Skip to main content

Bacterial Dye Release Measures in Response to Antimicrobial Peptides

Part of the Methods in Molecular Biology book series (MIMB,volume 2402)


Assessment of bacterial dye release following exposure to antimicrobial peptides (AMPs) provides a detailed understanding regarding their interaction with the inner and outer membrane of bacteria, and the leak of bacterial intracellular materials. This underpins the overall antimicrobial mechanism of these membrane-active peptides. DiSC3(5) is a membrane potential sensitive dye and can characterize the changes in bacterial membrane potential following exposure to AMPs (see Note 1). SYTOX Green is a nucleic acid stain that enters the cell upon loss of membrane integrity after exposure to AMPs and binds to DNA. SYTO9 is another nucleic acid stain, whereas propidium iodide (PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Both of these stains are widely used to monitor the viability of bacteria following exposure to AMPs. This chapter describes the methods of using these as bacterial dye release experiments for assessment of the antimicrobial mechanism of AMPs.

Key words

  • Antimicrobial peptides
  • Dye release
  • Bacterial membrane
  • Bactericidal
  • Bacteriostatic

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1843-1_21
  • Chapter length: 6 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   169.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1843-1
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Hardcover Book
USD   219.99
Price excludes VAT (USA)

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts PWK (2018) Molecular biology of the cell. W. W. Norton & Company, New York, NY

    Google Scholar 

  2. Felle H, Porter JS, Slayman CL, Kaback HR (1980) Quantitative measurements of membrane potential in Escherichia coli. Biochemistry 19(15):3585–3590

    CAS  CrossRef  Google Scholar 

  3. Hille B (1978) Ionic channels in excitable membranes. Current problems and biophysical approaches. Biophys J 22(2):283–294

    CAS  CrossRef  Google Scholar 

  4. Sperelakis N (2012) Cell physiology source book: essentials of membrane biophysics. Elsevier, Amsterdam

    Google Scholar 

  5. Yasir M, Dutta D, Hossain KR, Chen R, Ho KKK, Kuppusamy R, Clarke RJ, Kumar N, Willcox MDP (2019) Mechanism of action of surface immobilized antimicrobial peptides against pseudomonas aeruginosa. Front Microbiol 10:3053

    CrossRef  Google Scholar 

  6. Hilpert K, Elliott M, Jenssen H, Kindrachuk J, Fjell CD, Korner J, Winkler DF, Weaver LL, Henklein P, Ulrich AS, Chiang SH, Farmer SW, Pante N, Volkmer R, Hancock RE (2009) Screening and characterization of surface-tethered cationic peptides for antimicrobial activity. Chem Biol 16(1):58–69

    CAS  CrossRef  Google Scholar 

  7. Yasir M, Dutta D, Kumar N, Willcox MDP (2020) Interaction of the surface bound antimicrobial peptides melimine and Mel4 with Staphylococcus aureus. Biofouling 36:1019–1030

    CAS  PubMed  Google Scholar 

  8. Yasir M, Dutta D, Willcox MDP (2019) Comparative mode of action of the antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa. Sci Rep 9(1):7063

    CrossRef  Google Scholar 

  9. Yasir M, Dutta D, Willcox MDP (2019) Mode of action of the antimicrobial peptide Mel4 is independent of Staphylococcus aureus cell membrane permeability. PLoS One 14(7):e0215703

    CAS  CrossRef  Google Scholar 

  10. Dutta D, Cole N, Willcox M (2012) Factors influencing bacterial adhesion to contact lenses. Mol Vis 18:14–21

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Dutta D, Cole N, Kumar N, Willcox MDP (2013) Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses. Investig Ophthalmol Vis Sci 54(1):175–182

    CAS  CrossRef  Google Scholar 

  12. Larson D (2015) Clinical chemistry - e-book: fundamentals and laboratory techniques. Elsevier Health Sciences, Amsterdam

    Google Scholar 

Download references


The work is supported by the start-up fund from Aston University provided to Dr Debarun Dutta. Srikanth Dumpati acknowledges support from Marie Curie Multiply Co-fund fellowship, European Commission Research Executive Agency.

Transparency Declaration

The corresponding author affirms that this manuscript is an honest, accurate, and transparent account of the report produced.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Debarun Dutta .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Dumpati, S., Dutta, D. (2022). Bacterial Dye Release Measures in Response to Antimicrobial Peptides. In: Cranfield, C.G. (eds) Membrane Lipids. Methods in Molecular Biology, vol 2402. Humana, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1842-4

  • Online ISBN: 978-1-0716-1843-1

  • eBook Packages: Springer Protocols