Download Stains and types of staining techniques

Identification of Microbes: Stains and
types of staining techniques
What is staining?

Colored compound is used to develop a contrast between the specimen and the
background. A process of imparting colour to the cell is known as staining.
What is the difference between stain and dye?

A dye is colouring agent used for general purposes and a stain is used for any
biological specimen staining. Also dye is crude and stain is purified form.

Dyes are the textile colouring agents that have been prepared with lesser
specifications and they may contain
the impurities . Stains are the biological
colouring agents that are more pure and prepared with greater care and specification.
Why stain cells?
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Cells are stain to reveal the size and shape of microorganisms.
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The most basic reason that cells are stained is to enhance visualization of the cell or
certain cellular components under a microscope.
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Cells may also be stained to highlight metabolic processes.
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To differentiate between live and dead cells in a sample.
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Cells may also be enumerated by staining cells to determine biomass in an
environment of interest.
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Cells are stained to demonstrate the presence of internal and external structures.
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Cells are stained to distinguish between different types of organisms.
Chemistry of Dye
Definition of Dye
A dye is a coloured substance that has an affinity to the substrate to which it is being applied.
The dye is generally applied in an aqueous solution, and requires a mordant to improve the
fastness of the dye on the fiber.
Chemically a dye (stain) may be defined as an organic unsaturated cyclic compound with
chromophore and auxochrome group. The colour is usually due to chromophore and dyeing
property of salt formation is due to auxochrome.
A chemical possessing only chromophore group may be a good chromogen (Colored
compound) but may not be a good stain/dye unless and until it has an auxochrome group.
Without an auxochrome group the chromogen is not able to bind to cells or tissues or fibers.
The ability of a stain to bind to macromolecular cellular components depend on the electrical
charge found on the chromogen portion as well as on the cellular components to be stained.
Both dyes and pigments appear to be colored because they absorb some wavelengths of light
more than others. In contrast with a dye, a pigment generally is insoluble, and has no affinity
for the substrate. Some dyes can be precipitated with an inert salt to produce a lake pigment,
and based on the salt used they could be aluminum lake, calcium lake or barium lake
pigments.
Staining Methods in Microbiology
Simple Stains
They are also referred as monochrome stains, since only one dye is employed for the
colouration of bacterial smear (The act of taking bacteria taken from a lesion or area,
spreading them on a slide, and staining them for microscopic examination.). The surface of a
bacterial cell has an overall acidic characteristic because of large amount of carboxyl groups
located on the cell surface due to acidic amino acids. Therefore, when ionization of carboxyl
groups takes place it imparts negative charge to the cell surface as per the following equation.
COOH → COO- + H+
H+ is removed and the surface of the bacteria becomes negatively charged and a positively
charged dye like (methylene blue) attaches to the negatively surface and gives it a coloured
appearance.
Methylene blue chloride → Methylene Blue++ Cl‑
Negative Stains
Negative staining is a technique (used mainly in electron microscopy) by which bacterial
cells are not stained, but are made visible against dark background.
Acidic dyes like eosin and nigrosin are employed for this method. Though, this staining
technique is not very popular, it has an advantage over the direct or positive staining methods
for the study of morphology of cells. This is because of the fact that the cells do not receive
vigorous physical or chemical treatments.
The colouring power of acidic dye e.g. eosin in sodium eosinate is having negative charge,
therefore, it does not combine with the negatively charged bacterial cell surface. On the other
hand, it forms a deposit around the cell, resulting into appearance of bacterial cell colourless
against dark background. Some suitable negative stains include ammonium molybdate,
uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide
and auroglucothionate. These have been chosen because they scatter electrons well and also
adsorb to biological matter well. The method is used to view viruses, bacteria, bacterial
flagella, biological membrane structures and proteins or protein aggregates, which all have a
low electron-scattering power.
Differential Stains
Staining procedure which differentiates or distinguishes between types of bacteria is termed
as differential staining technique. Methods for simple staining impart same colour to all
bacteria and other biological material, may be slight variation in shade. On the other hand,
