STAINING AND

MOUNTING
TECHNIQUE
-Analyn H. Hubilla-
BSED-BIOSCI
 STAINING

■ It is a process of imparting colour to the cell. Colored

compound is used to develop a contrast between the
specimen and the background.
■ It is simply means coloring of the micro organisms
with the dye that emphasizes  and elucidate different
important structures of microorganisms including
bacteria, virus, protozoa and etc.
■ The process of adding a dye to a bacterial culture.
 What is the difference between stain and
dye?
STAIN
■ Is used for any biological
specimen staining
■ are the biological colouring
agents that are more pure and
prepared with greater care and
specification.
DYE
■ A dye is colouring agent used
for general purposes
■ is crude and stain is purified
form
■ are the textile colouring agents
that have been prepared with
lesser specifications and they
may contain  the impurities  
Stain Types
 Iodine is one of the more commonly available stains
and is used to identify starch in a variety of samples. It
will stain carbohydrates in plants and animal specimens
brown or blue-black. Glycogen will show as red.

 Methylene Blue is an alkaline stain useful in

identifying acidic cell nuclei and DNA in animal,
bacteria or blood samples. It’s also useful in
aquariums to prevent the spread of fungal infections
in fish.
 Eosin Y is an acidic stain which stains pink for alkaline
cells (cytoplasm, for example). It colors red for blood
cells, cytoplasm and cell membranes. Eosin's most
important medical uses are in blood and bone-marrow
testing, including the PAP smear. 

 Gram's Stain is one of the most frequently used

processes in identifying bacteria – used daily in hospitals.
It is a primary test that quickly and cost effectively
divides bacteria into one of two types: Gram positive or
Gram negative.
Why stain cells?

■ Cells are stain to reveal the size and shape of microorganisms.

■ The most basic reason that cells are stained is to enhance visualization of
the cell or certain cellular components under a microscope.
■ Cells may also be stained to highlight metabolic processes.
■ To differentiate between live and dead cells in a sample.
■ Cells may also be enumerated by staining cells to determine biomass in an
environment of interest.
■ Cells are stained to demonstrate the presence of  internal and external
structures.
■ Cells are stained to distinguish between different types of organisms.
3 Types of Staining
Procedures

 Simple Staining (shapes and arrangements)

 Differential Staining (Gram reactions)
 Special Staining (Capsule, flagella, spores)
(1) Simple staining techniques
■ Simple = only one dye is used during the staining procedure
■ Staining can be performed with basic dyes such as crystal violet or
methylene blue, positively charged dyes that are attracted to the
negatively charged materials of the microbial cytoplasm. Such a
procedure is the simple positive staining.
■ An alternative is to use a dye such as nigrosin or Congo red, acidic,
negatively charged (acidic) dyes. They are repelled by the negatively
charged cytoplasm and gather around the cells, leaving the cells clear
and unstained. This technique is called the simple negative staining
technique.
 ■ Simple positive staining procedure

1. Add one loopful of the sample (mixture of microorganisms) onto a glass slide.
2. Allow it to air-dry.
3. Heat-fix the specimen on the glass slide, unless the specimen is heat-fixed, the
bacterial smear will wash away during the staining procedure.
4. Flood slide with crystal violet and wait 1 min. or safranin and wait 3-4min.
5. Wash the smear with tap water to remove the excess of stain.
6. Blot dray, than add cederwood oil (immersion oil) and examine under a
microscope.
■ Simple negative staining
procedure
1. Place a small drop of nigrosin at the end of
the slide.
2. Place a loopful of sample ( mixture of
microorganisms) and mix with drop of
nigrosin.
3. Using the edge of another slide, spread the
drop out across the slide.
4. Allow to air dray.
5. Place one drop of immersion oil and and
examine under a microscope.
 The results of simple staining procedures
Simple positive staining: all bacteria are colored,
Simple negative staining: background is dark, bacteria are without any color
(2) Differential staining techniques

