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J Glob Infect Dis. 2017 Jul-Sep;9(3):93-101. doi: 10.4103/jgid.jgid_91_16.

Standardization and Classification of In vitro Biofilm Formation by Clinical Isolates of Staphylococcus aureus.

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Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Department of Community Medicine, Division of Biostatistics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Department of Biochemistry, Institute of Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.



Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofilm exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However, till date, consensus guidelines for in vitro biofilm quantitation and categorization criterion for the bacterial isolates based on biofilm-forming capacity are lacking. Therefore, it was intended to standardize in vitro biofilm formation by clinical isolates of S. aureus and then to classify them on the basis of their biofilm-forming capacity.


A study was conducted for biofilm quantitation by tissue culture plate (TCP) assay employing 61 strains of S. aureus isolated from clinical samples during May 2015- December 2015 wherein several factors influencing the biofilm formation were optimized. Therefore, it was intended to propose a biofilm classification criteria based on the standard deviation multiples of the control differentiating them into non, low, medium, and high biofilm formers.


Brain-heart infusion broth was found to be more effective in biofilm formation compared to trypticase soy broth. Heat fixation was more effective than chemical fixation. Although, individually, glucose, sucrose, and sodium chloride (NaCl) had no significant effect on biofilm formation, a statistically significant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (P= 0.037).


The present study puts forth a standardized in vitro TCP assay for biofilm biomass quantitation and categorization criteria for clinical isolates of S. aureus based on their biofilm-forming capacity. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacterium.


Biofilm; brain-heart infusion broth; tissue culture plate method; trypticase soy broth

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