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The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity
Author(s) -
Rodrigues Ubaldina M.,
Kroll R.G.
Publication year - 1985
Publication title -
journal of applied bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 0021-8847
DOI - 10.1111/j.1365-2672.1985.tb03351.x
Subject(s) - acridine orange , gram , raw milk , bacteria , sonication , biology , chromatography , staining , chemistry , food science , genetics
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.