differential staining methods impart distinctive colour only to certain types of bacteria.
The basic principle underlying this differentiation is due to the different chemical and
physical properties of cell and as a result, they react differently with the staining reagents.
Differential staining procedure utilizes more than one stain. In some techniques the stains are
applied separately, while in other as combination. There are two most important differential
stains, namely, (A) Gram stain and (B) Acid-fast stain.
(A) Gram Stain
Gram stain is one of the most important and widely used differential stains. It has great
taxonomic significance and is often the first step in the identification of an unknown
prokaryotic organism. This technique divides bacteria into two groups (i) Gram positive those
which retain primary dye like crystal violet and appear deep violet in colour and (ii) Gram
negative, which lose the primary dye on application of decolourizer and take the colour of
counterstain like safranin or basic fuchsin.
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of
cell wall), which stains purple while gram-negative bacteria have a thinner layer (10% of cell
wall), which stains pink. Gram-negative bacteria also have an additional outer membrane
which contains lipids. There are four basic steps of the Gram stain, which include applying a
primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the
addition of a trapping agent (Gram’s iodine), rapid decolorization with alcohol or acetone,
and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since it
will more intensely stain anaerobic bacteria but it is much less commonly employed as a
counterstain.
Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions.
These ions penetrate through the cell wall and cell membrane of both gram-positive and
gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial
cells and stains the cells purple.
Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and
iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a
mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore
color the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell
membrane. A gram-negative cell will lose its outer membrane and the lipopolysaccharide
layer is left exposed. The CV–I complexes are washed from the gram-negative cell along
with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an
ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell
due to the multilayered nature of its peptidoglycan. After decolorization, the gram-positive
cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is
usually positively charged safranin or basic fuchsin, is applied last to give decolorized gramnegative bacteria a pink or red color.
Acid Fast Stains
Acid fast staining is another widely used differential stain-ing procedure in bacteriology. This
stain was developed by Paul Ehrlich in 1882, during his work on etiology of tuber culosis (5).
Some bacteria resist decolourization by both acid and alcohol and hence they are referred as
acid-fast organisms. Acid alcohol is very intensive decolourizer. This staining technique
divides bacteria into two groups (i) acid-fast and (ii) non acid-fast. This procedure is
extensively used in the diagnosis of tuberculosis and leprosy.
Acid-fastness property in certain Mycobacteria and some species of Nocardia is correlated
with their high lipid content. Due to high lipid content of cell wall, in some cases 60% (w/w),
acid-fast cells have relatively low permeability to dye and hence it is difficult to stain them.
For the staining of these bacteria, penetration of primary dye is facilitated with the use of 5%
aqueous phenol which acts as a chemical intensifier. In addition, heat is also applied which
acts as a physical intensifier. Once these cells are stained, it is difficult to decolourize
Ziehl-Neelsen method:
Ziehl and Neelsen independently proposed acid fast stain, in 1882-1883 is commonly used
today. The staining reagents are much more stable than those described by Ehrlich.
The procedure for staining is as follows. Prepare a smear and fix it by gentle heat. Flood the
smear with carbol fuchsin (S19) and heat the slide from below till steam rise for 5 minutes.
Do not boil and ensure that stain does not dry out. Allow the’ slide to cool for 5 minutes to
prevent the breakage of slide in the subsequent prevent step. Wash well with water.
Decolourize the smear till red colour no longer comes out in 20% sulphuric acid. Wash with
water. Counterstain with 1% aqueous solution of malachite green or Loeffler’s methylene
blue (S18) for 15-20 seconds. Wash, blot dry and examine under oil-immersion objective
Endospore staining
Bacterial endosporesare metabolically inactive, highly resistant structures produced by some
bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria
can remain in this suspended state until conditions become favorable and they can germinate
and return to their vegetative state. The primary stain applied is malachite green, which
stains both vegetative cells and endospores.
penetrate the endospore.
Heat is applied to help the primary stain
The cells are then decolorized with water, which removes the
malachite green from the vegetative cell but not the endospore. Safraninis then applied to
counterstain any cells which have been decolorized. At the end of the staining process,
vegetative cells will be pink, and endospores will be dark green.