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Related Experiment Videos

Simple method for quantifying viable bacterial numbers in sputum

A Pye1, R A Stockley, S L Hill

  • 1Lung Immunobiochemical Research Laboratory, General Hospital, Birmingham.

Journal of Clinical Pathology
|August 1, 1995
PubMed
Summary
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A new method using dithiothreitol effectively quantifies viable bacteria in sputum. This simple technique improves bacterial recovery and ensures accurate results for respiratory sample analysis.

Area of Science:

  • Microbiology
  • Clinical Diagnostics

Background:

  • Accurate quantification of viable bacteria in sputum is crucial for diagnosing respiratory infections.
  • Existing methods for sputum culture may lack simplicity or reproducibility.

Purpose of the Study:

  • To develop a straightforward and quantitative culture method for determining viable bacterial counts in expectorated sputum.
  • To optimize sputum homogenization and sample handling for improved bacterial recovery and reduced variation.

Main Methods:

  • Sputum samples were homogenized using dithiothreitol (DTT), sterile saline, or glass beads to compare bacterial recovery.
  • The effect of DTT on bacterial viability was assessed over time.
  • Sampled volumes (10 microliters) were dispensed using a precision pipette and spread with a 'hockey stick' for even colony distribution.

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Main Results:

  • Dithiothreitol homogenization significantly increased viable bacterial recovery compared to glass beads or saline.
  • DTT did not adversely affect bacterial viability in culture broths.
  • Using a precision pipette for 10 microliter sample volumes minimized variation (<16%) and ensured even bacterial colony distribution on agar plates.

Conclusions:

  • The developed method offers a simple, quick, and accurate technique for quantifying viable bacteria in sputum.
  • A small sample aliquot is representative of the entire sputum sample, simplifying the process.