STAIN

AND ST A N IN G T EC HN IQ U E

PRIYA DIXIT
TM E N T OF B IOTE CH NOLOGY
DEPAR
KNOW
ERA UNIVERSITY, LUC
  INTRODUCTION
• Bacteria are microscopic organisms.

• They are also colorless for the most part.

• In order to visualize them to study their structure, shape and other structural characteristics, it becomes necessary to make them more easily visible.

• This means that the structures have to be contrasted from their environment so that they can be seen easily.

• Stain: Stain is a dye used to color the living or dead organelles.

• Stains and dyes are frequently used in histology (the study of tissue under the microscope) and in the medical fields of histopathology, hematology,
and cytopathology that focus on the study and diagnoses of disease at a microscopic level.

• In vivo staining (also called vital staining or intravital staining) is the process of dyeing living tissues. By causing certain cells or structures to take on
contrasting colour(s), their form (morphology) or position within a cell or tissue can be readily seen and studied. The usual purpose is to reveal
cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are
taking place within cells or tissues.

• In vitro staining involves colouring cells or structures that have been removed from their biological context. Certain stains are often combined to reveal
more details and features than a single stain alone. Combined with specific protocols for fixation and sample preparation, scientists and physicians can
use these standard techniques as consistent, repeatable diagnostic tools. A counterstain is stain that makes cells or structures more visible, when not
completely visible with the principal stain.
STAINS AND DYES
A dye is a general-purpose coloring agent, whereas a stain is used for coloring biological material.
 A stain is an organic compound containing a benzene ring plus a chromophore and an auxochrome group.
Chromophore is a chemical group that imparts color to benzene.
Auxochrome group is a chemical compound that conveys the property of ionization of chromogen (ability to form salts) and bind to
fibers or tissues.

TYPES:
ACIDIC: Negatively charged acid radicals imparts color in eosin, acid fuchsine, malachite green, nigrosin, Indian ink.

BASIC: Positively charged basic radicals combines with negatively charged particles in cytoplasm and gives color. Ex:
Haematoxillin, methylene blue, crystal violet, gention violet.

NEUTRAL: Both positively and negatively charged imparts different colors to different components. Ex: Geimsa’s stain,
Leishman’s stain, Wright’s stain.
Requirements for staining Stain:
• Stain- Majority of the stains used for staining bacteria are of the basic type as nucleic acid of bacterial cells attract the positive ions, e.G. Methylene blue,
crystal violet. Acidic stains are used for background staining.

• Mordant – It is a chemical that forms an insoluble complex with the stain and fixes it or causes the stain to penetrate more deeply into the cell. These
are used in indirect staining. For example, gram’s iodine in gram staining and phenol in ziehl neelson’s staining.

• Accentuater – It is a chemical which when added to a stain to make the reaction more selective and intense. For example, potassium hydroxide added
in loeffler’s methylene blue.

• Decolorizer – It is a chemical used to remove the excess stain in indirect regressive staining. For example, ethanol in gram’s staining.
OBJECTIVES OF STAINING

• Improves visibiltiy by greater contrast between the organism and the background, differentiate various morphological
types (by shape, size, arrangement, etc.).

• Determine the staining characteristic of organism and, at times, direct diagnosis of disease, and demonstrate the purity of
culture.

• observe certain structures (flagella, capsules, endospores, etc.),

Bacterial smear preparation:
Smear - Is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope.
Method:

• Aseptically a small sample of the culture is spread over a slide surface.

• This is then allowed to air dry.

• The next step is heat fixation to help the cells adhere to the slide surface.
• The smear is now ready for staining.
Tissue sections:
 The sections being embedded in paraffin.

 It is necessary to remove the paraffin so that a watery stain may penetrate.

 The paraffin is first removed with xylene, the xylene is then removed with alcohol and the alcohol is replaced with
water.
 The staining is then done.
SMEAR FIXATION:
1) HEAT FIXATION
 Pass air-dried smears through a flame two or three times.
 Do not overheat.
 Allow slide to cool before staining.
2) METHANOL FIXATION
 Place air-dried smears in a coplin jar with methanol for one minute. Alternatively, flood smear with methanol for 1
minute.
 Drain slides and allow to dry before staining.
Histological staining –
Process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution,
producing coloration of the active tissue component.
Micro anatomical or histologic staining is used to demonstrate the general relationship of tissues and cells with differentiation of
nucleus and cytoplasm

Histochemical staining (Histochemistry) –

Various constituents of tissues are studied through chemical reactions that will permit microscopic localization of a specific tissue
substance.

