|
|
|
Dyes and staining:
The terms dyes and stains are generally used interchangeably by the biologists, but they are not the same. Dye is a colouring agent which is used for the general purpose while, stain is a biological colouring agent and is manufactured with greater care under the strict specifications. Natural dyes predominated during the early part of the century, but, at present only few are being used.
Dye has been defined by Conn (8) as, an organic compound containing both chromophoric and auxochromic groups, linked to the benzene rings, in which chromophoric group is responsible for colouring property and the salt forming auxochromes for dying proper y.
|
.In the dye the colour is associated with some atomic groupings called as chromophorese. The examples of these groups include C = C (ethenyl), C = 0 (carbonyl), C=N (amino), C = S (thiocarbonyl), N = N (azo), N =0 (nitroso) and NO2 (nitro). The intensity of colour depends upon the number of such groups. Compounds of benzene containing chromophore radicals are called chromogens. Such compounds, though coloured, are not dyes, since they do not have the affinity for or the ability to unite with tissue.
To be a dye, a compound must contain not only the chromophore group, but also the additional group(s) called auxochrome(s). These auxiliary groups are responsible for imparting the property of electrolytic dissociation i.e., the separation of the dye molecule into its components or atoms and to form salts with either acid or alkali.
|
A more practical classification of dyes for cytologists is the one, which is based on the chemical behaviour 'of the dye, namely, acidic, basic and neutral. Acidic or anionic dyes are those dye salts in which the dye ion is an anion. Thus,acid dyes ionize to give the dye portion of the molecule, a negative electrical charge. They are usually sodium and sometime potassium, calcium and ammonium salts of colour acids. Conversely, the basic dyes are the salts in which the dye ion is cation and hence positively charged.
|
|
The common anions in these dyes are chloride, sulfate, acetate and oxalate. Neutral dyes are complex salts of a dye acid with, a dye base, i.e., eosinate of methylene blue.
Numerous methods are available in literature for staining of subcellular structures of cell, yet our knowledge on the mechanism of staining is in its infancy. This is mainly because of the large variation in the physical and chemical properties of the cell. The histological data suggest that new covalent compounds are not formed during the staining with the dye salts. Furthermore, the absorption spectrum of the dye in the stained cell is similar to that of the dye in the staining reagent.
|
|
|
Extensive washing of the stained cells results into the removal of the dye and the destained cells are again stained indicating that there is no change in the substrate or the dye during the staining procedure. The strong chemical bonding between the dye molecule and substrate is unlikely.
However, weaker chemical bonds are not excluded. Electrical charges are definitely involved in some of the staining procedures. According to the physical theory most of the substrate(s) in the staining techniques are solid, which provide opportunity for the adsorption of the dye molecule involving solely the physical process. However, the chemical theory is more popular.
|
A difference in response to staining indicates a chemical dif¬ference in the substrate regardless of the nature of the mecha¬nism. Variation in the chemical composition of the cell has been found to produce variation in the staining even though, a stain is taken up solely by a physical process as observed in the staining of fat globules. Thus, the .process of staining may involve ion-exchange reaction(s) between the stain molecule and the active site(s) at the surface or within the cell. Acid dyes are generally found to stain basic cell components and basic dyes generally stain acidic cell components.
This topic is very well covered by Lamanna and Mallette (9) and Clark (10).
To day, a wide range of dyes are available to the bacterio¬logists because of the synthetic manufacturing processes. They are used with various modifications in the basic staining techniques, routinly used in bacteriology. They are also used as indicator in the chemical reactions. The range of pH and the change in colour of some of the commonly used dyes is shown in Table I-I.
Useful pH range and colour change of some indicator dyes
Indicator dye |
Useful pH range |
Colour change acid to alkaline |
Cresol red (pK1) |
02.-1.8 |
Red-Yellow |
Thymol blue (pK1) |
1.2-.2.8 |
Red-yellow |
Bromophenol blue |
3.0-4.6 |
Yellow-Blue |
Bromocresol green |
3.8-5.4 |
Yellow-Blue |
Methyl red |
4.4-6.0 |
Red-Yellow |
Chlorophenol red |
4.8-6.4 |
Yellow-Red |
Bromocresol purple |
5.2-6.8 |
Yellow-Purple |
Bromothymol blue |
6.0-7.6 |
Yellow-Blue |
Phenol red |
6.8-8.4 |
Yellow-Red |
Gresol red (pK2) |
7.2-8.8 |
Yellow-Red |
Thymol blue (pK2) |
8.0-9.6 |
Yellow-Blue |
Phenolphthalein |
8.3-10.0 |
Yellow-Red |
Tolyl red |
10.0-11.6 |
Red-yellow |
Parazo orange |
11.0-12.6 |
Yellow-Orange |
Acyl blue |
12.0-13.6 |
Red-Bule |
|
|
