Hi Imran,

These bacteria that we had seem to only give problems when the cells are stressed.  We don't routinely use antibiotics, but some people use pen/strep - I don't remember the exact concentration.  We also tried gentamycin when we had this problem, but it is a bit toxic to the cells.  If you have problems with fungal contamination you can use amphotericin B.

best,

peggy

I write this additional to Tims recent post (topic: "failing scale up in insect cell culture") to keep you informed about our contamination problem and maybe get some feedback about similar observations:

in our media (Sf900III SFM from invitrogen/ Lot: 1211602) we noticed some precipitation in the flasks, which accumulates as "smear" at some points of the bottom.Its already visible, when we get the media out of the fridge, but the media is not turbid!

By examining under the microscope we see small oval and round particles which move back and forth and partly form larger "aggregates",they grow very slow,and in the cultures they only appear rarely and cells grow as usual.They only seem to affect the expression in High Five cells, Virusamplification and Expression in Sf9 still works despite the contamination.

I complained about the media at invitrogen and they immediately replaced the 60flasks and also a High Five stock, which I grew up with this media.Now I´m still waiting for the analysis in the QC-lab of invitrogen.

I´m extremely interested if someone else has seen something similar.If you find precipitates in your media- please investigate under the microscope and tell me your opinion: I would be only too pleased, to know what it is!!

looking forward to your reply!

a question to Sabine: what does the DSMZ charge for the identification?

Astrid

Dear Astrid and others,

I was really glad to see that other people have the same problems since I started to doubt my skills XD

Since 3 to 4 months, my colleauge and I try to express secreted proteins in baculovirus-infected Hi5 cells. We use different virus stocks for different proteins and reproduced the virus stocks from different stages (new bacmid preps or new transfection of Sf9 cells with old or new bacmid or reproduction by infection of Sf9 with "old" virus stocks). We both thawed Sf9 or Hi5 or infected or changed everything you could think of. I even thought that L-glutamine could be the problem since we were using PAA L-glutamine and then changed to L-glutamine by Life Technologies.

Then we even bought new Hi5 cells from Life Technologies (Lot: 1264798). I tried to thaw these cells but it was really difficult. There were almost no cells alive. Therefore I separated the floaters and managed within a week or so to grow the cells to put them into a shaker flask.

Finally, I tried to express the protein in the new Hi5 cells but again: no protein in the cell culture supernatant but small round moving dots. These "paricles" are very small but visible. In the beginning I thought, it would be cell debris or budded virus or something like that. But now I am pretty sure that this is a contamination. Now, that I thought about all these statements here and what we have tried and done, I am convinced that the problem is the Hi5 medium. We have Express Five (Lot: 1236538) and I am not 100 % sure but I think that our problem occured with this lot of medium. We added 1 % Pen/Strep and 0.1 % Fungizone (both GIBCO) and sterile filtered the medium. I hope that will work at the moment until the medium is exchanged by another (working) lot.

Our hypothesis is that these contamination either takes up the secreted proteins or maybe produces proteases since only the production of secreted protein is affected (intracellular YFP is always there!).

So, I hope I could help someone with our experience ;) Good luck!

We have had contaminations with Sf9 cells for almost a year now - some occasional outbreaks and more paceful times.

I have found the descriptions from Peggy and Sabine most familiar.

We use Sf9 cells from three different origins: our own stocks from 10 years ago purchased from a local company and i don't know the actual sourse; stocks from neighbour lab and they do not know the sourse. and I ordered a new stock from BD Boosciences (nr.551407).

We do not use antibiotics in our medium.

We use ExCell media.

We reuse plastic erlenmeyer flasks (different volumes) for culture. We wash with 7x detergent, rinse thourughly with water and autoclave (40 min with water + 40 minutes dry).

The contamination problem occurs with all of these cells:
Sf9 cells grow nicely, the viability however does not exceed 90%. After achieving the exponential division phase they soon go completly comtaminated overnight, from 90% viable cells to white haze in the flask. I now think it is a similar bacterian contamination that das been proposed here. The contaminated culture smells like sour milk. When we kill the culture with Virkon-S it smells like vomit.

This white haze outbreak has a lag period of 1-3 weeks. We have brougt on a clean Sf9 culture from the neighbours and the viability starts to sabilize at 80-90 for a couple of weeks (the cells divide normally!) and then there is the outbreak overnight, very rapid!

The new BD Boosciences culture got contaminated 2 weeks after the culture start.