Example:
• Perls prussian blue reaction for hemoglobin
• Periodic acid schiff staining for carbohydrates
Immunohistochemical staining
- A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated
under the microscope, using a wide range of polyclonal or monoclonal fluorescent labeled or enzyme-labeled antibodies
TYPES OF STAINING TECHNIQUES

• SIMPLE STAINING (USE OF OF SINGLE STAIN)

• DIRECT - (POSITIVE)
• INDIRECT - (NEGATIVE)

DIFFERENTIAL STAINING (USE OF TWO CONTRASTING STAINS)

• SEPARATION INTO GROUPS (1. GRAM STAIN , 2. ACID FAST )

• VISUALIZATION OF STRUCTURES (1. FLAGELLA STAIN 2. CAPSULE STAIN 3. SPORE
STAIN )
 SIMPLE STAINING
• A staining method that uses only a single dye that which does not differentiate between different types of organisms
• There is only a single staining step and everything is stained with the same color.
• Simple stains are used to stain whole cells or to stain specific cellular components.
• Types of simple staining:
Direct / positive staining : stain object
Indirect / negative staining: stain background

DIRECT STAINING (POSITIVE STAINING)

➢A simple staining technique that stains the bacterial cells in a single color.

➢Many of the bacterial stains are basic chemicals; these basic dyes react with negatively charged bacterial cytoplasm
(opposite charges attract) and the organism becomes directly stained
➢Examples are methylene blue, crystal violet, and basic fuchsin.
Loeffler’s methylene blue:
It is generally the most useful, it shows the characteristic morphology of polymorphs, lymphocytes and other cells
more clearly than do stronger stains such as the gram stain or dilute carbol fuchsin.
Polychrome methylene blue:
This is made by allowing loeffler’s methylene blue to ‘ripen’ slowly.
The slow oxidation of the methylene blue forms a violet compound that gives the stain its polychrome properties.
The ripening takes 12 months or more to complete, or it may be ripened quickly by the addition of 1.0% potassium
carbonate (k2co3) to the stain.
It is also employed in mcfadyean’s reaction.
Incontrast to the blue staining of most structures by the methylene blue, the violet component stains acidic cell
structures red-purple , e.G. The acid capsular material of the anthrax bacillus in the mcfadyean reaction.
Dilute carbol fuchsin
Made by diluting ziehl-neelsen’s stain with 10-20 times its volume of water. Stain for 10-25 seconds and wash well
with water. Over-staining must be avoided, as this is an intense stain, and prolonged application colours the cell
protoplasm in addition to nuclei and bacteria.
Indirect staining (Negative staining)

In this staining process, instead of ells background is stained.

 Here, an acidic dye like nigrosin or indian ink is used. Acidic stain carries a negative charge and repelled by the bacteria, which also carry a
negative charge on their surface. Hence, an acidic dye do not stain bacteria, instead, it forms a deposit around the organism, leaving the organism
itself colorless or transparent upon examination.

Importance of fixing the smears

“Fixation accomplishes three things:

(1) It kills the organisms;

(2) It causes the organisms to adhere to the slide;
(3) It alters the organisms so that they more readily accept stains
(dyes).
Differential staining
Separation into groups (1. Gram stain , 2. Acid fast )
THE GRAM STAIN
In the late 1800’s, christian gram observed that some genera of bacteria retained a dye-iodine complex when rinsed with alcohol, while
other genera were easily decolorized with alcohol and could be then visualized by a contrasting counter stain.
This staining procedure defines two bacterial groups: those which retain the primary dyes (“positive by gram’s method” or “gram-
positive”) and those which are easily decolorized (“negative by gram’s method” or “gram-negative”). This is the starting point for bacterial
identification procedures.
The difference in dye retention is dependent on such physical properties as thickness, density, porosity, and integrity of the bacterial cell
wall, as well as its chemical composition.
Gram-positive bacteria have thick, dense, relatively non-porous walls, while gram-negative bacteria have thin walls surrounded by lipid-
rich membranes.
Some non-bacterial organisms with thick cell walls (e.G., Some yeasts) also stain gram-positive.
Gram-positive bacteria which have lost wall integrity through aging or physical or chemical damage may stain gram-negative.
Gram-positive cell walls