■ The differential staining techniques are based on the application of a set of

several different dyes which react differently with different types of
microorganism.
■ An example is the Gram staining technique. This procedure separates
bacteria into two groups: Gram positive bacteria and Gram negative bacteria.
■ 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.
■ 1. 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.
■ 2. 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.
■ 3. 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.
■ 4. 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 gram-
negative bacteria a pink or red color.
■ PROCEDURE

■ Crystal violet is first applied, followed by the mordant iodine, which fixes the
stain.
■ Then the slide is washed with alcohol, and the Gram positive bacteria retain the
crystal violet iodine stain; however, the Gram negative bacteria lose the stain.
The Gram negative bacteria subsequently stain with the safranin dye, the
counterstain, used next. These bacteria appear red under the oil immersion
lens, while Gram positive bacteria appear blue or purple, reflecting the crystal
violet retained during the washing step.
■ Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the
crystal violet-iodine complex that occurs during staining, while Gram-negative
cells have only a thin layer of peptidoglycan. Thus Gram-positive cells do not
decolorize with ethanol, and Gram negative cells do decolorize. This allows the
Gram-negative cells to accept the counter stain safranin.
Gram Stain Procedure
1. Place a drop of NaCl onto the glass slide.
2. Using a sterilized and cooled inoculation loop, obtain a very small sample
of a bacterial colony.
3. Gently mix the bacteria into the NaCl drop.
4. Let the bacterial sample air dry
5. Using slide holder, pass the dried slide through the flame of Bunsen
burner 3 or 4 times, smear side facing up.
6. Apply primary stain: Flood slide with crystal violet stain.
7. Rinse: After 1 minute, rinse the slide with water.
8. Apply mordant: Flood the slide with iodine.
9. Rinse: After 1 minute, rinse the slide with water.
10. Apply decolorizer: Flood slide with acetone alcohol.
11. After 10 or 15 seconds, rinse the slide with water. (Do not leave the
decolorizer on too long or it may remove stain from the Gram-positive cells as
well.)
12. Apply secondary stain (counterstain): Flood slide with safrinin.
13. Rinse: After 1 minute, rinse the slide with water.
14. Dry on air
15. Place the stained smear on the microscope stage smear side up, apply oil
directly to the smear and focus using the 100X objective
■ Results of the Gram staining procedure:

■ Gram-positive bacteria appear blue or purple Gram-negative cells will

appear pink to red
■ Examples of Gram-positive and Gram-negative bacteria:
■ Gram-positive bacteria: Staphylococcus aureus, Streptococcus
pyogenes, Corynebacterium diphteriae
■ Gram-negative bacteria: Neisseria meningitidis, Escherichia coli,
Pseudomonas aeruginosa
 Acid fast staining technique
■ It is another widely used differential stain­ing procedure in bacteriology
■ This technique differentiates species of Mycobacterium from other bacteria.
Because the cell wall is resistant to water-based stains, acid-fast organisms
require a special staining technique.
■ This stain was developed by Paul Ehrlich in 1882, during his work on etiology
of tuberculosis
■ 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.
■ This technique differentiates species of Mycobacterium from other
bacteria. Because the cell wall is resistant to water-based stains,
acid-fast organisms require a special staining technique.
■ Heat or a lipid solvent is used to carry the first stain,
carbolfuchsin, into the cells. Then the cells are washed with a
diluted acid alcohol solution.
■ Mycobacterium species resist the effect of the acid alcohol and
retain the carbolfuchsin stain (they appear bright red under the
microscope). Other bacteria lose the stain and take on the
subsequent methylene blue stain (they appear blue under the
microscope).
Ziehl-Neelsen method:
■ In 1882-1883, Ziehl and Neelsen independently proposed acid fast stain, in
The staining reagents are much more stable than those described by
Ehrlich.
■ PROCEDURE:
1. Prepare a smear and fix it by gentle heat.
2. Flood the smear with carbol fuchsin (S19) and heat the slide from below till
steam rise for 5 minutes.
3. Do not boil and ensure that stain does not dry out.
4. Allow the’ slide to cool for 5 minutes to prevent the breakage of slide in the
subsequent prevent step.
5. Wash well with water.
6. Decolourize the smear till red colour no longer comes out in 20%
sulphuric acid.
7. Wash with water.
8. Counterstain with 1% aqueous solution of malachite green or
Loeffler’s methylene blue (S18) for 15-20 seconds.
9. Wash, blot dry and examine under oil-immersion objective
The Ziehl-Neelsen staining procedure with
Kinyoun modification (acid fast technique)

