Isolation & staining of cell organelles

Cell disruption
The initial step of subcellular organelle purification is to rupture the cell wall, the
plasma membrane or both, as this is a prerequisite to fractionation techniques. Cells
can be disrupted in various ways, which include mechanical methods –
homogenization (shear), cell rupture by pressure, sonication, grinding; structural
damage – osmotic shock, freeze/ thaw; chemical methods – treatment with
detergents, solvents and enzymes.
Mechanical methods:
(a) Homogenization
Disruption of cells is a process known as homogenization. First the cells are
suspended in solution of appropriate pH and salt content (usually isotonic sucrose
buffer) or combination of salts similar in Composition to those in the cell interior.
Then the cells are broken by special pressurized tissue homogenizer, in which the
cells are forced through a very narrow space between the plunger and the vessel
wall. The cell solution must be kept at 0°C in order to preserve enzymes and other
constituents, after they release from the stabilizing forces of
the cell.
(b) Cell rupture by pressure
This way of disruption is particularly useful to disrupt microbial cells but also for
other cell types. The cells are forced through a narrow extrusion by applying high
pressure. A kind of disruption by repeated high velocity compression and expansion
is known as a “French press”. This device is very powerful and is applicable for
molecular dissociation.
(c) Sonication
Many cells are broken by exposing a cell suspension to highfrequency
sound (ultrasonic oscillations). The sonication is used to disrupt cells, particularly
tissue cultures cells, and to release subcellular organelles. It must be taken into
account that considerable heat is generated, which is the main disadvantage using
this method. The ultrasonic waves commonly break open the cell periphery and
leave the
cellular organelles intact.
(d) Grinding
Some types of cells (such as plant cells, microbes and yeasts),especially those which
cell periphery include in addition to their plasma membrane, complex layer (e.g.
chitin or other poly-saccharides), must be disrupted first by grinding. This method
includes simple mortar and pestle grinding using sand or glass beads. Great
efficiency can be achieved if cells are undergone rapid freezing in liquid nitrogen.

Structural damage:
(a) Osmotic shock
The advantages of this method are its simplicity, easiness of performance and
absence of shearing forces. The method is useful for osmotic sensitive cells and it is
a good for non-osmotic organelles such as ribosomes, etc. Essentially, the cells are
transferred from one medium to another – hypotonic and it leads to swelling and
finally the cells burst open,because of passing the water into the cells in a effort to
balance the osmotic pressure. However there are some disadvantages: the osmotic
sensitive organelles such as mitochondria, lysosomes, Golgi apparatus, endoplasmic
reticulum, etc might also burst. On the other hand bacteria and some tissues don’t
undergone to osmotic lysis and for these reasons the method is inapplicable.
(b) Freeze/thaw
This approach includes freezing, followed by thawing, which procedure is repeated
several times, e.g. 8-10 times. The method is based on the formation of ice crystals
during freezing, which broke the cell membranes.
Chemical methods:
(a) Detergents
The most useful chemical reagents for disrupting the cell membranes are detergents,
which are small amphipathic molecules that tend to form micelles in water. Because
of the properties of the membrane compounds the hydrophobic ends of detergents
bind to the hydrophobic regions of the membrane proteins, thereby displacing the
lipid molecules and disrupting the membranes. Mainly two types of detergents are
used: ionic (such as SDS – sodium dodecyl sulfate) and nonionic (such as Triton X-
100 and NP 40). The strong (ionic) detergents can solubilize the most hydrophobic
membrane proteins, but at the same time they unfold them. In this respect such
strong detergents are unusable for functional studies and for isolation of subcellular
membrane structures. In such case mild (nonionic) detergents are predominantly
used in a low concentrations (to affect only the plasma membrane).
(b) Organic solvents
Besides detergents there are other agents capable of solubilising membrane
structures, but their usage is not so widely spread. Many organic solvents like
toluene, acetone, ethyl acetate, etc. are applied to dissociate membrane compounds.
As they affect other intracellular membrane-bound structures, they are considered to
be less useful in practice.

