Dear Aidong,
Some thoughts...
Are you sure the problem is actually phage-related? I ask because you are still having problems using phage-resistant stains...
Acid-washing your glassware, and a long-dry autoclave sterilisation (as previously suggested) should really do the trick. A good *manual* wash-out of your incubators would also certainly not hurt.
Clean / replace any filters in the incubators *and* Air-con if you have it! It's amazing what can creep in there...
Residual detergent in glassware can cause bacterial lysis.
Also confirm that your autoclaves are actually reaching and maintaining their temperature during media / glassware sterilisation.
Apologies if you've already tried all this!
With regards, Antony.
Sent from my iPhone
> Dear Aidong,
>
> we never had that problem but I heard that only long dry(!) heat treatment of glassware destroys the phages.
> No need to say that you have to do it with ALL your glassware.
>
> On 3/2/2013 12:15 PM, aidong wrote:
>> Many thanks for your rapid inputs.
>>
>> We used to make glyerol stocks but now we do not since the protein expression is not stable. Our lab is about 10 students, who have to make their own proteins so our three large-scale shakers are very crowded every day. We did try a formaldehyde gas treatment one time during our new year leave, however, the situation did not seem to improve. Since that gas is much harder to handle, we only stick to ozone treatment. Intriguingly, our neighbor lab, also a crystallography lab, which constantly uses bacterial cultures although not as much as we do, is just fine.
>>
>> Chloroform/phenol treatment might be a good one because some of our constructs are just very difficult to get any transformed colonies while some others are so easy. We have used gel extraction method to purify our plasmids because we thought some phage DNA is contaminated. This method slightly improved our situations but not complete. But I am thinking the entire phages are simply there in plasmid preps purified through miniprep columns.
>>
>> Cheers
>>
>> Aidong
>>
>>
>> On Mar 2, 2013, at 7:30 PM, DUFF, Anthony wrote:
>>
>> Hi Aidong,
>>
>> some ideas...
>>
>>
>> Do you use glycerol stocks of transformed expression cells (phage risk), or do you do fresh transformations each time?
>>
>> Do you have many people working together, or do you have periods of bacterial growth separated by periods of inactivity?
>>
>> Do you have neighboring labs who may be working with, or infected with phage?
>>
>> Do you have any old stocks, liquid media, antibiotics, etc, that could be contaminated?
>>
>> sincerely,
>>
>> Anthony
>>
>> ________________________________________
>> From: CCP4 bulletin board [[log in to unmask]] on behalf of aidong [[log in to unmask]]
>> Sent: Saturday, 2 March 2013 7:50 PM
>> To: [log in to unmask]
>> Subject: [ccp4bb] laboratory phage infection
>>
>> Hi All,
>>
>> Really sorry for my non-CCP4 related question. Our lab mainly uses
>> bacterial cultures to produce targets proteins for crystallizations.
>> However, we have been struggling with phage infections to our
>> bacterial cultures for a quite long time. To control its devastating
>> effects, we have regularly been using large-dose ozone treatments on
>> the whole lab space. We have also tried anti-phage BL21/DE3 strains
>> from Sigma USA but found it was still not avoidable. The lab has been
>> maintained well hygienic and its outdoor environment is clean and
>> neat. We keep good ventilations, including windows and central AC
>> systems. However, phages are still eating up our cultures with very
>> low percentage of survivals. Therefore, this has been our big
>> headache. We wonder whether you have the same experience and how to
>> keep a lab free of those bugs. Your suggestions are deeply
>> appreciated. Thanks.
>>
>> Sincerely
>>
>> Aidong
>>
>> 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/
>>
>> 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/
>>
|