Dear All,
As we also work with phages in the lab, we unfortunately have some experience with this (although the phages we work with haven't been identified in our cultures).
I second Tassos suggestion to close down normal operations for a week or more and clean with the whole lab involved and not in a holiday. This has the advantage of more hands and more brains to think about where the contamination might be coming from - and helps to convince everybody the problem is serious.
In my opinion the most common sources are contaminated bacterial stocks, and stocks and equipment that get in contact with bacteria.
Throw away all IPTG and antibiotic solutions and make fresh from newly bought powder after the cleaning operation.
Clean all erlenmeyers that are used to grow bacteria with Virkon.
Gilson pipettes can get contaminated by aerosols and should be carefully cleaned with Virkon regularly.
- make sure they are never horizontal, especially with a tip attached: buy carrousels or similar, and don't leave them lying on the bench.
- we have decided to now only use commercially sterilised filter-tips for all bacterial and DNA work to try and avoid aerosols getting in to the pipettes; and normal, unsterilised tips for protein work.
We also keep bottles for bacterial media etc separate from other bottles and do not sterilise the second kind - the autoclave does not kill phages and can actually be a source for them...
If a culture "dies" and phage infection is suspected, be very careful with it. Don't open but throw it away in a place as far away as possible from the lab. Clean the erlenmeyer with Virkon and do not use it again for large-scale cultures. We reserve them for phage work, but you might want to just throw them away (or sterilise them at very high temperature, i.e. pyrrolysis). If you have EM, take a small sample from the culture and try to look for the phage type.
I also agree with Tassos that people can be a problem:
- the way they work has to be checked, controlled and supervised, but perhaps the biggest threat is their attitude that "I didn't have a problem, my cultures grew normally, so I am not going to change my habits or clean anything or throw anything away". Here the IP has to convince everyone of the seriousness of phage infections for the operations of the whole lab.
Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij
On 3 Mar 2013, at 10:09, aidong wrote:
> Dear Opher,
>
> It was also our idea how to get over it since several years ago. We did it based on a philosophy that any bacteria infected with a phage will not be infected by the same phage again. Therefore, we let our BL21/DE3 strain infected in the lab and when most were died, some cells were survived. We picked these to make the competent cells for expression constructs. We believe these actually contained phages. In order to avoid this bomb to blow up, we managed to get a commercial strain (TonA and TonB deletion) purchased from Sigma USA but found it useless. I wonder how you screened your phage-resistant strains.
>
> In addition, it is our regular practice to use bleach to extensively disinfect all surfaces, including benches, used flasks, instruments and floors. Would virkon work more efficiently in our case?
>
> Sincerely,
>
> Aidong
>
>
> On Mar 3, 2013, at 3:36 AM, Opher Gileadi wrote:
>
> Hi,
>
> We had a massive phage infection at the SGC in 2004; all our washing and sterilization efforts could only solve the problem temporarily. I then recovered a phage-resistant subclone of BL21(DE3), and prepared derivative strains with pRARE2 (the tRNA plasmid in Rosetta2) or other plasmids. We have expressed thousands of protein since and never had a recurrence of phage problems. Although the resistance is to T1 phages, it seems these are the most common lab infections.
>
> Write me if you want the strains.
>
> Opher
>
> Aidong Han, Ph.D
>
> Department of Biomedical Sciences
> School of Life Sciences
> Xiamen University
> 3 South Xiangan Road
> Xiangan, Xiamen 361102
> China
> Phone: 0592-218-8172 (O)
> 0592-218-8173 (L)
> Web: http://life.xmu.edu.cn/adhanlab/
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