, L3 etc.  The CES is a system that with a two-step protocol will harvest L1’s at the moment they hatch from the egg’s. The main two (2) steps in the protocol are :

Yes, you can use small volumes of a worms. Recommended is a minimum of 10 gravid adults and already laid eggs.  For up to ~1k of L1's to be harvested, the CES-700 is typically recommended.  For larger volumes of L1's the CES-5000 is more suitable. Please contact us for advice or check the comparison table below.

As a starting point, for most (single strain) assays the default kits (one Stabilizer/washing filter + one Harvest filter) will suffice.

The thing to keep in mind is that after the washing (first step of the protocol), the first filter (stabilizing filter) will become available again after some cleaning (0.5M NaOH and preferable using (40khz) small ultrasonic cleaner). However, the 2nd filter (Harvest Filter) will remain occupied for as long as you are ‘Harvesting’ L1’s. This can be as short as 15 minutes, however if you need very large number L1’s, this can be several hours or typically overnight. During this period of ‘harvesting’ you cannot process a second strain.

If you need to process several strains at once (concurrently versus sequentially), you would need an extra Harvest filter for each strain you want to process concurrently.

You typically don’t need to have extra Stabilizing filters as this filter can be cleaned relatively quick and then be used again for the next strain.

If the gravid adults are without food for a longer period, they will ultimately stop laying eggs. One reason we recommend to start with as ‘many eggs ’as possible as the gravid adults will stop laying eggs after some hours without food.

For optimal/high yield of L1’s the majority of the C. elegans culture should consists of gravid adults and as many eggs as possible when starting the protocol. When working from ‘NGM plates’ it is therefore recommended to wash the worms from the plates into a liquid (culture) with sufficient food (OP50/HB101) while sufficiently oxygenated and let the worms lay egg’s for at least 8 to 12 hours in order to maximize the number of eggs being available before starting the CES protocol.

If ‘food arrest’ is not required as part of the assay/synchronization, you could consider adding ‘freeze dried E. coli’ during the Harvest period, however this also means that the L1’s will also develop / grow (no food arrest). For some assays preventing food starvation may even be required and if large number of L1’s are to be harvested, adding food to the Harvest media is an option. The advantage of using freeze-dried E. coli is it will use less oxygen (if any) as the freeze-dried bacteria will first need to recover / hydrate and even if a limited number of bacteria survive the freeze-drying, the E. coli will have a long lag time before starting to become active and start consuming oxygen. There is a source for freeze-dried E. coli if you don’t want to make it yourself: https://www.biovirid.com/nematodes  Or you can make it yourself by freezing a concentrated pellet of bacteria as the freeze-thaw cycle will kill (die-off) most of the E. coli.

Most eggs are carried over as they tend to clump/stick together and therefor don’t get washed out in the first step of the CES protocol. This is also the reason to try and get as many egg’s in your culture before starting the CES protocol. This also implies that ‘Tween and/or Triton’, or comparable products, should not be used in preparing the culture before the CES protocol step as this will also prevent the eggs from sticking together.

At the Harvest stage, you will find that only the L1’s pass through. Egg sediment typically stays on top of the Harvest filter.

The first Stabilization filter apertures are approximately 20um. This means that nematodes smaller than L3-L4 and debris will pass through as long as it is smaller (in width) than approximately 20 um. This will therefor allow most L3 still to pass through if they are not older than 20-24 hours as they tend grow to approximately 20-22 um width on NGM Plates at room temperature, having food available. L3’s from a liquid culture are somewhat smaller (width) compared to NGM plate cultures. Even if not all L3 will pass through in the first 'wash step' then this is not a problem as the next filter (Harvest) aperture is approximately 10 um and therefor if any L2, L3 where still carried over, they will not be able to pass through the Harvest filter. Only new hatchlings (L1’s) will pass through in the 2nd step of Harvesting. Keep in mind that most eggs will not pass through as they tend to stick together. This however is what we want as we want as many egg's as possible to be transfered to the Harvest filter to have a quick start.

Yes, the CES can be used for life span assays. As a start it is required to have a 'tight' synchronized population of Adults , i.e. make sure when using the CES protocol for 'Adult separation' that there are no L3, L4 and young adults as they may not pass through the first (Stabilizing) filter as the aperture size of this filter is approximately 20um. L3 may still pass through, however this is not guaranteed as the average width of an L3 (from NGM plates) is estimated at approximately 20-22 μm. This is also why it is recommended to repeat the 'Adult separation' cycle every 24 hours. Any longer and this may cause off-springs to grow larges than ">20um width"  and the seperation may become less effective. You should test this maximum time window as this will also depend on temperature and food availability. The protocol for 'Adult Separation' can be found and downloaded below.

Preferred method is to use 0.5M NaOH (Sodium Hydroxide) and a small ultrasonic cleaner.   The ultrasonic cleaner will ensure the filters are truly clean, while speeding up the process of cleaning. This will typically also take care of scaling.  Do NOT use an acid to descale the filters.  Using an acid will cause coloring/stains of the filters and if done repeatedly it will ultimately damage the filters.  

Preferred method is to use 70% isopropyl alcohol, either use a spray or submerge the filters completely and afterwards let the filters dry in a laminar flow hood or rinse it afterwards with (sterile demi water) to get rid of any isopropyl alcohol left.

Nearly four years were spent on the journey to achieve perfect functionality.  Unlike traditional filters, the CES filters do not have 'round' or 'semi-round' pores/apertures however feature a special elongated design (slit) in order to prevent the filters from clogging while allowing L1’s to pass through with ease. In order to get the unique level of synchronization the CES provides, every single aperture in the filter needs to be ‘zero defect’.

There are currently NO commercial membranes available that meets the required specifications and therefor a different technology was needed. More information can be found on the following page: Why the CES filters are Special

Summary:

CES-700: if you need between ~10 and less than 1k ~ 2k L1's with a short Harvest window or up to 5k~10k L1's with a larger Harvest window,  the CES-700 will do the job. (this assumes ~1k gravid adults to be available to start with, (less is always possible)

CES-5000: if you need more than ~10k L1's with a short Harvest window or up to and excess of 100k or more per Harvest filter  (multiple Harvest filters in parallel are always possible) then the CES-5000 is the preferred system.

Please note that the length of the 'Harvest Window' has a huge impact on the number of L1's harvested. The 'Gravid : L1' ratio is approximately 3-5 L1's per hour per available gravid nematode, after a 'lag' time of 7-8 hours (the time the egg's need to develop and hatch), assuming there are 'no' egg's to start with. Adding Serotonin at the Harvest step will give an initial 'burst' of 10-15 egg's per gravid nematode that can be harvested approximately 7-8 hours later. Adding Serotonin is a known method of stimulating the egg production and is used to get to the 'magic' 1 million⁺ number of perfect synchronized  L1's. (See remarks CES protocol)