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 37³C, 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. ß

The Utilisation of Prebiotics and Synbiotics in Dogs

“…Similarly to what had already been observed by some authors in studies aimed at characterising human intestinal microbiota (Langendijk et al, 1995;Harmsen et al, 2000), Greetham et al (2002) and more recently Hooda et al (2012) highlighted the scant reliability of traditional culture methods for isolating and identifying dog intestinal microbiota.…”

ORIGINAL ARTICLE: Impact of inulin and a multispecies probiotic formulation on performance, microbial ecology and concomitant fermentation patterns in newly weaned piglets

“…Gram‐negative anaerobes were grown on Wilkins‐Chalgren agar with addition of G‐N anaerobe selective supplement (Oxoid). Wilkins‐Chalgren with colistin sulfate (8 mg/l, Sigma‐Aldrich, Germany), novobiocin sodium salt (8 mg/l, Sigma‐Aldrich, Germany) and polymyxin B sulfate (16 mg/l, Sigma‐Aldrich, Germany) was used for clostridia (Schwartz et al., 1972; Fujisawa et al., 1995; Harmsen et al., 1999). All media listed above were incubated for 72 h at 37 °C in the anaerobic workstation mentioned.…”

Fermentable Carbohydrate Restriction Reduces Luminal Bifidobacteria and Gastrointestinal Symptoms in Patients with Irritable Bowel Syndrome

“…Stool samples were analyzed for luminal microbiota using fluorescence in situ hybridization (FISH) using a widely adopted protocol (33). The bacteria were fixed, suspended, and stored at 2208C until analysis.…”