Gram-positive bacteria normally have cell walls that are thick and composed primarily of peptidoglycan.
Peptidoglycan in gram-positive bacteria often contains a peptide interbridge.
In addition, gram-positive cell walls usually contain large amounts of teichoic acids , polymers of glycerol or ribitol joined by phosphate groups.
Amino acids such as d -alanine or sugars such as glucose are attached to the glycerol and ribitol groups.
The teichoic acids are covalently connected to the peptidoglycan itself or to plasma membrane lipids; in the latter case, they are called lipoteichoic acids.
Teichoic acids appear to extend to the surface of the peptidoglycan.
Peptidoglycan is a polysaccharide made of two glucose derivatives, n-acetylglucosamine (nag) and n-acetylmuramic acid (nam), alternated in long chains.
The chains are cross-linked to one another by a tetrapeptide that extends off the nam sugar unit, allowing a lattice-like structure to form.
The four amino acids that compose the tetrapeptide are: l-alanine, d-glutamine, l-lysine or meso-diaminopimelic acid (dpa), and d-alanine.
Because they are negatively charged, they help give the gram-positive cell wall its negative charge.

The functions of techoic acids are still unclear, but they may be important in maintaining the structure of the wall.

Teichoic acids are not present in gram-negative bacteria.

The periplasmic space of gram-positive bacteria lies between the plasma membrane and the cell wall, and is smaller than that of gram-negative bacteria.

The periplasm has relatively few proteins; this is probably because the peptidoglycan sac is porous and any proteins secreted by the cell usually pass through it.

Enzymes secreted by gram-positive bacteria are called exoenzymes

.
• They often serve to degrade polymeric nutrients that would otherwise be too large for transport across the plasma Membrane.

• Those proteins that remain in the periplasmic space are usually attached to the plasma membrane.

• Staphylococci and most other gram-positive bacteria have a layer of proteins on the surface of the peptidoglycan.

• These proteins are involved in interactions of the cell with its environment.

• Some are noncovalently attached by binding to the peptidoglycan, teichoic acids, or other receptors.
Gram-negative cell walls

• Even a brief inspection of shows that gram-negative cell walls are much more complex than gram-positive walls.
• The thin peptidoglycan layer next to the plasma membrane and bounded on either side by the periplasmic space usually constitutes only 5 to 10% of the wall weight.
• In e. Coli, it is about 2 nm thick and contains only one or two sheets of peptidoglycan.
• The periplasmic space of gram-negative bacteria is also strikingly different from that of gram-positive bacteria.
• It ranges in width from 1 nm to as great as 71 nm.
• Some recent studies indicate that it may constitute about 20 to 40% of the total cell volume, and it is usually 30 to 70 nm wide. When cell walls are disrupted carefully or removed without
disturbing the underlying plasma membrane, periplasmic enzymes and other proteins are released and may be easily studied.

• Some periplasmic proteins participate in nutrient acquisition—for example, hydrolytic enzymes and transport proteins.
• Some periplasmic proteins are involved in energy conservation.
• For example, the denitrifying bacteria, which convert nitrate to nitrogen gas, and bacteria that use inorganic molecules as energy sources (chemolithotrophs) have electron transport proteins
in their periplasm.

• Other periplasmic proteins are involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell.
GRAM STAINING – REQUIREMENTS
• The specimen is mounted and heat fixed on a slide before proceeding to stain it.
• The reagents required are:
➢crystal violet (the primary stain)
➢iodine solution (the mordant)
➢decolorizer (ethanol)
➢safranin (the counter stain)
➢Water (preferably in a squirt bottle)

Procedure
1. The bacteria are first stained with the basic dye crystal violet (primary stain). Both gram-positive and gram-negative bacteria become
directly stained and appear purple after this step.
2. The bacteria are then treated with gram's iodine solution (mordant). This allows the stain to be retained better by forming an insoluble
crystal violet-iodine complex, called as ‘iodine lake’. Both gram-positive and gram-negative bacteria remain purple after this step.
3. Gram's decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the
crystal violet-iodine complex while gram-negative are decolorized.
4. Finally, the counter stain safranin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not
affected by the counter stain. Gram-negative bacteria, that are now colorless, become directly stained by the safranin. Thus, gram-positive
appear purple, and gram-negative appear pink.
 MECHANISM OF GRAM STAINING

 The difference between gram-positive and gram-negative bacteria is thought to be due to the physical nature of their cell walls.
 If the cell wall is removed from gram-positive bacteria, they stain gram negative.
 Furthermore, genetically wall-less bacteria such as the mycoplasmas also stain gram negative.
 During the procedure, bacteria are first stained with crystal violet and next treated with iodine to promote dye retention.
 When bacteria are treated with ethanol in the decolorization step, the alcohol is thought to shrink the pores of the thick peptidoglycan found in gram- positive bacteria, causing
the peptidoglycan to act as a permeability barrier that prevents loss of crystal violet.

