Resazurine

The constitution of resazurin is not known with certainty. It contains one oxygen atom more than resorufin, and the two are evidently closely related from the ease with which resazurin may be transformed into resorufin. The two also yield the same dioxyphenazoxin on reduction. It is probable that resazurin is expressed by the formula — 

Acetyl resazurin, Ci2HeN04 C2Hs0)j forms long red needles M.P. 222°. 

Resazurin ethyl ether, Ci2H8N04(C2Ho), forms dark red needles M.P. 215°. 

This compound is formed by action of nitrous acid on an ethereal orcin solution, and is identical with the dyestuff obtained by Liebermann by action of nitrous acid on orcin in presence of sulphuric acid. 

Resorufamin is only obtained in small quantity. It is a base forming salts which are easily soluble, and the solutions exhibit practically the same fluorescence as the alkaline solution of reso-rufin. 

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The methylene blue and resazurin reduction methods indirectly measure bacterial densities in milk and cream in terms of the time interval required, after starting incubation, for a dye—milk mixture to change color (methylene blue, from blue to white resazurin, from blue through purple and mauve to... 

An even simpler and perhaps more effective approach to the problem might be application of the resazurin test as applied in the milk industry for indirect estimation of bacterial population. Proctor and Greenlie (34) have suggested this application, and Wolford (38) has worked with it in a limited way. The test is based upon a color change involved in the reduction of the dye. The time required for reduction decreases with increase in bacterial numbers. Intensive investigation of this method and its application with respect to increasing its sensitivity, and correlation of reaction rates with bacterial population and quality characteristics of the product, might be of considerable value. 

Membrane Integrity (CTO) membrane results in the conversion of resazurin into a fluorescent resorufin product ... 

Alamar Blue (AB) Live cells resazurin reduction to red fluorescent dye resorufin Cell metabolism No [33, 34]... 

The formation of sodium oxalate, when sodium acetate served as the sole carbon source, gave valid support to this mechanism. The presence and action of the dehydrogenating enzyme system was evidenced by incorporating into the medium the dye, Resazurin (118). In the course of dehydrogenation this interagent was discolored. It behaves as follows ... 

This indicator possesses the advantage that it may be used under aerobic conditions, in contrast to dyes such as methylene blue (115). Resazurin has also been applied in the study of the dehydrogenation of isopropanol and sorbitol (103), (118). 

The relationship of the dicarboxylic amino acids, aspartic and glutamic acids to this process has also been studied (106). This investigation has been facilitated by a quantitative method for the codetermination (23) of the dehydrogenation indicator, resazurin, and its reduction product, resorufin. The wood destroying molds used were Trameles cinnabarina and Lentinus lepideus. 

The metabolism studies with cyanide present showed no dehydrogenation whatsoever of the substrate. It is thus considered likely that the resazurin and the resorufin interact with some metal bearing system, possibly the cytochromes participating in the hydrogen transfer. Although the resazurin (or resorufin) may interact in a system several steps removed from the dehydrogenation of the particular substrates, the relative rates of reduction of the indicator are still comparable with the relative oxidation rates of the substrates. 

Part 28. Application of Resazurin to the Study of Dehydrogenations by Certain Merulii and Pomes annosus. Arch. Biochem. 9, 439 (1946). 

DeFelip and coworkers use sodium resazurin as a dye to give a rapid assay in three to six hours 33. 

Table 7.1 Absence of C /polyvinylpyrrolidonc (C60/PVP) complex influence (5mg/ml) on cellular viability in cell culture MA-104. Endpoint - resazurin reduction...

However, fullerene C60-modified surface is adequate for the adhesion and normal growth of cells in culture. Cells of the line MA-104 in the Eagle-MEM medium formed on fullerene film a normal monolayer. Cellular viability was assessed with the resazurin (Alamar Blue) reduction test. The dye resazurin is reduced by mitochondrial dehydrogenases of viable cells into the fluorescent product resorufin (maximum X = 530 nm, max X = 590 nm). The intensity of... 

