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Related Experiment Videos

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity.

U M Rodrigues, R G Kroll

    The Journal of Applied Bacteriology
    |December 1, 1985
    PubMed
    Summary
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    The direct epifluorescent filter technique (DEFT) effectively differentiates and enumerates viable bacteria in raw milk. Modified DEFT enhances sensitivity and accuracy for assessing hygienic status and product quality.

    Area of Science:

    • Microbiology
    • Food Science
    • Analytical Chemistry

    Background:

    • Accurate bacterial enumeration is crucial for assessing food safety and quality.
    • Traditional culture-based methods can be time-consuming and may underestimate viable but non-culturable bacteria.
    • The direct epifluorescent filter technique (DEFT) offers a rapid alternative for bacterial counting.

    Purpose of the Study:

    • To modify and enhance the DEFT method for improved bacterial differentiation and enumeration in raw milk.
    • To assess the correlation between modified DEFT counts and traditional colony counts.
    • To evaluate the impact of procedural modifications on DEFT sensitivity and speed.

    Main Methods:

    • A modified Gram-staining procedure with acridine orange counterstain was employed for DEFT.

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  • Stainless steel membrane filters and citrate-NaOH buffer were used to process larger sample volumes (10 ml).
  • Sonication was utilized for bacterial clump dispersal in pure cultures and raw milk samples.

    • Main Results:

    • DEFT successfully differentiated viable Gram-negative and all Gram-positive bacteria.
    • High correlations were observed between DEFT counts and colony counts for Gram-negative bacteria (r = 0.94 for 10 ml samples).
    • Processing 10 ml samples increased DEFT sensitivity five-fold and improved the correlation with colony counts (r = 0.90).
    • Procedural modifications reduced preincubation time and enhanced bacterial clump dispersal via sonication.

    Conclusions:

    • The modified DEFT method provides a sensitive and accurate approach for enumerating viable bacteria in raw milk.
    • DEFT offers a valuable tool for evaluating the hygienic status and predicting the keeping quality of dairy products.
    • Sonication-assisted DEFT yields bacterial counts that better reflect the overall microbial load compared to colony-forming units.