Are you sure that you have contamination with a fungus? It doesn't look like that. This greenish, fluorescent behaviour reminds much mure on Pseudomonas contaminants. In that respect I like to support the ideas of my colleagues Andrew, Norma and others. Please follow the advice to start all your cultures with a single, checked colony, isolated from solid medium and never ever subculture over more than one experiment. Start with a new colony. This always helps.

Godd luck - Johannes.

Hi there,

Have a go with amphotericin B or nystatin which are specific antimycotics. For info:

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/6/a9528dat.pdf

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/n6261pis.pdf

Thank you very much, Dominique - I have ordered Nystatin and will use it at 50 mg/l.

Dear Dominique, could you recommend non-polyene antimicotics (without interaction with cholesterol)?

@Andrei : cycloheximide inhibits specifically eukaryotic protein synthesis.

I have some additional questions. How long and what temp are you culturing? I quite never heard about anything fungal that can grow fast enough to outgrow escherichia in a culture. Did you ever use this system and not have a pellet like this and a non-transparent  medium? If not than would you consider this to be the effect  of the protein you are expressing? If its mildly toxic to the cells it may cause them to die and effectively burst clouding your medium and producing strange pellets but still not have enough toxicity to stop culture growth.

@Dominique : Thanks a lot for real help! Cycloheximide is very suitable but with limited activity spectrum. Question is about antibiotics in qrowth media for gram-negative bacteria in presence of sterols (specifically about E. Coli, not eucaryots) - so polyene antibiotics was not suitable. Maybe you could link to publications or other peoples about such antibiotics? Thank in advance

Hi Tomasz, the incubation is approximately 24 hours. I cannot shorten on this, since growth is slow at the low temperatures I use for induction, and this seems to be exploited by something forming a black pellet (not sure if fungus). Sometimes the pellet is light-brown, so different stuff is growing in every batch. :) :/ I never had this contamination before on small scale (5 - 50 ml) but it is not easy to sterilize several 100 ml of medium by filtration and autoclaving is difficult due to my situation (small lab, no autoclave, and S2) and M9 tends to precipitate during autoclaving. Some universities (like ours) do not invest in air condition in their labs (...) and in summer the high air-humidity gives fungi perfect circumstances to thrive. So I suspect/suppose it is a fungus, but have no hard evidence to support that.

If the use of nystatin solves the issue, I will know more.

@Andrei: so you are actually looking for antibacterial agents specific against Gram-, right?

@Dominique : No, it should be reversal - against all species excluding gram-negative . Water-soluble antibiotics are prefered , high cell density culture suitable if possible. Cycloheximide is perfect but probably need complimentary compound - look for activity spectum in file

I still believe it is not a contamination. You probably start with a small pre-culture and add it to the big one for expression. This is such a huge number of cells that they would outcompete any contaminants. Especially that 24 h is not a lot for a fungi. I was growing a lot of Mycobacterium and always the pelet looked like this + medium was never fully transparent. Nevertheless, when I had some fungal contaminations in mammalian cell culture, I used Fungizone(which I guess is Amphotericin B in commercial name) and it successfully eliminated the fungi. For the future, you could also make yourself a vector with a hygromycin resistance casette. It provides also selection against eukaryotes, including fungi.

OK Tomasz, sounds interesting... I also thought that maybe it is another bacterium. I do not think it is caused by my construct, which forms inclusion bodies and is not toxic. At least I have never encountered such a strange black pellet. As an alternative, I am trying with carbenicillin, which keeps up selection longer than ampicillin.

@Andrei: OK so apart from specific inhibitors of eukaryotic protein synthesis(cycloheximide,anisomycin...), you may also try azoles (like fluconazole)  and allylamines(non polyene molecules targeting cholesterol biosynthesis).

I used to add to my E. coli OP50 liquid cultures 400 mM concentration of nystatin, after autoclaving the culture medium prior adding molecules that can be affected by the process (like antibiotics, nystatin, etc.).

Your fungus looks like a non opened Agaricus bisporus, common mushroom. Spores tend to float around everywhere, a good praxys when handling liquid cultures is to open the bottles close to a bunsen fire so the hot air will make spores float avoiding contamination.

To avoid this contamination in the future maintain aseptic conditions as much. Wearing labcoats, gloves, cleaning material, autoclaving, not eating at the workplace, etc.

@José Luis : 400mM nystatin is a lot; usual working concentration is 50µg/mL which is roughly 50µM.