1. Place a drop of NaCl onto the glass slide.

2. Using a sterilized and cooled inoculation loop, obtain a very small sample of a
bacterial colony.
3. Gently mix the bacteria into the NaCl drop.
4. Let the bacterial sample air dry
5. Using slide holder, pass the dried slide through the flame of Bunsen burner 3
or 4 times, smear side facing up.
6. Flood slides with Kinyoun carbolfuchsin for 5 minutes.
7. Rinse gently with water until the water flows off clear.
8. Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds.
9. Rinse gently with water until the water flows off clear.
10. Flood slides with methylene blue for 1 minutes.
11. Rinse gently with water until the water flows off clear.
12. Allow slides to air dry before viewing.

Results of the Ziehl-Neelsen staining procedure: acid fast bacteria appear bright
red non-acid fast bacteria appear blue
Examples of acid fast and non-acid fast bacteria:
Acid fast bacteria: Mycobacterium tuberculosis, Mycobacterium avium
Non-acid fast bacteria: Escherichia coli, Staphylococcus aureus, etc.
■ Other staining techniques are designed to identify various bacterial
structures of importance. For instance, a special staining technique
highlights the flagella of bacteria by coating the flagella with dyes or
metals to increase their width. Flagella so stained can then be
observed. A special staining technique is also used to examine
bacterial spores. Malachite green is used with heat to force the stain
into the cells and give them color. A counterstain, safranin, is then
used to give color to the nonsporeforming bacteria. At the end of the
procedure, spores stain green and other cells stain red.
(3) Special Staining

■ To isolate the specific part of bacteria.

■ Examples:
– Staining of flagella
– Staining of endospores
– Staining of capsule
Endospore staining
■ Bacterial endospores are 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.
■ Heat is applied to help the primary stain penetrate the endospore. The cells are
then decolorized with water, which removes the malachite green from the
vegetative cell but not the endospore.  
■ Safranin is 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.
Capsule staining
■ Bacterial capsules play a role in adherence, protection (they inhibit ingestion
and killing by phagocytes), securing nutrients, and cell-to-cell recognition.
■ PRINCIPLE: Chemically, the capsular material is a polysaccharide, a
glycoprotein or a polypeptide. Capsule staining is more difficult than other
types of differential staining procedures because the capsular materials are
water soluble and may be dislodged and removed with vigorous washing.
Bacterial smears should not be heated, because the resulting cell shrinkage
may create a clear zone around the organism, an artifact that can be
mistaken for a capsule. The capsule is non-ionic, so that the dyes commonly
used will not bind to it. Two dyes, one acidic and one basic, are used to stain
the background and the cell wall, respectively.
■ Negative staining methods contrast a translucent, darker colored,
background with stained cells but an unstained capsule. The
background is formed with india ink or nigrosin or congo red.
■ A positive capsule stain requires a mordant that precipitates the
capsule. By counterstaining with dyes like crystal violet, methylene
blue or carbolfuchsin, bacterial cell wall takes up the dye. Capsules
appear colourless with stained cells against dark background.
■ Capsules are fragile and can be diminished, desiccated, distorted,
or destroyed by heating. A drop of serum can be used during
smearing to enhance the size of the capsule and make it more
easily observed with a typical compound light microscope.
■ Procedure of the capsule staining
1. Using sterile technique, add a loopful of bacterial culture to tube with 1 ml
NaCl.
2. Add one drop of carbolfuchsin into the tube and mix gently.
3. Heat the mixture under flame 1min.
4. Place a drop of mixture onto the glass slide.
5. Place a drop of nigrosin onto the same glass slide next to drop of mixture of
bacteria and the dye.
6. Use the other slide to drag the nigrosin-cell mixture into a thin film along the
first slide.
7. Allow to air dry for 5-7 minutes (do not heat fix).
8. Examine the smear microscopically (100X) for the presence of encapsulated
cells as indicated by clear zones surrounding the cells.
■ Results of the capsule staining procedure:
Encapsulated bacteria: Clear halos (capsules) are observed around pink
bacteria against dark background Bacteria without capsules: pink
bacteria with no clear halos
■ Examples of encapsulated and non-encapsulated bacteria:
■ Encapsulated bacteria:
Bacillus anthracis, Klebsiella pneumoniae, Streptococcus
pneumoniae, Neisseria meningitidis, Clostridium perfringens;
■ Non-encapsulated bacteria:
Neisseria gonorrhoreae, etc.
Summary of the staining techniques:
■MOUNTING
There are four common ways to mount
a microscope slide 
1. Dry mount - requires no water (slide, object, coverslip); usually used
for inanimate objects that don't require water to live.
In a dry mount, the specimen is placed directly on the slide. A cover slip
may be used to keep the specimen in place and to help protect the
objective lens. Dry mounts are suitable for specimens such as samples of
pollen, hair, feathers or plant materials.
■ Place slide on a flat surface.
■ Lay specimen on top of slide (use as thin of a specimen as possible - 1
cell layer thick is best).
■ Place coverslip slowly on top of specimen as flat as possible. 
■ In some cases, staining is unnecessary, for example when microorganisms are
very large or when motility is to be studied, and a drop of the microorganisms
can be placed directly on the slide and observed. A preparation such as this is
called a wet mount.

