Standard method of bacterial identification could be used in addition to the use of tissue culture with phage typing.

Hello Mohamed

Quantification of biofilm formation capacity

It was performed by the technique of polystyrene microplate wells (colorimetric method) described by Al-Shuneigat et al. cited in Rojas et al (2012). an adjusted bacterial suspension to a 0.5% Mc Farland was prepared tryptic soy broth. 20 uL of this suspension was dispensed into each well, 180 uL of more 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 buffer solution (PBS, pH 7.2). 200 L 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 mL of ethanol was added to 95% to solubilize the dye adhering to the walls, quantifying their optical density (OD) as an estimate of the ability of biofilm formation, the measurement was performed at 490 nm using a reader micro ELISA (Stat Fax 303 Plus) (24).

Each isolate was evaluated in quadruplicate, including in each trial eight uninoculated interpreted these as negative controls and additional eight wells with isolated known for their high capacities of biofilms holding bacterioteca Diagnostic Laboratory Bacteriological (four wells were introduced wells Klebsiella pneumoniae and Pseudomonas aeruginosa 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 of the positive controls as follows: the arithmetic mean () was calculated from the OD 490nm obtained positive controls and negative controls for use in the following three ways:

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

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

PC2: DOc (+) Your majesty / 2 + VM

Where they stand for:

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

strong, moderate, weak and not forming: From VM values, PC1 and PC2 obtained ranges for the classification of the capacity of biofilm formation in four categories were established. Interpreted as follows: will not be forming when the values are located between DOc (-) to figures ≤ PC1, weakly forming when the values are> PC1 ≤ VM, moderately forming when the values are> VM ≤ PC2 and strongly forming with values> PC2.

This technique helps you to quantify the formation of biofilms in a simple way.

Dear Mohamed,

Crystal violet staining is one of the simplest method to monitor biofilm formation in vitro. You can find the details of the assay on the link given below.  If you just want to monitor biofilm formation, CV staining is good enough.

http://www.ncbi.nlm.nih.gov/pubmed/21307833

You can use this protocol, normally work for most bacteria.

Biofilm formation assay

The amount of biofilm formed by the different strains was determined using a semi-quantita-tive adherence assay in 96-well polystyrene microtiter plates (BD Biosciences) as previouslydescribed [23,26,28]. Briefly, 20μl of stock cultures were inoculated into 5 ml (selective) BHImedium and grown to the end-exponential growth phase in a shaking incubator at 37°C. Cul-tures were subsequently diluted to an OD600of 0.005 (5.x106CFU/ml) in fresh BHI mediumwhether or not supplemented with 4% NaCl or 1% glucose. 200μl of the diluted cultures ofbacteria were pipetted into sterile 96-well polystyrene microtiter plates and incubated over-night at 37°C without shaking.After incubation, the wells were rinsed 3 times with phosphate-buffered saline (PBS) anddried afterwards. The adhered material was stained with 200μl of a 1% (w/v) crystal violet(Sigma) solution for 10 min, and subsequently, the wells were washed 3 times with water andagain dried. For quantification, 160μl of 30% (v/v) acetic acid solution was added to each wellto dissolve the crystal violet. The OD595of the dissolved stain was measured in a multipurposeUV/VIS plate reader (VICTOR3TM; PerkinElmer)

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation. Available from: https://www.researchgate.net/publication/291518077_The_Possible_Role_of_Staphylococcus_epidermidis_LPxTG_Surface_Protein_SesC_in_Biofilm_Formation [accessed Dec 19, 2016].

Article The Possible Role of Staphylococcus epidermidis LPxTG Surfac...

G'day Issa,

I have published two methods to identify biofilms (crystal violet assay and XTT assay).  Both are very easy and I have included in the manuscript how to quantify you biofilm.  Hope this is helpful. Thanks.

Cheers,

hafiz

Thank you Dear Sir. Mohd Hafiz Arzmi

With my respect for your help me .

Thank you my all Dears, Basil Mathao

Thank you my all Dears,Marielsa Gil and

Dear Laleh Khodaparast

thank for your help me 

with my all respect