Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

Abstract

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37°C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable.