A simple, rapid method for enumerating total viable and metabolically active bacteria in groundwater.

Area of Science:

• Microbiology
• Environmental Science
• Biotechnology

Background:

• Accurate enumeration of microbial cells in environmental samples is crucial for ecological and health assessments.
• Existing methods for microbial cell quantification in groundwater can be labor-intensive and may lack precision.

Purpose of the Study:

• To develop and validate a new staining procedure for the accurate enumeration of viable and metabolically active cells in groundwater.
• To combine enzymatic activity detection with DNA-specific fluorescence for enhanced cell characterization.

Main Methods:

• A novel staining procedure utilizing 2-(p-iodophenyl)-3-p-(nitrophenyl)-5 phenyltetrazolium chloride (INT) for enzymatic dehydrogenation.
• Integration of INT with the DNA-specific fluorochrome 4',6'-diamidino-2-phenylindole (DAPI).
• Application of the filter-transfer-freeze technique for sample preparation and microscopic analysis.

Main Results:

• The combined staining procedure successfully produced a pink intracellular formazan, indicating metabolic activity.
• The method allowed for the differentiation and enumeration of total viable and metabolically active cells.
• Statistically accurate cell counts were achieved in groundwater samples.

Conclusions:

• The developed staining procedure offers a reliable and statistically sound method for quantifying viable and metabolically active cells in groundwater.
• This technique enhances microbial cell enumeration in environmental studies.
• The combination of enzymatic and fluorescent staining provides a robust approach for microbial assessment.