Thus the dye-iodine complex is retained during the decolorization step and the bacteria remain purple.
In contrast, gram-negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores.
Alcohol treatment also may extract enough lipid from the outer membrane to increase the cell wall’s porosity further. For these reasons, alcohol more readily removes the crystal
violet-iodine complex from gram-negative bacteria. Thus gram-negative bacteria are easily stained red or pink by the counterstain safranin.
 ACID-FAST STAINING
Acid-fast staining is another important differential staining procedure.
It is most commonly used to identify mycobacterium tuberculosis and m. Leprae, the pathogens responsible for tuberculosis and leprosy, respectively.
These bacteria have cell walls with high lipid content, in particular mycolic acids—a group of branched-chain hydroxy lipids, which prevent dyes from readily binding to the cells.
However, m. Tuberculosis and M. Leprae can be stained by harsh procedures such as the ziehl-neelsen method, which uses heat and phenol to drive basic fuchsin into cells.
Once basic fuchsin has penetrated, m. Tuberculosis and M. Leprae are not easily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast.
Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.
Theory
 Once stained the acid fast bacterial cells resist decolorization with acidified organic solvents, e. G acid
alcohol and are therefore called ACID FAST.

➢ Acid fast staining property of the genus, mycobacteria, depends upon their lipid-rich cell walls which are
relatively impermeable to various basic dyes unless the dyes are combined with phenol.

➢ The exact method by which the stain is retained is unclear but it is thought that some of the stain becomes
trapped within the cell and some forms a complex with the mycolic acids. This is supported by the finding that
shorter chain mycolic acids or mycobacterial cells with disrupted cell walls stain weakly acid-fast, e.G.
Nocardia
•It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. So this method is also called ziehl-neelsen
staining techniques. Neelsen in 1883 used ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue.
Thus ziehl-neelsen staining techniques was developed.
•The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.
•This method is used for those microorganisms which are not staining by simple or gram staining method, particularly the member of genus mycobacterium,
are resistant and can only be visualized by acid-fast staining.

Principle of acid-fast stain

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the mycobacterial cell wall but by the application of heat, carbol
fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red. Then the smear is decolorized with decolorizing
agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents
the penetration of decolorizing solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized,
leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color
and appears blue while acid-fast cells retain the red color.
Procedure of acid-fast stain
• Prepare bacterial smear on clean and grease free slide, using sterile technique.
• Allow smear to air dry and then heat fix.
Alcohol-fixation: this is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained
immediately. M. Tuberculosis is killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. Tuberculosis whereas
alcohol-fixation is bactericidal.

• Cover the smear with carbol fuchsin stain.

• Heat the stain until vapour just begins to rise (i.E. About 60 C). Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
Heating the stain: great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly
fiammable chemicals have collected from previous staining. Only a small fiame should be applied under the slides using an ignited swab previously dampened
with a few drops of acid alcohol or 70% v/v ethanol or methanol. Do not use a large ethanol soaked swab because this is a fire risk.

• Wash off the stain with clean water.

Note: when the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.

• Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.E. Pale pink.
Caution: acid alcohol is fiammable, therefore use it with care well away from an open fiame.

• Wash well with clean water.

• Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin.
• Wash off the stain with clean water.
• Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
• Examine the smear microscopically, using the 100 X oil immersion objective.
Summary of Acid-Fast Stain

Cell colour
Application of Reagent
Acid fast Non-acid fast

Primary dye Carbol fuchsin Red Red

Decolorizer Acid alcohol Red Colorless

Counter stain Methylene blue Red Blue

 VISUALIZATION OF STRUCTURES
1. FLAGELLA STAIN
2. CAPSULE STAIN
3. SPORE STAIN

1. Flagella stain:
Principle: Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the Gram stain, or a simple
stain. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella
are critical to the identification of species of motile bacteria. The staining procedures require the use of a mordant so that the stain adheres
in layers to the flagella, allowing visualization.

