Detection of Lactobacillus helveticus Autolysis Using Gram Staining and Flow Cytometry

  • I. Jebavá Department of Dairy and Fat Technology,
  • J. Fiala Department of Fermentation Chemistry and Bioenginee¬ring,
  • A. Petříková Department of Dairy and Fat Technology,
  • P. Ulbrich Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Prague
  • M. Plocková Department of Dairy and Fat Technology,


In this study we combined a common method for autolysis detection - spectrophotometry of bacterial suspension in a buffer with Gram staining - and flow cytometry. After 4-h incubation in phosphate buffer the Gram-positive cells are colored like Gram-negative cells. A correlation between spectrophotometric observation and Gram staining was found. Using flow cytometry, the formation of two peaks of red fluorescence emitted by hexidium iodide (HI) was observed after 4-h incubation of the cell suspension in phosphate buffer. This indicates the presence of two cell groups with different capacity of binding HI to peptidoglycan as well as explains the observation by electron and light microscopy on Gram staining. A decrease in cell size during their cultivation in phosphate buffer was also observed.