José, from your experience, is it possible for a fungus to overgrow E. coli in 24 h? Even when the starting inoculum of E. coli is 0,1 OD600?

Dominique, you are right, I checked my notes and I used 25 micrograms per mL.

Hello, Peter.

Everytime I had contaminations of any fungus on a liquid medium they tend to form a threedimensional micelia (similar in structure to a dandelion flower). This looks more like the fruity body of a mushroom, that kind of growth should be imposible in 24h, and with limitant concentrations of oxygen. So I do not think is a mushroom, but it looks like one.

If your strain overproduces something, it might be that.

About the fluorescence, most peptides (like the used in LB medium) are autofluorescent on the green emission spectra. 

As I said, if you maintain aseptic conditions you should not usually have contaminations on your medium, even without antimicrobials and antimicotics on them.

I agree with Thomasz, no fungi can outgrow E.coli (Never).

Why did not you perform Gram's staining? You could have come up to some conclusion. 

Both of your centrifuge bottles show different color, are these cultures different?

Jose Luis is right in suggesting you to follow good laboratory practices because many times we do not realize that small things (precautions) can create wonders.

If you do not have an autoclave then you can use a pressure cooker for sterilizing glasswares and utensils. But mind it sterilization is the most critical thing.

You should fumigate your laboratory as per your convenience.  

The aseptic conditions in our lab are sure OK, I would say.

I meanhwile heard that the brown color might not even be a contamination, but could be E. coli in stationary phase changing color? The cultures are grown with enhanced oxygen supply, which I have never done before. It boosts the yield of protein and causes high cell densities even in minimal medium. Can E. coli switch to a metabolic state or secrete secondary metabolites which give the culture color?

I was hoping that any microbiologist or biologist with experience in E. coli cultivation (someone who has seen a pellet exactly like that before) could tell us what exactly is happening :)

Add actidione ( a common antifungal antibiotic) in the medium after sterilization and grow your E. coli. 

@Jai: Actidione is actually cycloheximide.

Exactly.......

Have you done Gram stains of the culture? I have never seen a fungal contaminant overtake an E. coli culture in such a short amount of time, and I have worked in some fungi-friendly environments...

I can´t wait to see a micrograph of your culture! You should "see" what´s happening there... If you cannot make a Gram stain, maybe a fresh preparation can tell you what kind of microorganism you have in your culture besides E. coli; from the microscopic analysis you can differentiate between mycelia, spores, yeasts or bacteria. Starting from that point,  you can plan what to do to "clean" your strain and to prevent further contamination. Of course, I agree with previous posts regarding good aseptic practice.

I think you should check the cell bank, the sterilization procedure and the integrity test of the air filter if your culture is done in fermenter. I have seen that behavior in the pellet in E. coli in the disruption debris.  

sub culture and identify the contaminant. Based on the organism , inhibitory ingredients can be added.

If there is contamination we can isolate E.coli in pure form on subculture onto solid medium by standard streaking method

I would also check your water. We had a problem once with our desalting apparatus and somehow a black precipitate occurred after autoclaving (not visible before). Either some kind of ferric oxide or some carbon residue..

Also you could do restreaking and 16S- and ITS-PCR and sequence it.

1. Streak out to single colonies (twice) to repurify your E. coli strain. 

2. Don't subculture from broth cultures more than once. Set up a starter culture from a single colony instead. 

3. Examine your sample microscopically. Gram-staining is fine, but a wet prep is quicker and easier, particularly if you have phase contrast. 

4. Fluorescent pigment is suggestive of Pseudomonas. It has a characteristic odour which I find sweet and sickly, quite different from the characteristic smell of E. coli. 

5. Depending on the genetic characteristics of your  E. coli strain, you might want to prepare some indicatory plates. MacConkey-lactose is classic, but quite a lot of lab strains are lac-negative. Beta-glucuronidase indicators are also good. 

Are you sure that you have contamination with a fungus? It doesn't look like that. This greenish, fluorescent behaviour reminds much mure on Pseudomonas contaminants. In that respect I like to support the ideas of my colleagues Andrew, Norma and others. Please follow the advice to start all your cultures with a single, checked colony, isolated from solid medium and never ever subculture over more than one experiment. Start with a new colony. This always helps.

Godd luck - Johannes.

Hello Peter,

By working with polluted water, I once had an infection by green algae. It looked like the pictures. This is extremely unlikely, but if that were the case, simply working in the dark prevent their development.