The sourse of the contamination has to come from our lab! The neighbours culture the cells at same conditions without any problems and with no need for antibiootics.

Sabine said their contamination was identified as Phyllobacterium myrsinacarum IAM 13584. How did you get rid of it?

Dear Olga,

our source of contamination was the outside and came in with the air, every year in spring, when flowering started.

Based on the identification which costed ca 250 Eur, we could convince the admin that we need money for an air inlet filter (which are routinely used for cell culture labs, but we had at that time a former organic chemistry lab)

Since then, there is no more problem, we use Powder ExCell420 for SF9 and ExCell405 for H5 and decontaminate our reused glass Erlenmeyer with Virkon and then wash in a dish washer and autoclave (whereas we bake every glass ware used for E.coli due to the phages) Viability is constantly about 95%

We never use antibiotic and I would not recommend to do so, as you lose control over the presence of contaminations in all your stocks.

As Peggy mentioned previously, we think these bacteria typically co-exist in almost undetectable amounts with insect cells, but as soon as these are sick or the load of bateria is unusually high (as in spring time) the natural equilibrium shifts to the bacteria.

Sorry Sabine, it seems i cannot read your post!

Dear Olga,

my internet  collapsed and the (long !) answer was lost.

Just in short: with the identification at DSMZ (ca 250 Eur) it was obvious, that the contamination is plant associated and came in with the air, pollen esp in spring time,

We installed an air-inlet filter, which is routinely present in cell culture labs, but at that time we had worked in a former chemistry lab.

With the filter installed, problems were gone.  I would not recommend to use antibiotic, you will lose control over the presence of contaminations!

We had a 2nd problem with medium, but now we use ExCell420 for SF9 and ExCell405 for H5.

Best
Sabine

Hi Sabine,

interestingly, my RSS feed showed your long posting although it's not displayed on the forum page. Here is your lost text:

Hi everyone,

I thought I would also post an update as this thread is still ongoing.  I also sent some of our contaminating bacteria to the DSMZ and they were identified as being most similar to Kocuria marina, which is a bacteria closely related to Micrococcus.  This identification was not confirmed in all of the tests, though, so it was not 100% conclusive.  I tried to isolate them and culture them alone so that there would not be any insect cells in the culture that I sent them, but as they grew so slowly it was difficult to do this.  In the end what I sent in looked like the same bacteria, but I can't guarantee it was not something else that started growing in there as well!

In the end we could not isolate the source of our contamination.  The only possibility left was that the commercial cell stocks we had came already with some small amount of these bacteria in them.  We compared with cells from a neighboring lab, but we could see these bacteria in their cultures as well upon very close inspection, even though we did these tests in fresh disposable flasks and did always a media control. This and the stories in this thread make me think that many people have stocks with this bacteria (or perhaps a similar type of bacteria) but never notice them, as they do not normally cause any problems.

In the end we got new stocks and the problem went away, but the weather also cooled down, so I suspect the root of the problem was not solved.  Normally our cells grow happily and maintain >95% viability, but over the summer with the high humidity we still occasionally see these bacterial infections, but never in our stocks, only after infecting with virus. (Or at least not at detectable levels in our stocks).  It is worse in Hi5 than in Sf9.  We try now to control the humidity in our cell culture room over the summer with a dehumidifier, and this helps.  It seems a bit illogical to me that the insect cells would prefer LOW humidity, since mammalian cells need high humidity, but for some reason this works.

So I can't really offer any recommendations except to try getting fresh stocks, and to keep the cells at low density (max of 4-5 x 10^6/mL), as this will prevent any additional stress.

best,

Peggy

Dear Peggy,

Would you have a video of these things/ be able to upload Hueseyin's video? (I know this is from some years ago now!) We have also been having contamination problems (almost exclusively with our Hi5 cells, our Sf21 seem to be far more robust), and have been going mad trying to isolate the source. We think it may be coming from our glassware/plasticware as the contaminations seem to happen essentially at random, and also when we use different stocks of media from different companies. There are no signs of microbial growth when we streak the media on agar, or incubate it in flasks without cells. The problem started in spring/summer last year, we seemed to get rid of it for a while (when we switched our glassware) but we are now seeing more contamination problems again with the warmer weather.

I can often see round, black dots that seem to jiggle back and forth under the microscope as you described (it is possible that these are cell debris moving under Brownian motion, but I get the feeling this is another type of movement, and that they are too many for this). I will look into the sequencing that you suggested.

Thanks ,

Rhian