Fluorescence Assay Determines Gram Sign in Living Bacteria

A bacterial Gram stain kit provides a novel one-step fluorescence assay, which can determine Gram sign in living bacteria in minutes, without the use of fixatives.

The kit includes mixtures of the green fluorescent syto 9 and red fluorescent hexidium iodide nucleic acid stains. The syto 9 stain labels both live Gram-negative and Gram-positive bacteria, whereas hexidium iodide preferentially labels live Gram-positive bacteria. In Gram-positive bacteria that are exposed to both dyes, the hexidium iodide stain effectively displaces the syto 9 stain. Thus, when a mixed population of live Gram-negative and Gram-positive bacteria is stained with this mixture of dyes, the Gram-negative bacteria fluoresce green and the Gram-positive bacteria fluoresce red. Dead bacteria do not exhibit predictable staining patterns.

This live BacLight bacterial Gram stain kit, developed by Molecular Probes (Invitrogen, Carlsbad, California, USA), overcomes several of the problems inherent in fixation-dependent procedures. The kit reagents exhibit low intrinsic background fluorescence. Gram-negative and Gram-positive organisms can be easily differentiated using any fluorescence microscope equipped with a standard fluorescein longpass optical filter set, or by flow cytometry. By using the kit, it is possible to mount stained bacteria on filter membranes and perform the direct epifluorescence filter technique (DEFT).

The original Gram stain involves a number of steps: heat fixation of the bacteria, a two-step staining protocol, alcohol extraction, and counterstaining. Over the years, several improved Gram-staining techniques were developed, but most involve cell fixation or cell-membrane permeabilization steps that kill the bacteria being tested. In addition, fixation steps can be a significant drawback if the bacteria need to be characterized further.



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