OP50 and bacillus contamination questions!

Model Organism: Worms Scientific Discussion
1

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:

http://i.imgur.com/nknrqIt.jpg?1

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!!

http://i.imgur.com/ruQLiF3.jpg

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.

2

Hi,

it would be useful to have a little more information here on a couple of things you mentioned in your post…

  1. re: the photographs. Are these plates innoculated with OP50 such that the whole (or more or less the whole) surface is covered or something completely different (it’s been a long day)?
    Have these plates been incubated @ 37 degrees?

  2. re: the large plates you pour and dry in the flow hood. When you incubate an empty plate @ 37 degrees do you see the same contamination?
    When you incubate an empty plate at room temperature do you see the contamination?

  3. re: making the agar. Do you stir the agar when or after you add the K buffer, Mg, Ca salts and cholesterol?

Steve

3

Identifying the source of contamination is a good starting point. The plates seem fine, since the contamination is restricted to the surface. So, it’s either 1) airborne or 2) present in the seeding solution (medium, incubator, or OP50 stock). Steve’s question 2 can resolve the source. Take a few unseeded plates and treat them in the same way as you would seeded plates (i.e., leave them uncovered in the hood for an hour if that’s your standard treatment). If the same contamination appears, then it’s airborne. Be sure you’re using a laminar flow and not a chemical hood [not to impugn your technique, but others have made this mistake]. UV sterilization is fairly effective against bacilli, and get your hood’s air flow tested.

If not airborne, then your seeding stock is the culprit. Inoculation from a single colony of OP50 is good technique, which leaves the LB medium or incubator. Bacillus spores are resistant to many sterilization techniques (e.g., heat and bleach), but filtration should work. Take a batch of LB, filter half of it, use a portion of each without OP50 inoculum, incubate as you would the OP50 culture, and use to seed plates. Perform the same experiment in a different incubator.

As for OP50-1, that’s a reasonable solution (assuming that your bacillus strain is not also resistant to streptomycin). The strain can be obtained from the Caenorhabditis Genetics Center (strain link at http://www.cgc.cbs.umn.edu/strain.php?id=11078).

Good luck,
Harold

4

Thank you very much both of you!!! I will try and go through all the questions and points:

@SteveH

  1. The plates are added concentrated OP50 from a stock (which we keep in the fridge) using a sterile pasteur pipet in the laminar flow hood.
    The plates are then left in the laminar flow hood to settle, that is to let them dry up a bit so everything is not floating around, but with the lids ON the plates!
    Sometimes I let the plates stand like this in the laminar hood overnight, but I have never put them in an incubator (neither 20-25C or 37C). Should I do that?
    So to answer your question, no the whole surface of the plates are not covered. OP50 is distributed as shown on the pictures.
    As to why I do this I do not know, it is not something I have been taught to do but just something I thought was best because it would increase the accessible
    surface area of the OP50 to the worms and thus maybe give higher yields.

So I am more than willing to hear how anyone else is making their plates, especially if it is also the relative large plates (15 cm diameter) or why my method may be flawed.

  1. that is a really simple control that I have not exactly done, thank you for that! As mentioned I have done some controls, one being adding OP50 (as described above)
    to a plate without adding anything else (no hypo treated eggs or live worms) and then store it together with the other plates in the 23C incubator. The plate did not get
    bacillus contamination but it did get the weird specks in the OP50 drops as seen on the pictures. to me that shows that I either introduce the bacillus spore in the step
    where I add the hypo treated eggs or that the spores are already there and only germinates when moist OP50 is added on top of them on the plates. That is why they only
    grow inside the OP50 when nothing else is added.

  2. When mixing the agar I do this under constant magnetic stirring so everything should be mixed fine. Though the thought that the additives have gone bad (except the cholesterol which is in EtOH) have crossed my mind earlier.

@Sperm or Egg?
Yes the restriction of the contamination to the surface of the plates indeed do point in the directions you mention, which is why we just did the chlorine bleach cleaning of the cell lab.
I am curios as to how you dry your plates if not by letting them stand with no lid on, in the laminar hood?
I will follow SteveH suggestion for the control with the plates.

We have UV light in our hood, but I was of the understanding that UV did nothing to bacillus spores?
Regardless the UV is running every night, but not when the plates are in there. maybe that is a mistake?
We have had the airflow and HEPA filter tested by our engineering department, and everything was okay.

