STAINING METHODS

INTRODUCTION

Staining is technique used in microscopy to enhance contrast in the microscopic image.

Stains and dyes are frequently used 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.
Bacteria have nearly the same refractive index as water, therefore, when they are
observed under a microscope they are opaque or nearly invisible to the naked eye.
Different types of staining methods are used to make the cells and their internal
structures more visible under the light microscope. Microscopes are of little use unless
the specimens for viewing are prepared properly. Microorganisms must be fixed &
stained to increase visibility, accentuate specific morphological features, and preserve
them for future use.
Stain

A stain is a substance that adheres to a cell, giving the cell color. The presence of color
gives the cells significant contrast so they are much more visible. Different stains have
different affinities for different organisms, or different parts of organisms. They are
used to differentiate different types of organisms or to view specific parts of organism.

It can be classified into the following types, depending upon its chemical nature and
the type of staining methods.
Based on chemical nature: There are three kinds of stain, acidic, basic and neutral,
depending upon the chemical nature of the stain.
Based on the staining method: There are four kinds of stain, viz. direct, indirect,
differential and selective stains.

Purpose
 Enables us to see the organism better: Microorganisms are very minute
creatures as well as appear transparent, so staining makes the specimen 9easy
to identify.
 Helps to differentiate organisms: Staining helps in distinguishing between
the two different groups of organisms, depending upon the colour retaining
ability of the cells (some microbes retain the colour of stain, while some don’t).
 To identify a particular structure: For further study of microorganisms, it is
also important to study the various internal and external structure of organism
like flagella, capsule, nucleus, spores etc.

Mechanism
Stains are organic compound composed of a benzene ring, a chromophore group and
an auxochrome group. benzene is a colourless solvent, and the chromophore group is
a molecule that imparts colour to the benzene. As a result, the compound formed is
called a ‘chromogen’. chromogen is not a stain in itself; it is just a coloured
compound.
The second part of the stain, the auxochrome, is a chemical group that ionizes the
chromogen i.e. it imparts a positive or negative charge to the chromogen group. As a
result, the auxochrome enables the ionized chromogen to bind to cells or tissue fibres
of opposite charge and thereby colour it.

PREPARATION OF BACTERIAL SMEAR

Microbial smear : It is a very small amount of microbial growth ( broth or solid ) spread
on a clean slide and drying by air .
FIXATION: It is the process by which the internal and external structures of cells and
microorganisms are preserved and fixed in position. It inactivates enzymes that might
disrupt cell morphology and toughens cell structures so that they do not change during
staining and observation. A microorganism usually is killed and attached firmly to the
microscope slide during fixation.

There are two fundamentally different types of fixation.

1. Heat fixation Bacterial smear will be gently showed in flame. This adequately
preserves overall morphology but not structures within cells.
2. Chemical fixation Chemical fixatives are used that will penetrate cells and react
with cellular components, usually proteins and lipids, to render them inactive, insoluble,
and immobile. Common fixative mixtures contain such components as ethanol, acetic
acid, mercuric chloride, formaldehyde, and glutaraldehyde.
Steps of microbial smear preparation:

1.Handle a clean slide by its edge, label the target place at the bottom side o the slide
by drawing a circle with a diameter of about 2 cm using a marker.

2.Sterile the loop until reaching the red heat.

3.If the bacterial culture was broth, shake the culture and transfer loopful of broth to
the center of the slide and spread over the target circle. While if the bacteria were
grown on solid medium, place loopful of water on the slide then transfer inoculums to
the water and homogenize the smear.

4.Sterile the loop.

5.Leave the smear to dry at room temperature (by air)

. 6.After drying, Pass the slide over the flame to fix the smear (avoid prolonged heating
of the slide).

Simple staining

When a single staining-reagent is used and all cells and their structures stain in the
same manner, the procedure is called simple staining procedure. This procedure is of
two types – positive and negative. In positive staining, the stain (e.g., methylene blue)
is basic (cationic) having positive charge and attaches to the surface of object that is
negatively charged. In negative staining, the stain (e.g., India ink, nigrosin) is acidic
(anionic) having negative charge and is repelled by the object that is negatively
charged, and thus fills the spaces between the objects resulting in indirect staining of
the object.
It determines the cell shape, size and arrangement of the microorganisms. It is a very quick or
simple method to perform and it makes the use of a single stain only. These are of two types,
namely direct and indirect staining.

