Estimation of the state of the bacterial cell wall by fluorescent In situ hybridization

Abstract

Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

FIG. 1

Epifluorescence micrographs of L. lactis IL1403 before infection with bacteriophage bIL66 (40× lens objective) (a and b), 30 min after infection (100× lens objective) (c and d), and 40 min after infection (100× lens objective) (e and f) and of an artificial mixture of fixed cultures of IL1403 and Lactobacillus helveticus CNRZ493, infected with their specific phages (100× lens objective) (g and h). Micrographs were taken without a filter for excitation light (a, c, e, and g), with a filter for fluorescein (b, d, and f), and with double exposure and filters for fluorescein and rhodamine (h).

FIG. 2

Kinetics of the HF of strain IL1403 after infection with bacteriophage bIL66. Open squares, ODs of noninfected culture; filled squares, ODs of infected culture; open circles, HFs of noninfected culture; filled circles, HFs of infected culture. Values are representative of results of five independent measurements.

FIG. 3

Kinetics of the HF of strain NCDO 712 after induction of prophage with MC. Open squares, ODs of noninduced culture; filled squares, ODs of induced culture; open circles, HFs of noninduced culture; filled circles, HFs of induced culture. Values are representative of results of five independent measurements.