Production of inactivated gram-positive and gram-negative species with preserved cellular morphology and integrity

J Microbiol Methods. 2021 May:184:106208. doi: 10.1016/j.mimet.2021.106208. Epub 2021 Mar 22.


There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.

Keywords: Bacterial ghosts; Bacterial inactivation; Beta-propiolactone; Heat-treatment; Pasteurization; Scanning electron microscopy; Standardized protocol.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Wall / drug effects
  • Cell Wall / genetics
  • Disinfectants / pharmacology*
  • Disinfection / instrumentation
  • Disinfection / methods*
  • Ethanol / pharmacology
  • Formaldehyde / pharmacology
  • Gram-Negative Bacteria / drug effects*
  • Gram-Negative Bacteria / genetics
  • Gram-Negative Bacteria / growth & development
  • Gram-Positive Bacteria / drug effects*
  • Gram-Positive Bacteria / genetics
  • Gram-Positive Bacteria / growth & development
  • Hot Temperature
  • Microbial Viability / drug effects
  • Propiolactone / pharmacology


  • Disinfectants
  • Formaldehyde
  • Ethanol
  • Propiolactone