The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity.
With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.
Rodrigues
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Kroll
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D000165:Acridine Orange; D000818:Animals; D001419:Bacteria / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D002417:Cattle; D005374:Filtration; D005516:Food Microbiology; D005840:Gentian Violet; D006090:Gram-Negative Bacteria / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000302:isolation & purification; D008892:Milk / Q000382:microbiology*; D010619:Phenazines; D013194:Staining and Labeling
C035863:Gram's stain; D010619:Phenazines; D000165:Acridine Orange; D005840:Gentian Violet