Vital+Fluorescent+Staining

__**Vital fluorescence staining **__


 * Basic Description: **

Staining is a technique most often used in microscopy cellular biology, so long with many other disciplines of science, in order to enhance the contrast in the microscopic image, highlighting structures in biological tissues for the purpose of being able to view them more clearly, often with the help of different microscopes. Fluorescence stains can be uses to define and examine certain bulk tissue, for example, highlighting muscle fibers or other connective tissue, colonies of cells; in order to classify different blood cells for instance, or for use in identifying organelles within individual cells ( Alberts et. al, 2008). This type of staining can serve many purposes; among those being qualifying or quantifying the presence of a specific compound, marking cells in flow cytometry or flagging proteins or nucleic acids in gel electrophoresis. It is a type of in vivo, or vital, staining in that it is the process of dyeing living tissues as opposed to in vitro staining methods. By causing certain cells or cellular structures to take on contrasting colors, their form or position withing a cell or tissue sample can be readily visible and further examined.


 * Purpose of Technique: **

The purpose of fluorescence stains is to reveal cytological details that might otherwise be overseen or not as apparent; another important use of this technique lies in that the staining is also capable of revealing where certain chemicals or chemical reactions are taking place within cells or tissues ( Alberts et. al 2008).


 * Origin and History: **

The exact origins of this exact technique are unknown, but the origins of staining in general is often credited to Austrian scientist, Camillo Golgi. Golgi is sought to have discovered a method of staining nervous tissue which could stain only a limited number of cells at random, but in their entirety. This enabled him and many more to follow to view the paths of nerve cells in the brain for the first time ( The Noble Prize in Physiology and Medicine, 2012). Fluorescence staining could have possibly arisen from this discovery as well, years to follow.


 * Recent Reserch: **

One study conducted by Thrane et. al ( 2008) sought to use vital fluorescent stains in order to detect some sort of stress factor or response in Pythium ultimum and Rhizoctonia solani after it had been in contact with viscosinamide from Pseudomonas fluorescens, DR54. Evidence for physiological changes in these and four other fungi was obtained using vital fluorescent staining with calcoflour white, nile red, systo 13, as well as a few other dyes. All of the isolates responded to viscosinamide treatment by exhibiting an increased branching, often times with hyphal swelling and an increased septation. The staining pattern indicated changes in the esterase activity, intracellular pH and mitochondrial organization and activity. Several of the fungi that were also stained showed with a reduced Nile red staining, indicating an overall decrease in hydrophobicity of the cell membranes, or walls. Furthermore, only Pythium were stainable with the nucleic acid stain, Syto 13 and showed changes in the nuclei in response to the viscosinamide. This study demonstrated that combinations of vital fluorescent stains are suitable for detailed studies of fungal stresses as induced by the viscosinamide (Thrane et. al 2008).

In another study conducted by Fuller et. al ( 2009), two differentially labeled bacterial strains were monitored during two bacterial transport experiments. Comamonas sp. Strain DA001 and Acidovorax sp. Strain OY-107 were grown and labeled with fluorescent stain TAMRA/SE or CFDA/SE. The fluorescently labeled cells and a bromide tracer were introduced into an aquifer. The results of this experiment showed that both CFDA/SE and TAMRA/SE were highly efficient at staining cells, with the latter stained cells often having a stronger fluorescence than the CFDA/SE stained cells by epifluorescence microscopy and hence were easier to detect and count under certain circumstances, such as the presence of large amounts of autofluorescing material ( Fuller et. al 2009).

In another study conducted using vital fluorescent stains, several different fluorescent mitochondrial dyes were tested as vital stains in motor nerve terminals and other cells in frog skeletal muscles. It was found that the 3,3 diethyloxadicarbocyanine iodide and 4-(4- diethylaminostyryl)- N-methylpyridinium iodide were most useful. Both dyes served to label motor nerve terminals with an extremely high reliability. Several control experiments showed that these dyes could be used to repeatedly observe neuromuscular junctions in living animals without having to worry about their affects on synaptic growth. Additional observations from this study concluded that these dyes may be useful for also determining the mitochondria content, and therefore oxidative capacity, of living muscle fibers ( Herrera and Banner, 2009).


 * References: **

Alberts, Bruce, et al. Molecular Biology of the Cell. New York: Garland Science, 2008. Print

The Nobel Prize in Physiology or Medicine 1906". Nobelprize.org. 8 Mar 2012 []

Thrane, C., Olsson, S., Nielsen, T. H. & Sorensen, J. (2008. Vital fluorescent stains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas fluorescens DR54. FEMS Microbiol Ecol 30, 11–23.

Fuller, M. E., S. H. Streger, R. K. Rothmel, B. J. Mailloux, J. A. Hall, T. C. Onstott, J. K. Fredrickson, D. L. Balkwill, and M. F. DeFlaun. 2009. Application of a Vital Fluorescent Staining Method for Simultaneous, Near-Real-Time Concentration Monitoring of Two Bacterial Strains in an Atlantic Coastal Plain Aquifer in Oyster, Virginia Appl. Environ. Microbiol. 66:4486-4496.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">A.A. Herrera, L.R. Banner The use and effects of vital fluorescent dyes: Observation of motor nerve terminals and satellite cells in living frog musclesJ. Neurocytol., 19 (2009), pp. 67–83