OP50 colony in how much ml broth

I have a very basic question. For general culturing purpose, in how much LB broth should i inoculate a single colony of OP50? I use a 50mm plate. How much is the “ideal” amount of cell density for maintaining the culture routinely?

We routinely use 20mL Universals filled with 10mL LB for OP50 cultures.

To start a new culture, we innoculate 10mL of LB with a colony from stock.

We use 60µL of an O/N culture for our 55mm NGM plates and start follow-on cultures (every 3 days) by transferring 60µL of the existing culture to a new LB universal. The OD600 is usually between 0.5 and 0.75.

If necessary, say for chemiotaxis assays or other experiments requiring more OP50, we grow up OP50 either in multiple universals… or in larger volumes of LB (for liquid culture of worms), spin down to pellet the OP50 and freeze the pellets.


How long do u use the culture before restarting…by restarting i mean start with the single colony… bcoz wen i had used it previously for a long time and i found tat cells begin to die if reuse the culture (mat like growth in broth) for a long time… and is it a good idea to do a liquid- liquid transfer of cultures??

there are obviously good reasons why liquid-liquid incoulations of new OP50 cultures might not be accepted practice as mentioned here;


However, we use a laminar flow hood when preparing new cultures and NGM/OP50 plates and regularly change our starter cultures (your other question) with freshly picked OP50 colonies from plates (every 2 weeks). Also, importantly, we check our NGM plates not just for worms but also for how the OP50 looks.

I think the main point is that sloppy technique increases the chances of contamination, good microbiological techniques minimise the risk of contamination. We are careful and we have healthy cultures (so far…).


ok thank u tat helped a lot…have u ever seen tat mat like growth in ur culture??? is it really dead OP50??? or is it somethin else??? just curious to know wat it is…

do you see these mats in an old OP50 culture that you have used multiple times to inoculate plates, or in new cultures that you have inoculated from a very old OP50 liquid culture?

The former could be dead cells and or contaminants and the latter most likely contamination.

Either way, it’s better to use fresh cultures and to change the stock regularly.


I’m not saying it’s necessarily best practice, but I would streak my bacteria (HB101 rather than OP50) and pick a single colony to inoculate 100 ml of B broth. This bottle would be left at 37 with the seal cracked for ~16 hours, and would become turbid. I would then store the streak plate and the bottle in the refrigerator and use the bottle to spot plates until it ran out or was significantly more than a month old. The bacteria settle out, so the bottle should be swirled before use. I would spot each 6cm plate with a single drop falling about 6 to 12 inches from a 10ml serological pipet (the volume of that drop depends on the shape of the pipet and the angle it’s held; the size of the lawn also depends on the distance the drop has fallen). 10cm plates would be spotted with one or two drops from a 25ml serological pipet which I would then spread around the plate with the end of the pipet. I had very good, very consistent results making lawns with these methods.

I do the same thing as Hillel but I innoculate 500mL of 2xYT. I keep the bottle until it runs out (can be a couple of months). It works well and is consistent.