I am producing C.elegans on a relative large scale to be able to purify proteins from them to be used for X-ray crystallographic studies, hence the larger than normal plates (see below).
Anyway my problem is a severe bacillus contamination as depicted below:
I also have a problem with weird specks or granules in my OP50 lawns. The specks appears after 1-2 days at 23C (or at 20C as well).
Does anyone know what they could be? Could it be bacillus in the OP50 or spores on the plates that germinates when in contact with the moist from the OP50?
NB this is a different plate than the one shown above!!
As mentioned we have been combating a quite severe bacillus contamination for a while now.
We had the contamination ID’ed (the one from the first picture not the one inside the OP50 lawns) and it was without any doubt bacillus and bacillus spores.
They were everywhere and they (the spores) could even survive rather harsh hypochlorite treatments of the worms where I change the hypochlorite
solution 8 times during a 10 min exposure or i use 2x as strong hypochlorite as normal (killed the eggs but not the spores).
The first pictures is actually from the 8x treatment.
Can bacillus spores really survive such strong hypochlorite treatments or am I maybe introducing them after the hypo treatment?
maybe via buffer or ddH2O or OP50?
I have been trying to figure out where the contamination originates from, and have tried different exclusions methods,
but nothing decisive have come from that.
We have just cleaned our whole Cell lab, flowhood, incubator etc. with chlorine and bleach
(in accordance to this paper) in an effort to kill the bacillus spores, the verdict is not
in on if it worked or not.
As mentioned we use the OP50 strain and not the OP50-1 strain (streptomycin resistance I believe) , would that be a way to go to combat bacillus?
if so do you know where can I obtain the OP50-1 strain/plasmid?
It should be mentioned that I am by no means a C.elegans expert, I am merely a crystallographer working with C.elegans so all help is much appreciated!
For reference here is my standard workflow for a 10 plates batch of C.elegans:
1Liter NGM media is used to cast 10xNGM plates (large plates:150mmX 20mm. CaCl2, MgSo4, KPO4 and Cholesterol is added after autoclaving
when media is about 55C during sterile conditions, no antibiotics are used, plates are poured manually)
I let the plates dry 1-2 hours in flow-hood before lids are put on. let them stand in hood until next day.
In the flow hood OP50 bacteria drops are deposited on the plates using sterile pasteur pipets. The OP50 Bacteria is grown in either fermentors or regular
shaking incubators using LB medium and sing a single colony for start ups everytime.
Worms from one large plate from last batch are harvested (I have used either S-basal or ddH2O for this), spin the worms down, wash them 3-4 times before
being exposed to a hypochlorite solution where the worm pellet is resuspended in 4 ml ddH2O or S-basal and then added premixed 0,5 ml KOH(5M) + 0,5 ml hypochlorite (10-15%).
The tube is vortex’ed for a fex sec and then again every 2 mins for a total of 10 mins. The hypo procedure is base on the one in the book
“C.elegans A practical approach” by I.A. Hope
After that I normally only have eggs left which are deposited on the NGM plates containing OP50 lawns.
The plates are then stored in a plastic box (cleaned in 70% EtOH) with a lid on and placed in a cell incubator at 23C for 5-7 days (depending on the number of eggs I got from the hypo)
The worms are harvested in S-basal if no contamination is present.