Differential Staining

Differential Stains use two or more stains and allow the cells to be categorized into
various groups or types. Both the techniques allow the observation of cell morphology,
or shape, but differential staining usually provides more information about the
characteristics of the cell wall (Thickness). Gram staining (or Gram’s method) is an
empirical method of differentiating bacterial species into two large groups (Gram-
positive and Gram-negative) based on the chemical and physical properties of their
cell wall.
Gram staining

Gram Staining is the common, important, and most used differential staining
techniques in microbiology, which was introduced by Danish Bacteriologist Hans
Christian Gram in 1884. This test differentiates the bacteria into Gram Positive and
Gram Negative Bacteria, which helps in the classification and differentiations of
microorganisms.
Principle of Gram Staining

When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant,
some of the bacteria are able to retain the primary stain and some are decolorized by
alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar
complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes
this thick cell wall to dehydrate and shrink which closes the pores in the cell wall and
prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal
Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive
bacteria and appears blue or purple in colour. In case of gram negative bacteria, cell
wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan
and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off.
When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls,
which allows the crystal violet-iodine complex to leach out of the cells. Then when
again stained with saffranin, they take the stain and appear red in color.
Materials Required:
Clean glass slides, inoculating loop, Bunsen burner, Bibulous paper ,Microscope ,Lens
paper
and lens cleaner, Immersion oil, Distilled water , 18 to 24 hour cultures of organisms
Reagents:
1. Primary Stain - Crystal Violet
2. Mordant - Grams Iodine
3. Decolourizer - Ethyl Alcohol
4. Secondary Stain - Saffranin

Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the
alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be
careful
not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with saffranin to counter counter-stain and let stand for 45 seconds.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
10. Blot dry the slide with bibulous paper.
11. View the smear using a light-microscope under oil-immersion.
Interpretation
Gram Positive: Blue/Purple Color
Gram Negative: Red Color
Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium, Corynebacterium,
Enterococcus, Gardnerella, Lactobacillus, Listeria, Mycoplasma, Nocardia,
Staphylococcus, Streptococcus, Streptomyces ,etc.
Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other
Enterobacteriaceae, Pseudomonas,Moraxella, Helicobacter, Stenotrophomonas,
Bdellovibrio, acetic acid bacteria, Legionella etc

This Photo by Unknown Author is licensed under

ACID-FAST STAIN

The Ziehl–Neelsen stain, also known as the acid-fast stain, widely used differential
staining procedure. The Ziehl – Neelsen stain was first described by two German
doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to
1894) a pathologist. In this type some bacteria resist decolourization by both acid and
alcohol and hence they are referred as acid-fast organisms. This staining technique
divides bacteria into two groups namely acid-fast and non acid-fast. This procedure is
extensively used in the diagnosis of tuberculosis and leprosy. Mycobacterium
tuberculosis is the most important of this group, as it is responsible for the disease
called tuberculosis (TB) along with some others of this genus
Principle

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These
are βhydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The
property of acid fastness is related to the carbon chain length of the mycolic acid found
in any particular species.
Ziehl- Neelsen Procedure

1. Make a smear. Air Dry. Heat Fix.

2. Flood smear with Carbol Fuchsin stain
3. Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate
the cell
wall
4. Cover flooded smear with filter paper
5. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed
6. Cool slide
7. Rinse with Distilled water
8. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains 3%
HCl and
95% ethanol, or you can decolorize with 20% H2SO4
9. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop by drop)
until
the red color stops streaming from the smear
10. Rinse with Distilled water
11. Add Loeffler’s Methylene Blue stain (counter stain). This stain adds blue color to
non-acid fast cells. Leave Loeffler’s Blue stain on smear for 1 minute
12. Rinse slide. Blot dry.
13. Use oil immersion objective to view.
Capsule staining
The purpose of the capsule stain is to reveal the presence of the bacterial capsule, the
watersoluble capsule of some bacterial cells is often difficult to see by standard simple
staining procedures or after the Gram stain. The capsule staining methods were
developed to visualize capsules and yield consistent and reliable results Capsule may
appear as clear halo when a fresh sample is stained by Grams or Leishman stain,
Negative staining- using - India ink, Nigrosin.