2. Wet mount - requires water (slide, water, object, coverslip); used to prepare

slides that hold living organisms (mobile or not).
■ the specimen is suspended in a drop of liquid (usually water) located between
slide and cover glass.
■ the water refractive index of the water improves the image quality and also
supports the specimen. In contrast to permanently mounted slides, wet
mounts can not be stored over extended time periods, as the water
evaporates.
■ For this reason, it is sometimes also referred to as a “temporary mount” to
contrast it from the “permanent mounts”, which can be stored over longer
times.
Different types of wet mounts
1. Using water from the natural habitat of the organism:

In the case of water organisms, such as algae or ciliates, the liquid water should
come directly from the sample. In this case the organism is immersed in its own
natural environment. The microscopist uses a dropper to place a drop of pond water
directly on the microscope slide.
2. Using 0.9% salt water:
In some cases water from the natural habitat may not be available. This is the
case when observing bacteria or molds grown on petri-dishes. Yoghurt bacteria, for
example, need to be diluted a lot before being able to observe them, otherwise they
are too dense to be observed as single cells. In this case it is necessary to mix some
salt (NaCl) into some water to ensure an optimal osmotic potential. This “physiological
saline”, as it is called, can be made by dissolving 9 grams of table salt (NaCl) in 1 liter
of water (or 0.9g Nacl in 100ml of water).
3. Using tap water: 
If one wants to observe non-living specimens, such as dust samples, sand grains, or
thin section cuts of plant material, then it is also possible to use regular tap water. These
specimens are not osmotically sensitive. If the specimen is observed without water, in a
dry condition, then the resolution and image quality may not be sufficiently high.
4. Using immersion oil: 
Some wet mounts are not made with water, but by using immersion oil. Immersion oil
is usually placed on top of the cover glass. In this case the specimen does not get into
contact with the oil. It is also possible to submerge the specimen in the oil, however.
Heat-fixed bacteria can be observed directly by placing a drop of immersion oil on the
specimen, without cover glass. The oil-immersion objective is then rotated directly into the
oil for observation.
5. Pure glycerin or glycerin-water mixtures: 
Glycerin has a strong tendency to withdraw water from the sample. For this reason it
also acts as a preservative. On the down side, the glycerin may therefore cause the
specimen to shrink and deform. Especially algae and other water organisms are sensitive
to dehydration. Other specimens, such as sectioned or microtomed plant material are not
as sensitive. The reason why glycerin is used is because of its high refractive index.
Advantages of a wet mount
■ Quick preparation: specimen fixation, dehydration and staining
are not necessary (but possible, if required). For this reason, wet
mounts are the first kind of mounts that students learn to make.
■ Few artifacts: If there is no chemical and physical processing of
the specimens before observation (no fixation), there are little
artifacts and the specimens appear in their natural condition.
■ Living and moving: It is possible to observe living and moving
organisms. It is also possible to observe certain processes of life,
such as feeding, cell division etc. (for water-based mounts)
■ Natural colors: The colors are natural and not faded. The colors
of permanently mounted specimens may fade over time.
Disadvantage of a wet mount
■ Movement: The advantage of observing movement can also be a