Jacques

Hi Peter Rehbein ,

Perhaps you have contaminant in one of these:

- Polluted culture media from the beginning (Sterilization, water (SDW)... etc.) and/or ;

- Working in aseptic conditions (Contamination during manipulation... etc) and/or ;

- Bad condition of conservation (Strain, Media, °T, Humidity, Darkness... etc.)

So you should check these scenarios one by one.

Good Luck !

why do you think it is a fungal contamination?!

anyway it is easy to check , subculture the samples on sabouraud dextrose agar or any other common media supported with a proper antibiotics, most common fungi will grow on such media while Ecoli can not grow....

good luck.............

Since the growth was too fast for a fungus, I think it was due to some faulty aseptic technique, already the preculture was affected... I have corrected this by sterile-filtering the preculture (which was never necessary before) and the contamination is now gone.

Thank you all for your advice from all over the world :)

Hi Peter,

I got the solution for you.

Use LabTIE OP50. They provide high quality OP50 in vials and pots. Its a monoculture and you get rid of the contamination problem we had in our lab before.

More info can be found here

https://www.labtie.com/112351-c-elegans-nutrition

Good luck!

https://www.labtie.com/112351-c-elegans-nutrition

Hey Peter, I have similar problems at the moment an just wanted to ask if you figured out what had happend?

Hi Nadine, I ended up cleaning the complete incubator and all surfaces of the benches with Peroxide solution and Isopropanol. Afterwards, the contamination did not occur again. Never found out what it was.

Best, Peter

Hey Peter, thanks for the quick response. We did the same with 70% ethanol and 1.2 % sodium hypochlorite and replaced every chemical, supplements etc. but still have the problem from time to time.

You could consider adding sterile filtration of your preculture Medium to the Workflow. Contamination does the Most harm in the preculture. The The inoculum for the main culture will then be Clean enough for a successful fermentation. Also, try to use autoinducing medium, as opening the flasks vor OD measurements can lead to contis as well...

I do not think it to be a fungal contamination. Did you get to see the fungi under the microscope. Could you send a light microscope photograph if possible. Secondly let me know what is the medium you are using and what are the components (not amount). May be I can help you better.

I think it was exhaustively described that it cannot be a fungus due to the fast growth. Must have been an environmental Bug, what exactly is not really important in the end.

Very true that it is not really important whatever it may be. Only thing that it has to be got rid off.

I disagree. Apparently, it is a problem more people are sharing than it should be ( we are talking about E. coli, fast growing and not very complicated to culture which are only some reason why it is so popular in molecular biology).

However, I have taken a microscopic image of one of our contaminated cultures. The dots look like spores to us ( could also be inclusion bodies of the protein but the morphology is not like E. coli). Maybe someone else has an idea. I would really appreciate that!

Apparently it appears like some insoluble crystalline growth.

It is interesting from a scientific perspective to find out what exact bug caused the contamination, however I still think it does not help much in solving the problem. The first and most important step must be to ensure proper aseptic handling. Without this ensured, the next conti might well be caused by a totally different bug, and you may needlessly spend time and effort looking at micrographs.

If the problem persists despite all efforts, maybe there is a problem with your cell banking. If you use cells from cryo-stocks your prepared yourself (with inadequate aseptic conditions), these have to be considered as possibly contaminated. Using them will give you contis again and again. Int this case, as pointed out above, streak out the cells until you get a single colony and start anew.

Another possibility is to use commercial aliquots of cells and transform fresh for every fermentation run or start a new cell banking from this commercial aliquot.

Besides the rationale some personal thoughts: If you are doing this inside your M.Sc. Thesis, you should consider going for the pragmatic approach and try to get back to your original research project as fast as possible - Nobody will ever give you credit for finding out which bug caused the contamination, but more likely for eliminating it and going on with your project. Scientific credit will always only come from your research project. Use your time wisely...

Best,

Peter

Also think about your pipettes.. if you have some sporulating bug, you can clean your surfaces as much as you want, as long as you sucked up spores into your pipettes. Also consider tyndallisation (sterilization, waiting for 24 h to give any spores a chance of outgrowth and another round of sterilization) of your media maybe, or at least check if everything stays sterile after autoclaving by incubating naked media and/or supplements at different temperatures.

Otherwise i agree with Peter, a master´s thesis is not the right time to solve general lab issues.

Good luck,

Florian

Dr. Andrew Jenkins

give good advice for what is you need to follow?