Yes the seeding stock have been my main suspect since the start. Because I am after large quantities of worms we go through our OP50 stocks rather quickly, so they are not left in the fridge more than 1-2 weeks.
Thus we grow OP50 in 10-20 liter batches or in 10L fermentors and the will only last for about 2-3 batches of 10xplates. They eat a lot!!
However we dont have any autoclavable cell harvesting tubes, so we clean the tubes in 70% ETOH before use each time, and try to do as much of the
handling in the laminar flow hood as possible. However bacillus spores are not readily killed by 70% EtOH so that may have no effect at all. But my small control experiments seem
to show that we introduce the contamination with the hypo treated worms/eggs step.

I have no clue but is it possible for either bacillus or bacillus spores (most likely spores I guess) to live inside the gut of the worms and thus be exposed or released
during hypochlorite treatment??

Your suggestion of filtration is simple but really good and quite easy to test!! I must admit I had not thought of that and I will do that right away.

Thank you for the link for the OP50-1 strain, I will see if they are willing to ship to Denmark.

Again thank you very much for your help! It means more than you think.

/Ruki

5

Hi,

from what you say in your second post I would make the following observations/comments/suggestions;

  1. concentrated OP50 stock…we have stocks of OP50 (grown as O/N shaking cultures) in our fridge. However, these are just cultures and are not centrifuged down to make the 50% preps that one might find frozen in the -20 and used for liquid culture for example. So you could try using a standard OP50 culture.
    I would suggest you add OP50 culture to your plates and spread it out over the surface with a bent glass Pasteur spreader. This will be absorbed quickly and so not slop around if the plates are moved after a few minutes. We use a homemade flow cabinet fitted with a HEPA filter for plate inoculation and that works fine. I guess with 150mm diameter plates you should be pipetting onto the agar surface about 225-250µL of OP50. the worms swim in the OP50 soup, so blobbing ‘picnic tables’ of of OP50 over the plate makes no sense…the worms have to move from
    blob to blob. The dried plates should then be incubated @ 37 degrees at least 8 hours and preferably O/N to allow the OP50 to establish.

  2. The pattern on your plates, blobs and ghosts is of course (now I look at it again) because the blobs have moved at some stage. The concentrated OP50 produces a thick blob and the place where the blob was has a few colonies in a ring.
    I’m still not convinced that these mysterious specks are not anything more than a result of poor mixing or too high a temperature during the NGM agar prep. Check your waterbath temp perhaps? As Harold said, checking the solutions added after the autoclaving step by filtering aliquots and mking parallel agar preps with filtered and unfiltered solutions might identify the rougue solution. a

Other suggestions/comments etc.

We routinely dry our plates with the lids half off in our Cat. 2 hood and have no contamination problems… use some test plates to check your HEPA.

After you bleach the worms and stop the reaction, what do you use to rinse the eggs? Have you tested your water on the plates?

But one point sticks with me, what was the strain identified from your sample sent to be tested? You say bacillus (which describes OP50), but do you mean Bacillus (which does not)? Knowing the identity may help track down where it is hiding.

Steve

6

Hi again

I have just been doing some small controls.

I took 7 large NGM plates that all have been drying in our laminar hood, and did the following:

1 plate added OP50 from our concentrated stock
1 plate added S-basal buffer
1 plate added Elga water
1 plate added autoclaved elga water
1 plate added added LB medium
1 empty plate

all above plates were placed in our 23C incubator

1 empty plate at 37C.

the plates have been incubating for a day now, and they all look perfectly fine so fare. I will give them a couple of days more to be sure.
It should be noted that we did clean our whole cell lab rather harshly in chlorine again yesterday, so we may have removed all the contamination that way.

Regarding the amount of OP50 need and the use of concentrated stocks we can simply not use any less, I wish we could because it takes forever to make but we need a lot of bio mass!
this youtube video shows one of our plates near saturationand keep in mind one batch is ten 150 mm plates. (sorry for the quality, youtube downgrades the quality a lot for some reason)

If I understand you correctly my home-brewed idea about how to deposit the OP50 on the plates makes no sense, thus I will try and cover the plates in OP50 instead and let them incubate ON at 37C before adding worms to it.

I wash my worms in either ddH2O or autoclaved S-basal buffer. I have seen no real change in using either, is one better than the other?

regarding the identification of our contamination it is of my understanding the OP50 is E.coli and therefore stains gram negative, the bacillus we identified stained gram positive and as such is not the same genus.

I will let you know how it all works. Again thank you for your help, both of you) I am always amazed when strangers take their time to really help out other ppl with the smallest things, for no other reason than kindness, cheers!

EDIT: forgot to say: the blobs and ghost you mention are not from the blobs moving that is where i added drops of highly concentrated c.elegans eggs. it was plate where I did a very harsh hypo
of a lot of worms to see how many eggs I could get and how fast I could reach a saturated plate. The blobs are OP50, and they do not move.

/Ruki