forget to add cholesterol, CaCl2, Pot Phos etc

Dear fellows
yesterday i made NGM plates and forget to add the chemicals (cholesterol, Pot Phos, Cacl2 etc)
Plates become solidify and I also paste OP50 bacteria. Now what to do with them…
can I use these plates or just throw them…

Secondly how to get rid of moisture and contamination… some times I felt water on NGM solid surface

Regards
Waqar

Hi,

yes, throw the plates away…if you poured like 200 plates then that’s a pain I know, but NGM missing all of these items gives you loads of problems particularly decreased brood size and delays in/lack of development. This compromises anything you might subsequently do with the worms you plate out. Plus, it’s not that much work to pour new plates is it?

Regarding moisture in / droplets on your plates…dry them out after they have cooled with the lids slightly open in a sterile cabinet until the moisture is driven off. The moisture content of NGM plates is such that plates dried for too short a period will ‘magically’ regain moisture when incubated at 37 degrees with OP50 and then RT with worms…so dry thoroughly.

As for contamination…it depends what the contamination is and when you see it…

Is it contamination of the NGM plates? (see link): http://forums.wormbase.org/index.php?topic=1709.msg3856#msg3856

Is it contamination of the OP50? Then start a new OP50 culture from a plate.

Is it mold spores? Then see the link above or trying cleaning your strain by bleaching.

However, a bit more info would be useful.

Steve

Thanks steve for your suggestions

Regards
waqar

Hi,

I just saw this post. I did similar mistakes but not exactly similar.

I added 5 times more than normal, the amount of KPO4, MgCl2, etc… in NGM Agar.
Can I use these plates? or I will throw them also?

Thanks.

Throw it away!