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Antimicrobial Agents and Chemotherapy, July 2005, p. 2612-2617, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2612-2617.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Microplate Alamar Blue Assay for Staphylococcus epidermidis Biofilm Susceptibility Testing

Robin K. Pettit,1,3* Christine A. Weber,1 Melissa J. Kean,1,3 Holger Hoffmann,1 George R. Pettit,1,2 Rui Tan,1 Kelly S. Franks,4 and Marilyn L. Horton5

Cancer Research Institute,1 Department of Chemistry and Biochemistry,2 School of Life Sciences, Arizona State University, Tempe, Arizona 85287,3 Bureau of State Laboratory Services, Arizona Department of Health Services, Phoenix, Arizona 85007,4 Phoenix Children's Hospital, Phoenix, Arizona 850165

Received 9 November 2004/ Returned for modification 3 January 2005/ Accepted 21 March 2005

Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidis ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 µg/ml (most >4,096 µg/ml), and four of the experimental compounds had MBICs of ≤128 µg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidis ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (r = 0.83 to 0.97) and with CFU/ml results (r = 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.

* Corresponding author. Mailing address: Cancer Research Institute, Arizona State University, Tempe, AZ 85287-2404. Phone: (480) 965-4907. Fax: (480) 965-8558. E-mail: pettitr{at}asu.edu.

Antimicrobial Agents and Chemotherapy, July 2005, p. 2612-2617, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2612-2617.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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