Evaluation of Different Microtiter Plate-Based Methods for the Quantitative Assessment of Staphylococcus aureus Biofilms

Walid F Elkhatib; Ahmed S Khairalla; Hossam M Ashour


Future Microbiol. 2014;9(6):725-735. 

In This Article

Abstract and Introduction


Aim: To quantitatively assess Staphylococcus aureus biofilms.

Materials & methods: In addition to the qualitative Congo red agar (CRA) method, we used the bioluminescence (BLM), safranine (SAF), crystal violet (CRV) and resazurin (RES) high-throughput microtiter plate-based quantitative assays.

Results: 60.47% (26/43) of S. aureus clinical isolates were weak biofilm producers. The CRA method detected positive-slime phenotypes (13.95%), but was unable to distinguish weak from negative producers. BLM assays demonstrated significant correlations with RES (highest), CRV and SAF (lowest). Lower coefficient of variation values indicate precision. BLM scored highest precision (coefficient of variation = 0.013) followed by RES, SAF and CRV.

Conclusion: BLM and RES detect live biomass in S. aureus biofilms (for physiological studies). SAF and CRV detect live/dead bacteria plus biofilm matrix (for monitoring overall biofilm architecture, not only its cell viability). Reliable assays are essential for effective biofilm therapy.


Biofilms are surface-associated microbial communities ensconced within an extracellular polymeric matrix.[1,2] Bacterial biofilms consist of three main elements namely microorganisms, matrix and surface. Consequently, mature biofilms require the simultaneous existence of such components to develop.[3] Microbial cells within biofilms are protected from the host immune system and acquire significantly high resistance to antibiotics.[4] The presence of these microbial communities is frequently associated with a variety of chronic diseases[5] and is also a cause of the notable spread of biofilm-associated nosocomial infections, mainly owing to the escalating use of implanted medical devices.[4,6] Eradication of these communities is uncommonly achievable resulting in serious and life-threatening consequences for patients. Gram-positive staphylococci are key pathogens in the hospital environment[7,8] leading to complicated treatment and considerably increased morbidity and mortality.[9–12]

For the quantitative assessment of bacterial biofilms, some high-throughput assays have been applied including the ATP bioluminescence (BLM) assay,[13] safranine (SAF) assay,[14] crystal violet (CRV) assay[13,15] and resazurin (RES) assay.[16] Furthermore, the Congo red agar (CRA) method was used as a qualitative technique for detection of slime-producing staphylococcal strains.[17] Although viable bacterial determination of the biofilms by laborious plate count methods has been reported, treatment of the biofilms should be evaluated via bacterial killing as well as removal of the matrix, as the slime layer can be easily re-colonized. Therefore, an optimum biofilm model should address both the matrix and its enclosed bacterial cells.[16] Evaluation of biofilm-associated infections and their prevalence in hospital and clinical settings requires rapid and precise quantitative assays that can replace tedious techniques of microscopic visualization such as scanning electron microscopy and confocal laser scanning microscopy.[13] Similarly, biofilm research on Staphylococcus aureus requires validated quantitative models that focus both on matrix and viable bacterial bioburden. Appropriate laboratory assays of S. aureus biofilms are important to develop novel strategies to combat their biofilms.[16] In this context, the present study has primarily addressed an in vitro comparative analysis on correlations and precisions of four microtiter plate-based assays implementing BLM, SAF, CRV and RES for determination and quantification of the biofilms in S. aureus clinical isolates.