Stains for Biology and Compound

Classification of Biological Dyes

2 4
3 2
4 2
5 2

Natural Dyes


Leuco Compound

  • Chromophores are easily reduced by combining with hydrogen at the double bonds.
  • Reduction of chromophore results in loss of color.
  • These decolorized dyes are known as Leuco compounds.
  • Use: they are used as an indicator of oxidation & reduction reactions.


  • Nitro group may be reduced to amino radical
  • Decolorization of p-rosaniline
  • Decolorisation of p-rosaniline

Theories of Staining

  • Staining solutions generally contain a low concentration of dye.
  • A very dilute staining solution acting for a relatively long period of time will, in general, produce much better results than a more concentrated solution acting for a short time interval.
  • Intensifiers: The addition of certain substances to staining solutions causes the organisms to take up the stain more deeply than would occur in their absence. These substances are spoken of as intensifiers.
  • Intensifies include acids, alkalies, aniline oil, phenol, etc.
  • Mordants: They are chemicals that have the ability to make dyes stain objects that they would not stain otherwise. These substances are given the name of mordants.
  • Tannic acid is example of a mordant.
  • Simple stains: A simple staining solution is one that contains only a single dye dissolved in the solvent. •Eg: dilute carbol fuchsin, methylene blue, and gentian violet or crystal violet.
  • Carbolfuchsin Stain: This stain is prepared by dissolving the dye basic fuchsin in a 5 per cent solution of phenol or carbolic acid.
  • The phenol is added as an intensifier. •The basic fuchsin is a mixture of the two dyes p-rosaniline and rosaniline.
  • Methylene Blue Stain: Methylene blue is tetramethyl thionine, a basic dye.
  • The chromophore is the thiazine group with one of the benzene rings having a quinoid structure.
  • Because of its strongly basic , nature it stains nucleuses and nucleic acid granules very intensely.
  • It is very useful in making a rapid survey of the bacterial population of milk.
  • This dye is usually preferred in staining smears for the diagnosis of diphtheria.
  • It is used in combination with eosin for staining blood films.
  • Crystal Violet Stain: Crystal violet is also a member of the triaminotriphenyl-methane group of compounds.
  • The dye is also known as methyl violet 10J5, gentian violet, hexa methyl violet, etc.
  • Differential Stains: Differential stains are composed of more than one dye.
  • In some of the staining techniques, the dyes are applied separately; in others they are mixed and applied in one solution.
  • Two most important differential stains used in bacteriology are the Gram stain and the acid fast stain.

Staining of Bacteria

Preparation of Smear

Viability of Fixed and Stained Organisms

  • It is generally stated that bacteria in dried, fixed, and stained smears are no longer viable and that no danger from infections is possible if pathogenic organisms are so treated.
  • Thurn (1914) reported that organisms are not necessarily killed in fixed and stained preparations. He found that cultures of Staphylococcus aureus, Eberthella typhosa, Escherichia coli, Bacillus anthracis, Vibrio comma, Corynebacterium diphtherias, and Saccharomyces cerevisiae smeared onto glass slides and fixed by passing three times through the flame, but not stained, still contained viable organisms.
  • On the other hand, B. anthracis survived 1 min. and B. mesentericus 3 min. of treatment with carbol fuchsin and both organisms survived 5 min. of treatment with methylene blue.
  • More recently Morton (1939) also showed that organisms are able to survive drying and fixation by heat.
  • In addition he reported that certain organisms are capable of surviving treatment with basic fuchsin, Hucker’s gentian violet, aqueous safranine, and methylene blue stains.