India ink

Commercially available India ink is used undiluted

Procedure
1. Place a loop full of India ink on the slide
2. A small portion of the culture is emulsified in the drop of ink
3. Place a clean cover slip over the preparation without bubbles. Press down gently
4. Examine under dry objective
Uses
India ink is used to demonstrate capsule which is seen as unstained halo around the
organisms
distributed in a black background eg. Cryptococcus.
Endospore Staining (Bartholomew and Mittwer’s Method ):
It differentiates the endospore from the vegetative cell and makes the
use of both acidic and basic stains.
Endospore: A term itself defines its meaning, in which endo stands for
inside and spore stands for a reproductive structure. Therefore,
endospores are the reproductive structures inherent to the cell. It acts like
a dormant spore, which can resist harsh physical and chemical conditions.
Endospores are commonly found in gram-positive bacteria.
Requirement
1. Cell suspension of endospore producing bacteria.
2. Malachite green stain.
3. Saffranin stain.
Procedure
1. Take a clean grease free slide and prepare a thick smear on a slide.
2. The smear is heat fixed by passing the slide from the flame for about 25 times.
3. The slide is allowed to cool.
4. Further the slide is treated with Malachite green stain and allowed it to react for
about
10 minutes.
5. After 10 minutes slide is given a water wash treatment.
6. Further the slide is treated with counter stain that is saffranin for about 30
seconds.
7. After 30 seconds the slide is water washed, air dried and observed under oil
immersion.
Mechanism
1. In this staining technique a longer heat treatment and prolonged staining
technique.
2. Endospore gets stained due to longer heat treatment, prolonged staining and
heavy
concentration of stain.
3. Here we pass he slide from flame for about 25 times in addition we use
concentrated
stain that is 7.6 % Malachite green for about 10 minutes.
4. This technique stains the cell as well as the endospore.
5. When we give water wash treatment the water acts as a weak decolorizing agent
and
decolorizes cytoplasm and not endospore
6. So here further we apply a counter stain that is Saffranin. 7. Due to application of
saffranin the cytoplasm gets stain in pink colour.
Observation The endospore appears green in colour as well as cytoplasm appears
pink in colour.
Flagella Staining (Liefson’s Method):
Bacteria have two types of locomotory organs and that are Flagella and pili.
Flagella are a thin, hair like structure made up protein called as flagellin. It sizes ranges
from 20 μ to 200 μ in length. Flagella is one of the most important locomotory organ.
It is mainly made up of three parts- 1) Basal body 2) Filament 3) Hook.
Flagella are generally present in rod shape bacteria and very few cocci shape
bacteria possess flagella. As flagella are very thin and hair like they cannot be easily
observed under microscope. So a special technique is design to increase thickness
of flagella as well as stain it. Due to this technique we can observe structure of flagella
easily under microscope.
Requirement : Flagellated cell culture slant, Leifson’s stain, 1 % Methylene blue,
Distilled water.
Procedure:
1.Take two hours old, flagellated cell culture slant and add two to three drops of sterile
Distilled water in the slant with the help of sterile pipette.
2. The distill water is added slowly without disturbing the growth of cells.
3. After addition of distill water incubated the slant for 20 minutes.
4. Then take a drop of suspension from the slant and place the drop on a clean slide
which is kept in slanting position.
5. The drop should flow slowly from one end of slide to other end to avoid folding of
flagella on cell.
6. Allow smear to air dry.
7. After air drying the slide is flooded with Leifson’s stain till a thin film of shinny surface
appear.
8. After this give a gentle stream of water wash treatment to a slide.
9. Treat the slide with 1 % methylene blue treatment for 1 minute.
10. Give the slide water wash treatment, air dry and observe under oil immersion lens.
Mechanism
In this procedure thickness of flagella is increase so it can be visible. The Leifson’s
stain is made up of tannic acid, basic fuschin stain prepared in alcohol base. When we
treat Leifson’s stain with cell the tannic acid get attach to the flagella and alcohol get
evaporated. After evaporation of alcohol the thickness of flagella is increased due to
deposition of tannic acid. Whereas Basic fuschin stain the Flagella. After Leifson’s
stain treatment cells are treated with Methylene blue stain. This Methylene blue stains
the cell.
Result
Flagella appear red in colour and bacterial cell appears blue in colour.

