I have tried YEB, YEM, LB mediums but still facing the problem. temrature: 28c; rpm: tried 190 and 230; antibiotic rifampisin(tried 10, 25 and 50mg/l) and kanamycin(50mg/l)
also I tried culture without antibiotic still I had the problem.
If you use YEP medium (yeast extract-1g, Peptone -- 1 g, Nacl-0.5 g for 100 ml medium) if you use 10 mg/ml rifampicin , use same temperature and try using 150 rpm shaking overnight you can avoid clump formation. As well use fresh culture for inoculation. The clump may form due to stress. so you can avoid old cultures for inoculation. If you face the same problem you can also try using half anibiotic concentration because agro is sensitive.
Clumping often happens when a medium is low in a nutrient other than carbon and energy (eg nitrogen, maybe something else), or very rich. Basically, the bugs have so much carbon & energy available that they make polysaccharide as a sort of external carbon storage. If you're only working at laboratory scale (it's always helpful to know that sort of thing) seek out mineral salts media for your strain. Some need biotin and/or L-glutamic acid, some can't use nitrate but most use ammonium salts. Have a look at what Bergey has to say - your University should have access.
The better you control their nutrients, the better control you have over your cultures.
If you don't want to change the medium, the addition of small quantities of detergents like tween-80 around 0.05 to 0.1 % may help. Normally this have no effect on the culture. Hope this help.
Hi Harunipriya
looks like a physical phenomenon, maybe caused of polyglucan capsules of bacteria, something that you observe in the formation of biofilms.
A physical solution would be, like Alexandro proposed, to use a detergent, I have very good experience in using Carbopol 934, an anionic detergent for filamentous funghis tom prevent the formation of bigger clusters
http://www.webpages.uidaho.edu/~bhe/pdfs/fungal.pdf
If this is not matching you can add a small amoun of a Glucanase, you have industrial mixtures for cereal industries that provide alpha and beta forms and xylanase to keep pumping circuits liquid and detsroy disturbing glucanes.
thanks for your replies ....actually I tried YEP medium also, but still the same problem, detergent suggestion is interesting will try it also, we are out of tween 80, using tween 20 instead will not be problematic, will it ?
I would suggest using a higher RPM, 250 minimum and even try 300. As far as application for transformation they are still useful even with clumping. Just spin down real quick with centrifuge and take liquid phase. Possibly check the plasmid quality with PCR or sequencing to confirm.
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