disadvantage. Due to the movement of the organisms it may be more
difficult to take pictures or to make drawings. There is a solution to this
problem: one can slow down ciliates and other protozoa by adding a solution
such as ProtoSlo, which increases the viscosity of the water.
■ Evaporation: The heat of the lamp causes the water to evaporate more
quickly. More water must be added under the cover glass from time to time.
■ Focus: Some organisms may swim vertically in the water and therefore
move in and out of focus. Here it is important not to use too much or too
little water. Too little water may squeeze the specimen between cover glass
and slide.
■ Storage: Wet mounts can not be stored over a longer time.
Materials and Method
For making a wet mount you need these materials:
■ Microscope slides
■ Cover glasses
■ The specimen to be observed: make sure that the specimen is sufficiently
small and thin. Thick specimens must either be cut (microtomed) into
sections, be squeezed or torn apart.
■ Water: take care that the osmotic potential of the water is compatible with
the specimen. For example, do not use fresh water with marine specimens,
and vice versa. Use pond water (and not tap water) for observing pond
organisms.
■ Droppers, pipette: these are for transferring the water
■ Tweezers: for handling the specimen, the cover glass and for adding water
If the specimen is already in water (algae, ciliates
etc.) then you can proceed the following way:
■ Place a small drop of sample fluid (containing the specimen) in the
center of the microscope slide.
■ Hold the cover glass on one side with the help of tweezers. Lower
the cover glass onto the water drop at an angle.
■ Then slowly lower the cover glass into the liquid. This will minimize
disturbing air bubbles.
■ Remove excess water with filter paper or tissue paper. The cover
glass should not float freely. The surface tension of the water should
hold it in place. Alternatively you can add more water using a pipette
or tweezers.
If the specimen is not in water:
■ Place a small drop of water (without specimen) in the center of the microscope
slide.
■ Place the specimen into the water.
■ Add some more water on top of the specimen and make sure that the specimen is
completely submerged. Otherwise there is the possibility for air bubbles forming
between cover glass and specimen. The remaining steps are the same as above.
■ Hold the cover glass on one side with the help of tweezers. Lower the cover glass
onto the water drop at an angle.
■ Then slowly lower the cover glass into the liquid. This will minimize disturbing air
bubbles.
■ Remove excess water with filter paper or tissue paper. The cover glass should not
float freely. The surface tension of the water should hold it in place. Alternatively
you can add more water using a pipette or tweezers.
A wet mount can also be prepared by placing a drop of culture on a cover slip (a glass cover
for a slide) and then inverting it over a hollowed out slide. This procedure is called the
hanging drop.

3. Section Mount
In a section mount, an extremely thin cross-section of a specimen is used. Using
a microtome, cut a thin slice of your selected specimen such as an onion, and
carefully set it on your slide. Then follow the instructions for a dry or wet mount. A
stain can often be applied directly to the specimen before covering with a cover slip.
Section mounts are suitable for useful for a wide variety of samples such as fruit,
vegetables and other solids that can be cut into small slices.
4. Smear
A smear is made by carefully smearing a thin layer of the specimen across a slide
and then applying a cover slip. Typically, a smear should be allowed to air dry before
applying a stain. 
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