Problems associated with the direct viable count procedure applied to gram-positive bacteria

Area of Science:

• Microbiology
• Molecular Biology
• Environmental Science

Background:

• Fluorescent in situ hybridization (FISH) using 16S rRNA probes is effective for identifying single bacterial cells.
• The direct viable count (DVC) procedure enhances ribosomal RNA for FISH but is limited to nalidixic acid-sensitive, gram-negative bacteria.
• Existing DVC methods are not suitable for diverse gram-positive bacteria.

Purpose of the Study:

• To develop a method for enumerating metabolically active, gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis).
• To combine substrate responsiveness with FISH for accurate identification and enumeration.
• To adapt the DVC procedure for gram-positive bacteria, overcoming limitations of existing methods.

Main Methods:

• Evaluated various low-nutrient and complex media, fluoroquinolone and beta-lactam antibiotics, antibiotic concentrations, combinations, temperatures, and incubation times.
• Tested the procedure on bacteria in different physiological states and in natural samples.
• Compared bacterial enumeration using plate counts and direct FISH.

Main Results:

• No single published DVC method was effective for all tested gram-positive species.
• An improved procedure was developed to identify physiological state, taxonomic identity, and enumerate viable gram-positive bacteria.
• Direct FISH combined with substrate responsiveness provided a viable enumeration method.

Conclusions:

• The developed method successfully enumerates viable gram-positive bacteria, providing physiological and taxonomic information.
• This approach expands the utility of FISH and DVC techniques for studying gram-positive bacteria.
• Applying DVC methods to entire ecosystems remains a significant challenge.