STAINING OF CELL ORGANELLES

Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic

image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological
tissues for viewing, often with the aid of different microscopes. Stains may be used to define and
examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations
(classifying different blood cells, for instance), or organelles within individual cells.

In biochemistry it involves adding a class-specific (DNA, proteins, lipids, carbohydrates) dye

to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent
tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry,
and to flag proteins or nucleic acids in gel electrophoresis

A few common stains :-

Different stains react or concentrate in different parts of a cell or tissue, and these properties are
used to advantage to reveal specific parts or areas. Some of the most common biological stains are
listed below. Unless otherwise marked, all of these dyes may be used with fixed cells and tissues; vital
dyes (suitable for use with living organisms) are noted.

Acridine orange : Acridine orange (AO) is a nucleic acid selective fluorescent cationic dye useful for
cell cycle determination. It is cell-permeable, and interacts with DNA and RNA by intercalation or
electrostatic attractions. When bound to DNA, it is very similar spectrally to fluorescein. Like
fluorescein, it is also useful as a non-specific stain for backlighting conventionally stained cells on the
surface of a solid sample of tissue (fluorescence backlighted staining).

Bismarck brown : Bismarck brown (also Bismarck brown Y or Manchester brown) imparts a yellow
colour to acid mucins. Bismarck brown may be used with live cells.

Crystal violet : Crystal violet, when combined with a suitable mordant, stains cell walls purple.
Crystal violet is an important component in Gram staining.

DAPI : DAPI is a fluorescent nuclear stain, excited by ultraviolet light and showing strong blue
fluorescence when bound to DNA. DAPI binds with A=T rich repeats of chromosomes. DAPI is also
not visible with regular transmission microscopy. It may be used in living or fixed cells. DAPI-stained
cells are especially appropriate for cell counting.[4]

Eosin : Eosin is most often used as a counterstain to haematoxylin, imparting a pink or red colour to
cytoplasmic material, cell membranes, and some extracellular structures. It also imparts a strong red
colour to red blood cells. Eosin may also be used as a counterstain in some variants of Gram staining,
and in many other protocols. There are actually two very closely related compounds commonly
referred to as eosin. Most often used is eosin Y (also known as eosin Y ws or eosin yellowish); it has a
very slightly yellowish cast. The other eosin compound is eosin B (eosin bluish or imperial red); it has
a very faint bluish cast. The two dyes are interchangeable, and the use of one or the other is more a
matter of preference and tradition.

Ethidium bromide : Ethidium bromide intercalates and stains DNA, providing a fluorescent red-
orange stain. Although it will not stain healthy cells, it can be used to identify cells that are in the final
stages of apoptosis - such cells have much more permeable membranes. Consequently, ethidium
bromide is often used as a marker for apoptosis in cells populations and to locate bands of DNA in gel
electrophoresis. The stain may also be used in conjunction with acridine orange (AO) in viable cell
counting. This EB/AO combined stain causes live cells to fluoresce green whilst apoptotic cells retain
the distinctive red-orange fluorescence.

Acid fuchsine : Acid fuchsine may be used to stain collagen, smooth muscle, or mitochondria. Acid
fuchsine is used as the nuclear and cytoplasmic stain in Mallory's trichrome method. Acid fuchsine
stains cytoplasm in some variants of Masson's trichrome. In Van Gieson's picro-fuchsine, acid fuchsine
imparts its red colour to collagen fibres. Acid fuchsine is also a traditional stain for mitochondria
(Altmann's method).

Haematoxylin : Haematoxylin (hematoxylin in North America) is a nuclear stain. Used with a

mordant, haematoxylin stains nuclei blue-violet or brown. It is most often used with eosin in H&E
(haematoxylin and eosin) staining—one of the most common procedures in histology.

Hoechst stains : Hoechst is a bis-benzimidazole derivative compound which binds to the minor
groove of DNA. Often used in fluorescence microscopy for DNA staining, Hoechst stains appear
yellow when dissolved in aqueous solutions and emit blue light under UV excitation. There are two
major types of Hoechst: Hoechst 33258 and Hoechst 33342. The two compounds are functionally
similar, but with a little difference in structure. Hoechst 33258 contains a terminal hydroxyl group and
is thus more soluble in aqueous solution, however this characteristics reduces its ability to penetrate the
plasma membrane. Hoechst 33342 contains a ethyl substitution on the terminal hydroxyl group (i.e.
an ethylether group) making it more hydrophobic for easier plasma membrane passage

Iodine : Iodine is used in chemistry as an indicator for starch. When starch is mixed with iodine in
solution, an intensely dark blue colour develops, representing a starch/iodine complex. Starch is a
substance common to most plant cells and so a weak iodine solution will stain starch present in the
cells. Iodine is one component in the staining technique known as Gram staining, used in
microbiology. Lugol's solution or Lugol's iodine (IKI) is a brown solution that turns black in the
presence of starches and can be used as a cell stain, making the cell nuclei more visible. Iodine is also
used as a mordant in Gram's staining, it enhances dye to enter through the pore present in the cell
wall/membrane.

Methyl green : Methyl green is used commonly with bright-field microscopes to dye the chromatin
of cells so that they are more easily viewed.

Methylene blue : Methylene blue is used to stain animal cells, such as human cheek cells, to make
their nuclei more observable. Also used to staining the blood film and used in cytology.

Neutral red : Neutral red (or toluylene red) stains Nissl substance red. It is usually used as a
counterstain in combination with other dyes.

Nile blue : Nile blue (or Nile blue A) stains nuclei blue. It may be used with living cells.

Nile red : Nile red (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric
acid. This produces a mix of Nile red and Nile blue. Nile red is a lipophilic stain; it will accumulate in
lipid globules inside cells, staining them red. Nile red can be used with living cells. It fluoresces
strongly when partitioned into lipids, but practically not at all in aqueous solution.

Rhodamine : Rhodamine is a protein specific fluorescent stain commonly used in fluorescence

microscopy.
Safranin : Safranin (or Safranin O) is a nuclear stain. It produces red nuclei, and is used primarily as
a counterstain. Safranin may also be used to give a yellow colour to collagen.

Stainability of tissues
Positive affinity for a specific stain may be designated by the suffix -philic. For example,
tissues that stain with an azure dye may be referred to as azurophilic. This may also be used for more
generalized staining properties, such as acidophilic for tissues that stain by acidic stains (most notably
eosin), basophilic when staining in basic dyes and amphophilic[5] when staining with either acid or
basic dyes. In contrast, Chromophobic tissues do not take up coloured dye readily.

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