Generally, to grow a biofilms, you inoculate a surface/well with your bacteria, allow some time for adherence (e.g. 90 mins), then remove the supernatent/looseley adhered cells, then allow the adhered ones to colonise and grow as a biofilm. Its a relatively straightforward process, but there are many factors to consider depending on what kind of biofilm you want to replicate.

Do you need a constant flow? Do you need to use a CDFF for example?

Are you looking at batch culture where media is removed daily, bi-dialy etc?

Do you need to supplement with vitamins/stress inducers etc etc?

In terms of visualising, you can set up a number of them, then do a crystal violet stain to check for growth and measure with absorbance, or you can use confocal microscopy to monitor daily or however frequently you want. Many stains are available - do a lit search. You may want to enumerate the biofilm too, so detach the cells from the surface and recover, dilute and plate out to count.

I am author on a paper that has a method for acrylic biofilms (I'm looking at oral biofilms), but the principle is the same for their development, but it can be modified as you see fit. See Cavalcanti et al., 2015 (https://www.ncbi.nlm.nih.gov/pubmed/25574582)

Hi, 

The straightforward way of growing E. Coli biofilm is to immerse the biofilm support/surface material in the growth medium inoculated (at least 10% V/V inoculum should be used) with E. Coli (I suppose a pure strain is being used here) for at least 12 hrs. Then the support materials with adhered seed cells can be transferred to fresh medium (a mild washing step may be there, but should be done under strictly aseptic conditions) for the biofilm to grow and mature. Cell counts can be performed microscopically by Crystal violet staining or using spectrophotometry for optical density determination. I think following a standard protocol for biofilm growth will be beneficial for you, as there are many available including on E. Coli. I am proving the link for some useful papers here:   

1. doi:10.1111/j.1365-2672.2008.03791.x

2. doi: 10.3791/2437

3. doi: 10.1128/JB.188.10.3572-3581.2006

 I hope these will be useful in your research on biofilms. There are many other papers available on E. Coli biofilms which you can search yourself.

Regards,

Shiladitya 

u can use safranin staining (0.1% dissolved in methanol) after growing the cells for 48 hours. inoculum can be 2X10^6 cells in LB medium.  the non adhered cells can be washed off with PBS and stained with safranin. after the stain dries one can dissolve the stain in 30% glacial acetic acid and od value can be checked spectrophotometrically

It depends on the type of biofilm you are trying to grow. I presume it is a single strain biofilm. Ideally I dilute a suspension of an overnight culture which is well into stationary phase 1/100 and then dispense the solution into a 96-well high bind or elisa plate with flat wells . After an appropriate temperature of incubation , the supernatant can be removed and the cells washed in PBS buffer. The biofilm (cells and EPS) can then be stained using 0.1% crystal violet which can be measured with a spectrophotemeter. The advantage in this technique is that you can have many replicates of your experiment. There is also the calgary biofilm device and other flow through biofilm devices. I wrote about some of the choices in this paper if you have access  ' Microbial biofilms: Biosurfactants as antibiofilm agents '  https://www.researchgate.net/publication/267733353_Microbial_biofilms_Biosurfactants_as_antibiofilm_agents

Article Microbial biofilms: Biosurfactants as antibiofilm agents

Hi Anil,

Below is a reference to an Introductory chapter on basic biofilm models and review article.

• Different Methods for Culturing Biofilms in Vitro in Biofilm Infections. Peterson et al 2010
• 10.1016/j.tim.2013.06.002

Good luck with your work

Dear Anil,

Maybe the following of help.

Concerning on how to monitor the biofilm growth, it can be considered to use tools that affect the biofilm formation (e.g. use of stains) or require destructive sampling of the biofilm. But also in-situ non-destructive tools can be considered. 1) Oxygen concentration or oxygen decrease rates can be assessed using optodes. 2) NMR and 3) optical coherence tomography (OCT). Attached are some papers that give information on such non-destructive tools. Optodes based imaging and OCT imaging are much lower in cost than e.g. confocal laser scanning microscopy (CLSM). 

Best regards, Hans

Thanks everyone for your time and sharing thoughts and information.