Identify Unknown Bug Found in Ampicillin Starter Culture

[Andy Resnick]

I'm hoping someone with more experience can help us identify these:

[PLAIN]http://img600.imageshack.us/img600/4836/1ugmlamp2crop2.jpg

This image was acquired at 40X phase contrast.

This summer, we have been trying to amplify a plasmid (provided by Monique) in E. coli. We have colonies on plates, the control plate is clear (LB agar + 50 ug/ml ampicillin), but our starter cultures have been... disagreeable.

The image above is from a starter culture obtained by innoculating LB broth + 10 ug/ml ampicillin with a single colony from the LB/ampicillin plate. The rods are (most likely) E. coli, but we don't know what those bulbous bugs are.

Unfortunately, there are bacteria that are naturally resistant to ampicillin- Pseudomonas and Klebsiella, both of which will appear similar to E coli- so we haven't ruled out contamination.

If anyone can identify what we have, I'd appreciate it.

 

[Evo]

I see measuring spoons, sunglasses, bowling pins, a spoon, a magnifying lens. Hope that helps.

 

[Ryan_m_b]

Unfortunately I can't identify what that is for you, recently I've had problems of my own with contamination (serial fungal infections in incubators; not cool). In spite of repeated attempts at using different antifungals in the media eventually I had to admit defeat, destroy everything, deep clean the lab (again) and defrost new stocks.

 

[Andy Resnick]

We think they are bacterial spores. We're trying to locate the source of contamination- hopefully it's not from the plasmid dot blot.

Bacterial spores are really hard to get rid of...

 

[lisab]

I see measuring spoons, sunglasses, bowling pins, a spoon, a magnifying lens. Hope that helps.

Aaaahahah...except for the sunglasses, that's what I saw, too !

 

[Andy Resnick]

We got confirmation that these are spores. It appears the original blot is ok, so the contamination is somewhere else (that's good news). The timing for this is pretty good, actually- I'm leaving for vacation at the end of today, so we had been planning on shutting everything down anyway.

What I found is that spores are not killed by EtOH, UV, quaternary ammonium compounds, etc- the only thing that seems to work is 10% bleach. I'll spray everything down and start fresh in 2 weeks.

 

[zomgwtf]

We got confirmation that these are spores. It appears the original blot is ok, so the contamination is somewhere else (that's good news). The timing for this is pretty good, actually- I'm leaving for vacation at the end of today, so we had been planning on shutting everything down anyway.

What I found is that spores are not killed by EtOH, UV, quaternary ammonium compounds, etc- the only thing that seems to work is 10% bleach. I'll spray everything down and start fresh in 2 weeks.

Well good thing you've found the contamination was not in the original. Also pretty good that this coincides with you're vacation. Hopefully things will be better next time.

 

[bobze]

Andy, sorry for the late reply. They are indeed spores. Or more precisely, they are live spore forming bacteria (not in a vegetative spore state).

My bet is probably on a Bacillus spp. as they are common environmental and skin organisms and spore formers. As you have surmised, spore forming bacteria can be incredibly tough to kill--Due to the nature of the bacterial spore itself.

You could be in luck though. A couple things can help us pin this down for you. Lots of those spore formers, like the Bacillus spp. I'm guessing it is, are gram positive rods--While your E. coli is a gram negative rod. GNR and GPRs have different native antibiotic resistances, which you can possibly use to your advantage.

If it is a Bacillus spp. you could add vancomycin to your media which will inhibit growth of the bacillus spp. (though it won't actually destroy the spores).

If you could, could you post up some gram stain results, a picture of how a colony looks at 24h incubation and a catalase result. That will help with identifying the organism and how you can successfully get rid of it. Short of that, if you know anyone with a Vitek or Microscan, I'd throw it on a ID plate and AST card and go from there.

Edit: And as an after thought, make sure everyone in your lab knows good fundamental sterile working techniques when working with cultures! Its always good for even seasoned veterans to brush up on their sterility practices. It might be beneficial for you hold a little "workshop", especially for any students (grad or undergrad) who will be doing cultures for you. Its easy to get into bad lab technique habits that make problems like contamination a life long drama.

 

[Ryan_m_b]

Edit: And as an after thought, make sure everyone in your lab knows good fundamental sterile working techniques when working with cultures! Its always good for even seasoned veterans to brush up on their sterility practices. It might be beneficial for you hold a little "workshop", especially for any students (grad or undergrad) who will be doing cultures for you. Its easy to get into bad lab technique habits that make problems like contamination a life long drama.

Tell me about it! I had a protracted battle with fungal infections in my lab a few months ago. After a thorough deep clean, destruction of stocks and restarting of experiments I still had to tell people off for things like opening the incubator and talking whilst nosing around.