ABSTRACT
Novel immunofluorescence resonance energy transfer (immuno-FRET) assays for both Bacillus cereus spores and Escherichia coli O157:H7 are reported. Both assays involve the use of dual (QSY-7 and Oregon Green 514-antibody)-labeled spores or vegetative bacteria, such that Oregon Green 514-labeled antibodies are quenched by proximal QSY-7 molecules that are covalently bound to the dual (Oregon Green 514 and QSY-7)-labeled cells. Upon introduction of unlabeled bacteria or spores, in the respective assays, an increase in fluorescence is observed in proportion to the numbers of unlabeled cells. This is due to migration of Oregon Green 514-labeled antibody from the dual-labeled cells to the unlabeled target cells as verified by fluorescence microscopy. Optimization of the QSY-7 surface density led to a B. cereus spore detection sensitivity of approximately 1.0 x 10(5) to 2.5 x 10(5) spores per milliliter and 3.5 x 10(5) cells per milliliter for E. coli using a conventional cuvette-based spectrofluorometer.
Subject(s)
Bacillus cereus/isolation & purification , Escherichia coli O157/isolation & purification , Fluorescent Antibody Technique , Spores, Bacterial/isolation & purification , Energy Transfer , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Food Microbiology , Microscopy, Fluorescence , Molecular Structure , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, FluorescenceABSTRACT
A technique for primary plastic embedding for light microscopy was utilized in evaluating plaque lesions in four patients with mycosis fungoides and compared to tissues from the same patients embedded in paraffin. Standard 3 mm punch biopsies were utilized. Specimens were fixed in B-5 and embedded in araldite epoxy resin. In specimens from all four patients, the cytological details of the mycosis cells were superior in the plastic embedded tissues. this practical and relatively inexpensive method may prove to be a sensitive indicator of mycosis cells in early lesions. This technique also permits the use of a variety of special stains, immunoperoxidase and electron microscopic analyses.
