Hello Regiane:

You need to grow the bacteria in a minimum amount of liquid media and put the slides inside a humid chamber so they do not desiccate. I have used 25 ul drops and spread them a little with the micropipet tip. Allow them to grow more than 24 h or more depending on which step of the biofilm formation you are interested in looking at. Bye

You could possibly use silane/ polylysine coated slides to improve adherence, depending on what bacteria you are using...

You can refer to these research papers:

1. Anti-biofilm potential of a glycolipid surfactant produced by a tropical marine strain of Serratia marcescens.

Dusane DH, Pawar VS, Nancharaiah YV, Venugopalan VP, Kumar AR, Zinjarde SS

Biofouling. 2011;27(6):645-54

2. Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms. Dusane DH, Nancharaiah YV, Zinjarde SS, Venugopalan VP Colloids and surfaces. B, Biointerfaces. 2010 Nov 1;81(1):242-8

3. Disruption of fungal and bacterial biofilms by lauroyl glucose. Dusane DH, Rajput JK, Kumar AR, Nancharaiah YV, Venugopalan VP, Zinjarde SS Letters in applied microbiology. 2008 Nov;47(5):374-9

4. Biofilm formation by a biotechnologically important tropical marine yeast isolate, Yarrowia lipolytica NCIM 3589. Dusane DH, Nancharaiah YV, Venugopalan VP, Kumar AR, Zinjarde SS Water science and technology : a journal of the International Association on Water Pollution Research. 2008;58(12):2467-75

All the best.

Grow bacteria on square cover slips submerged in growth media in 6-well microplates, or submerge microscope slides in 50 ml conical tubes with 20-30 ml of media. In both cases, you will have growth on both surfaces and can sterilize the outer surface with ethanol, and image the other.

Thanks a lot for your tips! I am growing S. epidermidis and biofilm is formed properly in plastic but not in my glass slides. I will try the procedures next week and let you all know. Once more, thanks and have a nice weekend!

I used to grow biofilm on glass by submerging microscope slides in 50 mL conical tubes with 25 mL of media. I was using an E. coli strain that expressed curli to attach to surfaces. I found that if the tubes are tilted with a slight agitation, the biofilm grows better.

You might use a drip flow biofilm reactor from BioSurface Technologies. Please, see for instance Goeres et al. A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor. Nature Protoc. 2009;4(5):783-8.

Albert Wijk, coating the glass with fibronectine will not cause problems to the confocal analysis?

hai regiane

you can have a look at the following research article

In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant

Staphylococcus aureus biofilms

Dhamodharan Bakkiyaraj and ShunmugiahThevar Karutha Pandian*

Claire Perrin and Shawn Lewenza, thanks a lot! I did as you both told and it worked. It was quite easy and staining also worked.

That's great Regiane. I'm happy to have been of some help.

Hi, I did similar analysis with V.Cholerae and what worked well for me is using glass coverslips in 50 ml centrifuge tubes, i ensure that my coverslips are stuck between walls of tubes. after that we used thick wax on flat glass slide to give proper height to coverslip so that we can examine biofilm in more depth and it worked great

Use glass bottomed petri-dishes or well plates or Labtak chamber slides, this very easy for biofilm microscopy studies and good for con-focal analysis. see the links below

http://www.nuncbrand.com/en/page.aspx?ID=229

http://glass-bottom-dishes.com/pages/product.html

Also grow the biofilm in shaker, it helps lot for the thick biofilm formation

Hi,

Please help me with protocol of microscopic studies of biofilm in glass bottomed well plates.

hi

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568995/pdf/nihms315401.pdf

Thank you for your help.This document  will be very useful.

For observing bacterial Biofilm under confocal Microscope Is it necessary for bacteria to have GFP?