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Flow cytometry evaluation of complement mediated bacterial membrane damage

F J Alvarado-Alemán1

  • 1Departamento de Medicina Geográfica, Hospital General Dr. Jesús Kumate, Cancún, Q. Roo, México.

Archives of Medical Research
|January 1, 1996
PubMed
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A novel flow cytometry method reveals that p-nitro blue chloride tetrazolium (NBT) causes a quenching effect on fluorescent bacteria. This NBT-laser beam quenching (NBT-LBQ) can assess complement-outer membrane interactions in bacteria.

Area of Science:

  • Microbiology
  • Immunology
  • Biotechnology

Background:

  • Complement-mediated bacterial killing is crucial for innate immunity.
  • Understanding complement-outer membrane interactions is key to developing new antibacterial strategies.
  • Specialized techniques are needed to study these complex interactions.

Purpose of the Study:

  • To introduce a novel flow cytometry technique for evaluating complement-outer membrane interactions.
  • To characterize the NBT-laser beam quenching (NBT-LBQ) effect in Neisseriae gonorrhoeae.
  • To assess the potential of NBT-LBQ for studying serum-sensitive microorganisms.

Main Methods:

  • Utilized flow cytometry with lucifer yellow-stained Neisseriae gonorrhoeae.
  • Applied the redox reaction of p-nitro blue chloride tetrazolium (NBT) leading to formazan precipitation.

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  • Analyzed fluorescence shifts using Kolmogorov-Smirnov summation curve analysis.
  • Compared results using normal complement versus heat-inactivated complement.

    • Main Results:

    • A distinct shift to lesser fluorescent channels was observed upon NBT exposure, termed NBT-laser beam quenching (NBT-LBQ).
    • Significant differences in NBT-LBQ were detected between microorganisms treated with normal complement and heat-inactivated complement.
    • The NBT-LBQ effect demonstrated a clear correlation with complement activity.

    Conclusions:

    • The NBT-LBQ technique offers a sensitive method for assessing complement-outer membrane interactions.
    • This flow cytometry-based approach has significant potential for evaluating the susceptibility of bacteria to complement.
    • NBT-LBQ provides a valuable tool for studying bacterial immune evasion mechanisms and developing novel therapeutics.