The procedure of Flagella Stain (Wet Mount Technique)

• Grow the organism to be stained at room temperature on blood agar for 16 to 24 hours.
• Add a small drop of water to a microscope slide.
• Dip a sterile inoculating loop into sterile water.
• Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water).
• Touch the loopful of motile cells to the drop of water on the slide. Note: agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell.

• Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet mount has barely enough liquid to fill the space under a coverslip. Small air spaces around the edge are
preferable.

• Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do not proceed with the stain.

• If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes. This allows the bacterial cells time to adhere either to the glass slide or to the coverslip.

• Gently apply 2 drops of ryu flagella stain (remel, lenexa, kansas) to the edge of the coverslip. The stain will flow by capillary action and mix with the cell suspension. Small air pockets
around the edge of the wet mount are useful in aiding the capillary action.

• After 5 to 10 minutes at room temperature, examine the cells for flagella.

• Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about halfway from the edge of the coverslip to the center of the mount.

• Focusing the microscope on the cells attached to the coverslip rather than on the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is
primarily on the slide rather than the coverslip.

Result Interpretation of Flagella Stain

1.Presence or absence of flagella
2.Number of flagella per cell
3.Location of flagella per cell

Limitation
Even with a specific stain, visualization of flagella requires an experienced laboratory scientist and is not considered an entry-level technique.
 Capsule staining:
Capsule stain is a type of differential stain which uses acidic and basic dyes to stain background & bacterial cells respectively so that presence of capsule is easily visualized. Capsule is synthesized in the cytoplasm and
secreted to the outside of the cell where it surrounds the bacterium. Most of the capsulated bacteria have a capsule made up of a polysaccharide layer but some bacteria have capsule made up of polypeptide, or glycoprotein.

• Capsules are associated with virulence in several microorganisms, including streptococcus pneumoniae and neisseria meningitides, because capsules resist phagocytosis thus evading the host immune system.

• Capsule staining is diagnostically useful since it is a virulent factor (e.G.Pneumococci).

• Bacterial capsules are non-ionic,so neither acidic nor basic stains will adhere to their surfaces.

• Capsules are demonstrated either by negative staining (nigrosine or india ink)or by special staining, e.G.Hiss’method,anthony’s method
Principle of Capsule Stain
Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic
stain (e.g., Nigrosine, congo red) and to stain the cell itself using a basic stain (e.g.,crystal violet, safranin, basic fuchsin, and methylene blue).

Various types of methods are available for the demonstration of the presence of a capsule. The results (stain of the cells, background, and capsule) depend on the type of method used. Two
commonly used methods are discussed here:

A. India ink method

In this method, two dyes, crystal violet, and India ink are used. The capsule is seen as a clear halo around the microorganism against the black background. This method is used for
demonstrating Cryptococcus.
•The background will be dark (color of India ink).
•The bacterial cells will be stained purple (bacterial cells take crystal violet-basic dyes as they are negatively charged).
•The capsule (if present) will appear clear against the dark background (capsule does not take any stain).
Materials and Reagents required
•Test bacteria: 36-48 hour culture of capsulated bacteria e.G. Klebsiella pneumoniae growing on a slant of EMB agar or culture of other capsulated bacteria and non-capsulated bacteria [note: growing klebsiella
pneumoniae in milk-based media (e.G. Skim milk) increase its capsule size, making it easier to visualize. ]
•Stain solutions: depending on the type of method used (crystal violet, india ink, nigrosin, copper sulfate, basic carbol fuschin solution, methylene blue solution, etc).
•Microscopic slides
•Inoculating loop
•Microscope with 100x objective lens (oil immersion)
•Immersion oil
•Gas burner
•Tissue paper
METHOD

1.Place a single drop of India ink on a clean microscope slide, adjacent to the frosted edge.
2.Using a flamed loop and sterile technique, remove some Klebsiella pneumoniae from culture tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of
organism, but try to avoid spreading the drop. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a
film. This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear.
3.Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry!  Heat will melt the capsule!
4.Saturate the slide with crystal violet for 1 minute and rinse slightly & very gently with water. Be cautious water may remove the capsule from the cell.
5.Let the slide air dry for a few minutes. DO NOT blot the slide! Blotting will remove the bacteria from the slide and/or distort the capsule.
6.Observe the slide under oil immersion.
Results: Look for purple cells surrounded by a clear halo on a dark background. The halo is the capsule. You may need to decrease the amount of light
in order to make the capsule easier to see.
B. Anthony’s stain method
In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain. There is no mordant in the capsule
staining procedure. A 20% copper sulfate solution serves a dual role as both the decolorizing agent and counterstain. It decolorizes the capsule by washing out the crystal violet, but
will not decolorize the cell. As the copper sulfate decolorizes the capsule, it also counterstains the capsule. Thus, the capsule appears as a faint blue halo around a purple cell.