Table 7.2 Cellular viability of MA-104 culture grown on fullerene on the surfaces (FoS) during 72 h. (resazurin reduction test)...

Clothier R, Starzec G, Pradel L et al. (2002) The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (Resazurin) assay. ATLA. 30 493-504. 

Strain K4 was tested in the absence of oxygen at various concentrations of glycerol (10,20,30,40 g L ).Two serum bottles were used with a modified Hungate technique one ofthe bottles contained resazurin and a reducing agent, namely cysteine-H Cl, and the other did not contain resazurin. The gas was analyzed by GC with TCD. 

Fermentation of lactose during the growth of micro-organisms in milk has a major effect on its redox potential. The decrease in the h of milk caused by the growth of lactic acid bacteria is shown in Figure 11.3. A rapid decrease in h occurs after the available 02 has been consumed by the bacteria. Therefore, the redox potential of cheese and fermented milk products is negative. Reduction of redox indicators (e.g. resazurin or... 

Both the neutral red and SRB staining methods present the advantages that they avoid some potential for artifacts related to chemical interference with the tetrazolium and resazurin assay chemistries used to measure cell metabolism and the signals are stable. The obvious and major disadvantage of... 

Resazurin is a chemical redox indicator that functions like the tetrazolium compounds. The resazurin reduction assay approach has successfully replaced tritiated thymidine incorporation for some HTS applications (Ahmed, Gogal, and Walsh 1994 Shum et al. 2008). Resazurin can be dissolved in physiological buffers resulting in an intense blue solution that is added directly to growing... 

For HTS applications, the incubation period required to generate adequate fluorescent signal above background is usually about 2 to 4 hr, depending on the number of cells per well and their metabolic activity. Although longer incubation periods have been used, caution should be observed to avoid artifacts caused by the potential for secondary chemical reduction of resorulin into a colorless non-fluorescent compound and the known toxic effects of resazurin exposure to cells. 

One example is the known interference by reducing compounds that affect the chemical conversion of substrate to a colored indicator. This is especially true for the tetrazolium assays (Ulukaya, Colakogullari, and Wood 2004 Chakrabarti et al. 2000 Pagliacci et al. 1993 Collier and Pritsos 2003). The growing list of interfering compounds includes ascorbic acid and sulfhydryl reagents such as glutathione, coenzyme A, dithiothreitol, etc. Similar interferences by compounds that affect the oxidation and reduction chemistry of cells are likely to cause artifacts with the resazurin reduction assay. Assays that measure markers of metabolism also can be influenced by the pH of the culture medium and other factors that may stimulate or stress the metabolic rates of cells. 

FIGURE 6.4 Comparison of effects of Alamar blue (resazurin) and GF-AFC reagents on viability of cells measured using a luminescent ATP assay. Resazurin or GF-AFC was incubated with 10,000 DU145 cells per well for 18 hr prior to measuring ATP as an indicator of cell viability. Alamar blue reagent is more toxic to cells. 

The recent identification of selective protease substrates to simultaneously measure markers of both viable and dead cells led to the development of optional methods for HTS that provide flexibility and added advantages (Niles et al. 2007a). The assay to measure viable cells is based on a cell-permeable protease substrate called glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC). The procedure is a homogeneous add-incubate-measure method that is faster, more sensitive, and less toxic to cells than the tetrazolium and resazurin reduction assays. The substrate can be prepared in an aqueous buffer and is added directly to samples containing cells. The substrate permeates viable cells where constitutive protease activity in the cytoplasm rapidly removes the amino acids, yielding free AFC. The amount of AFC released is directly proportional to viable cell numbers and shows improved sensitivity compared to the resazurin assay (Figure 6.5). The AFC is detected via a microplate fluo-rometer equipped with a (380- to 400-nm excitation/505-nm emission) filter set. 

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