(c) Enzymes
This approach is mainly used to digest the extracellular matrix structure (e.g. animal
tissues) and the cell walls (bacteria, fungi and plants). The choice of enzyme
depends on the specificity of the object. The aims of the treatment are directed to
obtain cell suspension from solid tissues or protoplasts from cells possessing rigid
walls. In the first case different proteolytic enzymes (collagenase, trypsin, elastase,
etc.) are applicable for animal tissues and others (cellulase, pectinase, etc) for plant
tissues. In the second case we use enzymes to digest some specific components of
the cell wall without (or less) effecting the membrane, e.g. cellulase for plant cells,
glucanase (Zymolyase, Lyticase or Gluzulase) for yeasts and lysozyme for bacteria.
Methods for cell fractionation
Centrifugation: Differential centrifugation
The most widely used method for cellular components fractionation is
differential centrifugation. Differential centrifugation is used for the first steps in
cell fractionation because it rapidly separates large membrane organelles from
smaller one. It is based on differences in speed at which organelles sediment to the
bottom of a centrifuge tube. The selection of velocity and duration of time depends
on different size weight, density and shape of studying organelles. This type of
centrifugation is known as velocity sedimentation.
• Obtaining of subcellular fractions by velocity sedimentation
Centrifugation at low centrifugal force results in sedimentation of the largest
subcellular particles – nuclei. Usually, nuclear pellet is obtained by centrifugation at
700 g, 10 min. The supernatant from this fraction is removed and loaded again on
another centrifuge tube and run at an increased centrifugal force – 10,000 - 15,000 g
for 10 min (or longer), which results in sedimentation of mitochondrial faction.
Microsomal fraction requires higher speed and longer time of centrifugation. The
appropriate parameters are 100,000 g, 30 min - 2 hr. The final supernatant includes
free ribosomes and soluble components of cytosol (both, small structures and
soluble molecules). Although this procedure allows separation of main subcellular
organelles, additional steps are needed to purify them.
• An example protocol for nuclei isolation
Different protocols exist in the literature for isolation of nuclei, but we shall
describe one explored in our laboratory, because it is applicable for many cell types
(e.g. hepatocytes and fibroblast cell culture). To preserve the structures, all
procedures have to be performed on ice or at 4°C. All buffers should contain PMSF
(a protease inhibitor).
PROTOCOL 1
1) The liver is homogenized in buffer A containing (146 mM sucrose, 100 mM KCl,
1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.0, 0.1 mM PMSF). The homogenate is
filtrated through 2-3 layers of wet cheesecloth to remove some cell aggregates.
2) Nonidet-40, or Triton X-100 is added (0.2 % final concentration) and the
homogenate is incubated 5 min on ice with continuing stirring.
3) The sample is placed on the centrifuge tube and run at 700 g for 5 min, at 4°C to
receive a crude nuclear fraction. Usually this fraction is contaminated with unbroken
cells and cell debris.
4) In the next step the pellet is re-suspended in buffer B containing (146 mM
sucrose, 100 mM KCl, 5 mM MgCl2, 0.5 mM CaCl2, 10 mM Tris-HCl, pH 7.7).
The solution is overlaid onto 1M sucrose cushion and is pelleted at 1600 g for 10-15
min, at 4°C.
5) The pellet is washed in the same buffer B and is sedimented at 1600 g for 10 min,
at 4°C. The final pellet consists of nuclei with satisfied purity.
 Separation of mitochondrial fraction into mitochondria, lysosomes and
peroxisomes
Mitochondria can be isolated either from the post-nuclear supernatant or
mitochondrial fraction of differential centrifugation, by single centrifugation, using
a sucrose gradient as well as hybrid Percoll-metrizamide gradient. Densities of
layers have been determined with higher and lower values, considering the density
of organelles have to be separated (see Table 1). Number of layers depends on the
desired sensitivity of separation. Commonly the “steps” of density depending on the
reagents are 1.9 – 1.25 g/ml in the layer on the bottom, 1.20 – 1.06 g/ml in the next
layers and about 1.06 – 1.04 g/ml in the upper layer .
PROTOCOL 2
(1) Place 2 ml of 35 % metrizamide on the bottom of a centrifuge tube and overlay 2
ml of 17 % metrizamide carefully.
(2) Overlay 5 ml of 6 % Percoll onto 17 % metrizamide layer.
(3) Gently fill the tube with post-nuclear supernatant leaving at least 5 mm free
space from the top of the tube (the volume of this layer should be about 4.75 ml).
Gradient should be prepared just before use and placed on ice.
(4) Centrifuge for a minimum 15 min at 50,500 g (20,000 rpm in Beckman SW-40
rotor), at 4°C. After centrifugation, a set of discrete bands should be observed.
(5) Remove about 1 – 3 ml of material from each interface with a Pasteur pipette.
Mitochondrial band should appear at the 17 / 35 % metrizamide interface.
(6) Collect Percoll / 17 % metrizamide interface (which contains lysosomes
contaminated with mitochondria) and adjust to 35 % metrizamide by mixing with
appropriate volume of 80 % metrizamide (e.g. 1.125 ml of collected interface with
0.875 ml of 80 % metrizamide).
(7) Place on the bottom of a centrifuge tube and after that overlay 17 %
metrizamide, followed by 5 % metrizamide solution. Supplement the tube with 0.25
M sucrose.
(8) Centrifuge the second gradient at 20,000 rpm for 15 min and collect interfaces.
Lysosomes will retain at the 5 / 17 % metrizamide interface and second
mitochondrial band may appear at the 17 / 35 % metrizamide interface.
• Separation of microsomal fraction, separation of macromolecules
Isolation of microsomal fraction, or “microsomes” can be separated from the post-
mitochondrial supernatant fraction by centrifugation at 100,000g. This fraction
consists mainly of plasma membranes, Goldgi vesicles and endoplasmic reticulum.
Fractionation of microsomes can be performed by density equilibration in a
continuous (or discontinuous) sucrose gradient centrifugation, where the sample is
layered on the top or at the bottom of gradient. Centrifugation of post-mitochondrial
supernatant in 0.44 M sucrose layered over 1.3 M sucrose at 100,000 g for about 7
hr will result in rough microsomal pellet and smooth microsomal band at the
sucrose-sucrose interface.