Materials and Reagents required

•Test bacteria: 36-48 hour culture of capsulated bacteria e.G. Klebsiella pneumoniae growing on a slant of EMB agar or culture of other capsulated bacteria and non-capsulated bacteria
[note: growing klebsiella pneumoniae in milk-based media (e.G. Skim milk) increase its capsule size, making it easier to visualize.]
•Stain solutions: depending on the type of method used (crystal violet, india ink, nigrosin, copper sulfate, basic carbol fuschin solution, methylene blue solution, etc).
•Microscopic slides
•Inoculating loop
•Microscope with 100x objective lens (oil immersion)
•Immersion oil
•Gas burner
•Tissue paper
Method:
1.Place a single drop of crystal violet on a clean microscope slide, adjacent to the frosted edge.
2.Using a flamed loop and sterile technique, add three loopful of test bacterium (any capsulated bacteria such as Klebsiella pneumoniae, Streptococcus pneumoniae) from broth culture. If
you are adding bacteria from a culture plate make sure that there are no large clumps of the organism, but try to avoid spreading the drop.
3.Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film. This is done by contacting the drop of
crystal violet with the clean microscope slide and using the capillary action of the dye/ slide to spread the crystal violet across the smear.
4.Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry!  Heat will melt the capsule!
5.Tilt the slide and rinse with 20% copper sulfate solution. DO NOT RINSE WITH WATER! Water will remove the capsule from the cell.
6.Let the slide air dry for a few minutes. DO NOT blot the slide! Blotting will remove the bacteria from the slide and/or distort the capsule.
7.Observe the slide under oil immersion.
Results: Look for purple cells surrounded by a clear or faint blue halo on transparent background. The halo is the capsule. You may need to decrease the amount of light in order to make the
capsule easier to see.
 Points to remember
• Clean your microscope with lens cleaner, removing all oil from lenses.

• Dispose of staining waste and slides in designated waste containers.

• Be cautious while handling the slide, since the organisms have not been killed.

Hiss Method
• The capsule is nonionic in nature so it doesn’t get stain by a acidic stain but a basic stain, such as crystal violet, stains the cell as well as the capsule.
• This is followed by treatment with hypertonic solution 20% Copper sulphate solution, which serves dual role of both the decolorizing agent and counter stain.
• Copper sulphate solution, being hypertonic, causes diffusion of stain towards outer surface of cell.
• After drying of slide, the stain which is not passed from the capsular layer during diffusion retains in the capsular layer. Copper sulphate then decolorizes the capsule.
• Capsule appears as a faint blue halo around a purple cell.
Endospore staining
An endospore is a dormant, tough, and non-reproductive structure produced by some bacteria in the phylum firmicutes.
In 1922, dorner published a method for staining endospores. Shaeffer and fulton modified dorner’s method in 1933 to make the process faster the endospore stain is a differential stain which selectively stains
bacterial endospores. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. 
• Spores are normally impervious to stains.

• Under the light microscope endospores have a high light refractivity indicative of high protein content.

Endospores can be stained by:

• Modified zeihl-nelson’s method using 0.25-0.5% sulphuric acid as decolorizing agent,

• Barthelomew-mittwar’s method

• Schaeffer- fulton stain technique.

Principle of 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.
In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for
cellular material, so vegetative cells may be decolourized with water. 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.
Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may
be spherical or elliptical.
Reagents used for Endospore Staining
Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)
0.5 gm of malachite green
100 ml of distilled water
Decolorizing agent
Tap water or Distilled Water
Counter Stain: Safranin
Stock solution (2.5% (wt/vol) alcoholic solution)
2.5 gm of safranin O
100 ml of 95% ethanol
Procedure of endospore stain
• Take a clean grease free slide and make smear using sterile technique.

• Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide.

• Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required. Alternatively, the slide may be steamed over a container of
boiling water.

• Wash the slide in tap water.

• Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.

• Examine the slide under microscope for the presence of endospores. Endospores are bright green and vegetative cells are brownish red to pink.

Result of endospore staining:
Endospores: endospores are bright green.
Vegetative cells: vegetative cells are brownish red to pink.

Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical.

