Jose - For the most part, it's difficult to not have biofilms grow.  If I understand your system, the keys to early success will be -

1) Not over-feeding them.  The richer the growth media, the faster the planktonic organisms in your system will grow, making any surface adherent organisms very starved and therefore disadvantaged.  In the real world, organism don't have the benefit of endless carbohydrate, amino acids, and vitamins.  If I were starting from scratch, I'd work with a minimal media.  From there, the first modification to growth conditions you could make is varying the ratio of carbon to nitrogen.  Carbon rich, nitrogen poor media in some instances can promote the production of extracellular polysaccharides, which will help establish the film.

2) Pick realistic hydrodynamic conditions.  The faster the flow rate over the surface, and the higher the turnover of fresh media in your system, the sparser but in general the tougher the biofilms will be that you form.

Mostly, my advice is to jump in and try it.  Find a very favorable surface for your first attempts and see what happens.  Good luck!

You can have different options as surface, such as stainless steel, glass, polypropylene and some specific polymer surfaces. These surfaces are cut in a certain size for example 1 cm2 in total. These surfaces can be called coupons.

These coupons are placed in a petri plate. Then covered with enough media.

This media is inoculated with starting culture of the bacteria, I prefer to use 0.05 at OD600 as final concentrations. Then the coupons are incubated for 24 hr. This incubation is same as growing agar plates in an incubator. Based on the interaction of the bacteria to the surface this incubation time can be longer. 

Counting the bacteria: Wash the coupons to get rid of planktonic cells in the media. PBS is a good option. but some researchers also use 0.85% NaCl solution.

When you are handling the coupons, be very careful because biofilm is a complete structure. it is easy to slide of the surface in big parts.

After washing place the coupons in conical tubes and covered them with enough PBS.  Then you should use sonication bath (bench top, small ones) and vortexing to break off the cells from the surface. Now all the cells are in the supernatant phase. Take the supernatant pellet it. Then do a CFU count with drop plate method.

For the size of the biofilm; it depends on the size of the coupons you are using. you can have bigger coupons with bigger biofilms on it.

Jose - I agree with the above.  A couple of points I'd strongly suggest, having made plenty of mistakes in biofilm work previously.

First, and most importantly, your culture conditions should mimic as closely as possible the environment you're trying to understand.  It's straightforward to get a biofilm of some kind to grow in a tube, on a coupon or pin, or even in a flow cell.  For your conclusions to really hope for some relevance to the real world system that you're hoping to understand, you should think about the fluid dynamics (e.g., slow flowing, fast flowing, stagnant, etc.) and the available nutrients.  Most biofilms of any medical, industrial, or ecological importance are living off of nutrients at concentrations much different than media that you'll find on your shelf. 

Reproducing the organism's natural habitat (e.g., a river bed, a pharmaceutical plan water system) is really the most important and time consuming part of the work.  Once you've figured out how to convince a microorganism that it's not in a laboratory, but in the real world, the remainder of the work - such as how to quantify adhesion, growth, etc. - becomes a lot easier.

Good luck!

Thanks John, my aim is to screen different organic polymers as a surface for E. coli biofilm growth (with industrial purposes). 

Once I have the overnight culture and transfer to the substrate I will use, what growing conditions should I use iniciate E. coli biofilm growth? Media, Tº, oxygen concentration, etc.

Best regards.

José Luis,  on the last question you have done. The E. coli is not a demanding microorganism grows well in an oven at 35-37 ° C temperature and aerobically.

Excuse me because my English is very bad.

I hope to help

Marielsa, I know about the conditions to grow and maintain E. coli, but you need certain environmentan and feeding conditions to elicit biofilm formation, and that is what I want to know.

Which growing conditions do I need to provoke biofilm formation (and maintenance) on E. coli, in vitro.

Jose - For the most part, it's difficult to not have biofilms grow.  If I understand your system, the keys to early success will be -

1) Not over-feeding them.  The richer the growth media, the faster the planktonic organisms in your system will grow, making any surface adherent organisms very starved and therefore disadvantaged.  In the real world, organism don't have the benefit of endless carbohydrate, amino acids, and vitamins.  If I were starting from scratch, I'd work with a minimal media.  From there, the first modification to growth conditions you could make is varying the ratio of carbon to nitrogen.  Carbon rich, nitrogen poor media in some instances can promote the production of extracellular polysaccharides, which will help establish the film.

2) Pick realistic hydrodynamic conditions.  The faster the flow rate over the surface, and the higher the turnover of fresh media in your system, the sparser but in general the tougher the biofilms will be that you form.