PROTOCOL 3
1) Make a crude extract from yeast (1.2 – 1.3 g wet weight) and mix up to 10 ml
with buffered mannitol (0.3 M mannitol in 50 mM Tris-HCl, pH 7.2). Then adjust
the density with 3 ml of 62 % (w/v) sucrose in buffered mannitol (BM)
2) In a 40 ml centrifuge tube prepare a discontinuous gradient as follow: place the
suspension on the bottom and load 2-ml portions of 7, 6, 5, 4, 3, 2 and 1 % (w/v)
sucrose in BM. Overlay the gradient with 2 ml BM.
3) Centrifuge the tube in a swing-out rotor at 11,000 g for 1 hr at 4°C. A layer,
consisting of low-density vesicles (LDV), forms on top of the gradient. On the
bottom of the tube a pellet of membranes including as well plasma membranes is
obtained.
4) Carefully remove LDV layer with a syringe. Because there is no contamination, it
is not necessary to wash it before examination.
5) Discard the remainder of the gradient and wash the surface of the pellet gently
with BM.
6) Suspend the pellet in 10 ml of BM and homogenize the mixture in a Teflon-glass
hand homogenizer (0.1 ml clearance).
7) Dilute the suspension up to 30 ml with BM and incubate it for at least 20 min in
an ice-water mixture.
8) Add 9 ml of 62 % (w/v) sucrose in BM to the suspension and centrifuge it at
11,000 g, for 30 min in a swing-out rotor.
9) Remove again the fraction of small LDV population, which appears on top of the
gradient, and homogenize the membrane fraction in 2 ml of BM.

PROTOCOL 4
The suspension of membrane fraction must be proceeded immediately to
obtain purified plasma membranes using a modification of the sucrose density-
gradient method of Hossack:
1) Dilute the 2 ml suspension of membranes with a further 2 ml BM (see Protocol 3)
and place on the bottom of a centrifuge tube.
2) Load a discontinuous gradient of sucrose solutions over the suspension in the
similar way as in Protocol 3. Prepare the gradient starting with two 5-ml aliquots of
62 % and then 50 % (w/v) sucrose in BM, followed by 3-ml aliquots of 45, 40, 35,
30, 20 and 10 % (w/v) sucrose in BM.
3) Centrifuge the tube at 24,000 g for 90 min, 4°C in a swingout rotor. After
centrifugation several bands of membranous material can be seen in the gradient and
they can be removed by peristaltic pump.
4) Discard the remaining supernatant liquid and re-suspend the pellet in 35 ml of
BM, centrifuge and re-suspend again the pellet in 2 ml of BM.
5) Correct density to a refractometer value, equivalent of 30 % (w/v) sucrose, using
62 % (w/v) sucrose in BM.
6) Overlay onto this suspension 2-ml aliquots (2 % step decreases in concentration)
starting from 21 % (w/v) to 1 % (w/v) sucrose in 50 mM Tris-HCl buffer, pH 7.2.
7) Centrifuge the gradient at 11,000 g for 4.5 hr, 4°C in a swingout rotor. The
obtained pellet contains purified plasma membranes.
A modification of discontinuous gradient might be used to obtain the Goldgi
fraction from rat liver tissue:

PROTOCOL 5
1) Rat liver tissue is homogenized in 3 vol. of 52 % sucrose,
containing 0.1 M sodium phosphate pH 7.1 and homogenate is filtrated through two
double layers of wetted cheese cloth.
2) The filtrate is adjusted to 43.7 % sucrose by adding 0.25 M sucrose and 15 ml is
pipetted into a cellulose nitrate tube (these tubes are preferred because the interfaces
are clearly visible).
3) Sequentially 10-ml aliquots of 38.7 %, 36 %, 33 % and 12 ml of 28 % sucrose
solutions are overlaid onto the homogenate (this is best done running the solutions
down the site of the tube).
4) The gradients are centrifuged at 100,000 g for 55 min. Distinct bands at the 28/33
% and 33/36 % sucrose interfaces appear. The fractions are collected, diluted with
an equal volume of water and re-centrifuged for 10 min at 16,000 g.
5) The Golgi fraction is collected from the supernatant by centrifugation at 180,000
g for 60 min. The pellet formed is suspended in neutralized 0.25 M sucrose (using
hand-driven pestle).
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.

-sd/-
Sanjeeb K. Dey Baidya
Asst. Professor & HoD
Dept. of Zoology
Patkai Ch. College

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