Endospore staining by dorner’s method

• Carbolfuchsin stain
0.3 gm of basic fuchsin
10 ml of ethanol, 95% (vol/vol)
5 ml of phenol, heat-melted crystals
95 ml of distilled water
Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water.
Mix and let stand for several days. Filter before use.

Decolorizing solvent (acid-alcohol)

97 ml of ethanol, 95% (vol/vol)
3 ml of hydrochloric acid (concentrated)
Counterstain (Nigrosin solution)
10 gm of nigrosin
100 ml of distilled water
Procedure
1.Take a clean grease free slide and make smear using sterile technique. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the
slide.
2.Saturate the blotting paper with carbolfuchsin and steam for 5 to 10 minutes, keeping the paper moist and adding more dye as required. Alternatively, the slides may be steamed over a
container of boiling water.
3.Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute; rinse with tap water and blot dry.
4.Further take a drop of nigrosine on one end of a slide and make a thin film of a stain all over the smear with the help of other slide.
5.Allow the film of Nigrosin to air dry.
6.After air drying observe the slide under oil immersion.

Vegetative cells are colorless, endospores are red, and the background is black.
Examples of Endospore Staining
Positive
Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum spp, Sporolactobacillus spp, Sporosarcina spp, etc.
Negative
E. coli, Salmonella spp, etc.
Zeihl-nelson’s method
What is ziehl-neelsen staining?
• The ziehl-neelsen staining technique is a differential staining technique that was initially developed by ziehl and modified later by neelsen, hence the name ziehl-neelsen stain.

• Neelsen used carbol-fuschin from ziehl’s experiment, with heat and added a decolorizing agent using acid-alcohol and a counterstain using methylene blue dye, thus developing the ziehl-neelsen technique of
staining.

• The use of acid-alcohol in the technique earned it the name acid-fast stain and the application of heat in the technique gives it the name the hot method of acid-fast staining which is a synonymous name for
the ziehl-neelsen staining technique.

• This technique is used on microorganisms that are not easily stained by basic stains such as negative staining or  gram staining. One of the most complex micro-organisms that require harsh treatment of the
ziehl-neelsen compounds is the mycobacterium spp.

