As mentioned above there is no standard test but many use the minimum biofilm inhibitory concentration (MBIC).

Is the infection topical?

You are right, standard MIC or MBC would not indicate the same inhibitory concentration for antibiotics. This has been proven by many years of research.

The MBIC can be performed using a high bind (or tissue culture) 96 well plate with 0.1% crystal violet as a measure of biofilm formation.

You can approach this test in two ways. The first is to add a series of antibiotics (usually double dilutions) transversly across the 96 well plate in duplicate or triplicates and add the Pseudomonas biofilm forming culture on top, incubating in a static environment. Or you can cultivate the Pseudomonas biofilm first then add the antibiotic (this test is more relevant ).

To prepare a seed biofilm solution:

1. growPseudomonas overnight to stationary phase

2. Take an aliquot of this suspension and dilute 1/50 dilution in sterile half strength media such as nutrient broth. This is the biofilm seed culture.

3. Add 150/100 ul of this supension to flat bottomed (high bind) 96 well plates

4. Add series of antibiotic concentrations and incubate static overnight or 48 hours if biofilms are not present in the positive control wells.

5. At the same time, make control wells of just Pseuomonas culture for comparisons and also controls of just culture media with antibiotic.

6. After incubation period, aspirate supernatant, leave plates to air-dry, fix with 10 or 20 ul methanol, aspirate after a few minutes, wash 2 or 3 times gently with phosphate buffered saline then stain with 50 ul of 0.1% crystal violet.

7. After a few minutes the crystal violet is aspirated and the wells are washed with PBS and air dried.

8. You can hold the plate up to the light at this stage and to check for biofilm inhibition. The crystal violet can be solubilised with 30% acetic acid transferred to another 96 well plate and the OD read at 570nm. Compare with control and blank wells.

The other point is that it is really difficult to treat biofilm infections with antibiotics alone because it demands high therapeutic doses of antibiotic. Other reserchers have reported successes in treating biofilms using: combinations of antibiotics, and biosurfactants; using biosurfactant based antibiotics or using combinations of antibiotics and dilutions of acetic acid (vinegar).

Article Microtiter Dish Biofilm Formation Assay

Hi Andres,

Check out the ESCMID guidelines, they might be of help.

Article ESCMID∗ guideline for the diagnosis and treatment of biofilm...

Dear Andres,

Technically antibiotic/antimicrobial resistance of biofilm is not measured. Bacteria which possess EPS can form biofilms. So, you have to determine the antimicrobial resistance/sensitivity profile of Pseudomonas aerogenosa, using standard methods. For this a number of different antibiotics (of variable potencies/concentrations) will be used against your bacterium. Its resistance/sensitivity will be the measure of biofilm resistance/sensitivity.

Sometime lower concentrations do not kill the bacterium/bacteria but may stop the production of EPS, which will automatically stop biofilm formation. This I am telling about the lab experiments, this can not be applied on patients as such. As there are many other factors which should be considered and the experiment must need elaboration after the above mentioned preliminary experiment.

Hi.

May be this article can help you

With thanks

Hello,

Basically to check the antimicrobial resistance for biofilm, there are two ways of carrying out the experiment

* Treatment of antibiotics in the preformed biofilm and check the amount of distortion observed in the biofilm.

* You can add the antibiotics in different concentrations to the planktonic cells and then check their ability for biofilm formation.

# Have a control treatment with planktonic cells. This will give a statistical significance for the effect on the biofilm.

# Make sure to maintain the initial cell density (in terms of absorbance) for all the samples.

# Perform the experiments in triplicates.

Manobala. T