Mostly, my advice is to jump in and try it.  Find a very favorable surface for your first attempts and see what happens.  Good luck!

Dear Jose Luis Brioso Then he placed a technique that has helped me both biofilm formation of S. aureus to  enterobacteria and non-fermenting Gram negative Bacilli. As some signs are distorted in this format I leave two link where the technique appears in Spanish in both works the same technique was used but different modes of interpretation.

Quantification of biofilm formation capacity

It was performed by the technique of polystyrene microplate wells (colorimetric method) described by Al-Shuneigat et al. quoted Rojas et al (2012). an adjusted bacterial suspension to a 0.5% Mc Farland prepared in tryptic soy broth. 20 μL of this suspension was dispensed into each well plus 180 mL of trypticase soy broth with 0.25% glucose (1:10 dilution) and then incubated in a humid chamber for 24 hours at 37 ° C. After the incubation, each well were performed two washes with sterile saline phosphate buffer solution (PBS, pH 7.2). 200 μL of crystal violet solution were added 0.01% maintained at room temperature for 30 min and then two new washes with PBS solution were made. Following this 200 μL of ethanol 95% was added to solubilize the dye adhering to the walls, quantifying the optical density (OD) as an estimate of the biofilm formation capacity, the measurement was performed at 490 nm using a reader micro ELISA (Stat Fax 303 Plus).

Each isolate was evaluated in quadruplicate in each trial including eight uninoculated interpreted these as negative controls plus eight wells isolated known for their high capacities of biofilm formation holding bacterioteca Diagnostic Laboratory Bacteriological (four wells were introduced wells Klebsiella pneumoniae and Pseudomonas aeruginosa for four wells), the latter are interpreted as positive controls.

For the classification of each isolated in their ability to form biofilms, mathematical equations containing the OD of the negative control wells as quoted by Rojas et al were used, plus, a variant that includes the OD for the positive controls as follows: the arithmetic mean () of the OD obtained at 490 nm of the positive controls and negative controls for use in the following three formulas are calculated:

VM: DOC (+) - Doc (-) / 2

PC1: VM - DOc (-) / 2 + DOc (-)

PC2: DOc(+) –VM/2 + VM

Where stands for:

VM: middle value; DOc (+) :() of the optical density at 490 nm of the positive controls; DOc (-): () optical density at 490 nm of the negative controls; PC1: first cutoff; PC2: cutoff second.

strong, moderate, weak and no forming: From the values of VM, PC1 and PC2 obtained ranges for the classification of the ability to form biofilms in four categories were established. Interpreted as follows: will not be forming when the values are located between DOC (-) to figures ≤ PC1, loosely forming when the values are> PC1 ≤ VM, moderately forming when the values are> VM ≤ PC2 and strongly forming values> PC2.

excuse me again because my English is very bad.

https://www.researchgate.net/profile/Marielsa_Gil?ev=hdr_xprf&_sg=gddwgJnJiPcxv7sianxrqw9tj5cIMFGzuzCJWNeris8mT-_8avWEola9-O3djAJZ

https://www.researchgate.net/profile/Marielsa_Gil/publications

I hope you find it useful.

Marielsa Gil

Jose,

Use Tissue culture plates (96-well or 24-well). If the biofilm growth is poor, try adding 1% glucose to your system - it helps a lot.

quantification:

1. You can stain your biofilm with 0.1% crystal violet for 40 minutes (room temperature). Wash unbound dye with water two or three times. The use 95% ethanol  to solubilize Crystal violet and quantify it via absorbance at 595 nm.

2. Plate counting: detach biofilm by vigorous pipetting; then plate.

3. ATP viability assay (use BacTiter Glo ATP viability kit). Just wash your biofilm and then add 50 ul of the kit, per well (dry well).

4. Use tetrazolium or resazurin to quantify cells via their metabolism.

Good luck.

Hello José,

Have you found your answers and protocol of choice? My project involves growing biofilms on sponges and different foams. I am starting with E. Coli, so I am interested in your feedback. My problem is that the foam I use retains fluorscent dies and xstal violet. So, I try to quantitate the amount of bacteria by determining the protein quantity using a BCA test. I first destroy my foam in 2% SDS and then sonicate. I have to use a negative control because the foam also keeps the proteins present in the media. To know if I have a biofilm, I guess that my only solution is to perform microscopy, unless someone has another idea. Regarding the importance of the media to favor biofilm construction, I have also read that a minimal media is better, however if depends on the hydrophobicity nature of the surface.

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First of all, be sure you choose an E. coli strain able to produce biofilm in the conditions you are going to do your experiments. Also, the sponge must be sterilized.