• Mycobacterium, actinomycetes, norcadia, isospora, cryptosporidium, and some fungi contain a thick cell wall made up of lipoidal complexes known as mycolic acid.
•Principle
Mycolic acid is difficult to stain and therefore simple stains like gram staining can not penetrate the thick cell wall of these organisms.
of the Ziehl-Neelsen Staining
• The
• Ziehl-Neelsen
They require harsherstain uses basic
treatments fuchsin
to allow and phenol
stain penetration forcompounds
identificationto stain
and the cell wall
examination of Mycobacterium
and hence species. or the hot method of acid-fast stain.
the use of the ziehl-neelsen
•Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuschin and phenol allows penetration through the bacterial cell wall for
visualization.
•Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable. These are ß-hydroxycarboxylic acids made
up of 90 carbon atoms that define the acid-fastness of the bacteria.
•Use of Carbol-fuschin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. addition of acid alcohol along with
the application of heat forms a strong complex that can not be easily washed off with solvents.
•The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin.
•While non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and take up the counterstain dye of methylene blue and appear blue
•This technique has been used in the identification of Mycobacterium tuberculosis and Mycobacterium leprae.
Reagents used in the ziehl-neelsen stain
1.Carbol-fuschin (primary dye)
2.20% sulphuric acid or acid-alcohol (decolorizer)
3.Methylene blue dye (counterstain) or malachite green
•Preparation of reagents
1. Carbol fuschin
• Distilled water- 100ml
• Basic fuschin- 1g
• Ethyl alcohol (100% ethanol)- 10ml
• Phenol crystals- 5ml
2. Acid alcohol (3% hydrochloric acid in 95% ethyl alcohol)
• Ethyl alcohol- 95 ml
• Distilled water- 2 ml
• Concentrated hydrochloric acid- 3 ml
3. 0.25% methylene blue in 1% acetic acid
• Methylene blue- 0.25g
• Distilled water- 99ml
• Acetic acid- 1ml
Procedure
• On a clean sterile microscopic slide, make the smear of the sample culture and heat fix the smear over blue heat.
• Over the smear, pour and flood the smear with carbol fuschin and heat gently until it produces fumes.
• Allow it to stand for 5 minutes and wash it off with gently flowing tap water.
• Add 20% sulphuric acid and leave it for 1-2 minutes. Repeat this step until the smear appears pink in color.
• Wash off the acid with water.
• Flood the smear with methylene blue dye and leave it for 2-3 minutes and wash with water.
• Air dry and examine the stain under the oil immersion lens.
Results and interpretation
• Acid-fast bacteria retain the primary dye, carbol-fuschin, and stain pink.
• Non-acid fat bacteria take up the methylene blue dye and appear blue.
Applications of ziehl-neelsen staining
• Used for examination and identification of mycobacterium species.
• Used to differentiate between acid-fast and non-acid fast bacilli
• Used for the identification of some fungal species such as cryptosporidium.
• Limitations of ziehl-neelsen staining
• It can only be used to identify acid-fast bacilli.
• The physical morphology of the organism is distorted.
Schaeffer-fulton method
•Malachite green isused to stain the endospores (primary stain)
•The malachite green is forced to permeate the spore wall by heating(mordant).
•Washing with water remove stain from vegetative cells, but no tfromspore wall.
•The endospores thus retain the primary dye while the vegetative cells lose the primary stain and take there dcolor of secondary stain(safranin).
•In basic laboratories, the simplest endospore staining technique is the schaeffer-fulton technique because of its easy and it is rapid to identify the bacteria. It applies the use of malachite green dye (alkaline solution with a ph of
11-11.2 and its a water-soluble dye) along with the use of steamed-heat which softens the endospore covering allowing penetration of the dye into the spore. The malachite green dye binds to the spore mildly and if washed
with water, without fixing, it easily washes away, and that’s why the application of steamed heat is important to allow the dye to penetrate the endospore. Water is used as a decolorizing agent, to was away from the malachite
dye from vegetative forms. Lastly, the use of a counterstain, safranin reagent, also knows as the secondary stain, is to stain the vegetative forms of the underdeveloped furmigates vegetative forms after the malachite dye has
been washed away by the decolorizing agent (water).
•Malachite green dye and safranin work well in bacteria because of the alkaline nature of the malachite green reagents which are charged positively while the cytoplasm of the bacterial cell is basophilic hence there is an
attraction between the malachite green dye with the bacterial cell, making it easier to absorb the dye. Visualization of the cells under the microscope will show the appearance of pink-red stain for The vegetative cell forms,
which take up the counterstain while the endospores will appear as green dotted particles (ellipses), having taken up the malachite green dye.
•Reagents
•Malachite green dye
•Water (decoloriser)
•Safranin
Procedure
•Equipment: glass slide, inoculation loop, bunsen burner
•Preparation of microscope slide (adapted for all other endospore staining techniques)
1.Clean the glass slide (with visible circles), with alcohol to remove any stains.
2.Using a sterile inoculation loop, put two small drops of water in each circle.
3.Aseptically, open the tube with a bacteria culture and flame it at the top and collect a loopful of the bacterial culture from the tube,  flame the tube again and close.
4.Smear the bacterial culture in the drop of water on the slide.
5.Air dry till its completely dry.
6.Heat-fix the slide with smear facing up, by running it over the blue flame 3-4 times nb: do not flame the side with the bacteria
7.Leave to cool and then start to stain.
Staining procedure:
8.Cover the smears with a piece of absorbent paper.
9.Place the slide over a staining rack, that has a beaker/water bath of steaming water.
10.
Flood the absorbent paper with malachite green and let it steam for 3-5 minutes. Remove the stained absorbent paper carefully and discard and allow it to cool for 1-2 minutes.
11.
Gently rinse the slide with tap water by tilting the slide to allow the water to flow over the smeared stain. This is to remove the extra dye present on the slide on both sides and to also remove extra
dye staining any vegetative forms in the heat-fixed smear.
12.
Add the counterstain, safranin for 1 minute.
13.
Rinse the slide with water, on both sides to remove the safranin reagent.
14.
Ensure the bottom of the slide is dry before placing it on the stage of the microscope to view with the oil immersion lens, at 1000x for maximum magnification.

Result
•The vegetative forms will take up the pink/red stain from safranin while the endospores will stain green, from the malachite green dye.
Interpretation of results
•The vegetative forms stain pink/red because they take up the counterstain (safranin) while the endospores take up the green from the malachite green.
• This is because, during smearing and heat fixing, the malachite green penetrates into the endospore with the help of the heat from the steam, and during the water-rinse, the dye is not easily washed away.
• And for the vegetative forms, the dye is easily washed away because of their fragile outer covering, hence they take up the last stain which is the counterstain, hence they appear pink-red.
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