Peritrichous flagella
STAINING OF METACHROMATIC GRANULE- ALBERT LAYBOURN METHOD
The aim of Albert stain is to identify bacteria that contain special
structures known as metachromatic granules. Albert stain distinctly identifies
metachromatic granules that are found in Corynebacterium diphtheriae.
Corynebacteria are gram-positive, non-spore forming, non-motile
bacilli that contain metachromatic (Volutin) granules which are
intracellular inclusion bodies, found in the cytoplasmic membrane of some
bacterial cells for storage of complexed inorganic polyphosphate (poly-P)
and enzymes. When these granules are subjected to stain with methylene
blue dye, they appear reddish-purple color and not the blue dye.
Basically, this bacteria is initially cultured in selective media either a
Loeffler agar or Mueller-Miller tellurite agar, or Tinsdale tellurite agar, and its
colonize isolated to prepare liquid culture that is then used for staining.
Albert stain only acts as a confirmatory stain for the bacteria. Being a
differential stain, it can only stain volutin granules, hence bacteria without
these granules can not be stained nor identified with this technique.

Principle of Albert Staining

Albert staining technique aims at detecting the presence of metachromatic
granulated bodies of Corynebacterium diphtheriae. Albert stain is made up
of two staining solutions; designated as Albert Solution 1 and Albert
Solution 2, their compositions being;
Albert Solution 1:
 toluidine blue, malachite green, glacial acetic acid, and alcohol
Albert solution 2:
 Iodine and Potassium iodide in water
To use Albert’s staining solutions, each of the two solutions must be
prepared effectively with the right percentages of components in order to
demonstrate the granules with the right color after staining.
Albert staining solution 1 acts as the staining solution while Albert solution
2 acts as the mordant, i.e an ion element that binds and holds a chemical
dye, to make it stuck on the micro-organism.
Procedure of Albert Staining
Reagents:
Albert staining Solution 1
Albert staining solution 2
Distilled water
A. Staining:
1. Aseptically, take a loopful culture of Corneybacterium diphtheriae
2. Make a smear at the center of a clean sterile glass slide
3. Heat fix the smear, gently
4. On a staining rack, place the smeared glass slide.
5. Add Albert staining Solution 1 into the smear and leave it for 3-5
minutes
6. Wash the smeared slide with gently flowing tap water
B. Mordanting
1. Add Albert staining solution 2 and leave it for 1 minute
2. Wash the slide with gently flowing tap water.
3. Blot to dry the smeared glass slide
4. Add cedarwood oil on the smear
5. Then observe under a microscope by oil immersion at 1000x
Result
The metachromatic granules stain bluish black while the rest of the
microbial cell stains green.

lactophenol Cotton Blue (LPCB) Staining

 Lactophenol Cotton Blue (LPCB) Staining is a simple histological
staining method used for the microscopic examination and identification of
fungi.
Principle of Lactophenol Cotton Blue (LPCB) Staining
Lactophenol Cotton Blue (LPCB) Staining method works on the principle of
aiding the identification of the fungal cell walls.
 Fungi are eukaryotic organisms with both macroscopic and
microscopic characteristics.
 The fungal spore cell wall is made up of chitin of which the components
of the Lactophenol Cotton Blue solution stains for identification.
 The lactophenol cotton blue solution acts as a mounting solution as
well as a staining agent.
 The solution is clear and blue in color and it is made up of a
combination of three main reagents:
 Phenol: It acts as a disinfectant by killing any living organisms
 Lactic acid: To preserve the fungal structures
 Cotton blue: To stain or give color to the chitin on the fungal
cell wall and other fungal structures
 The stain will give the fungi a blue-colored appearance of the fungal
spores and structures, such as hyphae.

Procedure of Lactophenol Cotton Blue (LPCB) Staining

1. On a clean microscopic glass slide, add a drop of 70% ethanol
2. Add the fungal specimen to the drop of alcohol using a sterile
mounter such as an inoculation loop (from solid medium),
depending on the sample of use.
3. Tease the fungal sample of the alcohol using a needle mounter, to
ensure the sample mixes well with the alcohol.
4. Using a dropper or pipette, add one or two drops of Lactophenol
Cotton Blue Solution (prepared above) before the ethanol dries off.
5. Carefully cover the stain with a clean sterile coverslip without
making air bubbles to the stain.
6. Examine the stain microscopically at 40X, to observe for fungal
spores and other fungal structures.

Results and Interpretation

Fungal spores, hyphae, and fruiting structures stain blue while the
background stains pale blue.
For example,
 Aspergillus niger stains the hyphae and fruiting structures a delicate
blue with a pale blue background.
 Trichophyton mentagrophytes also stains the hyphae and fruiting
structures a delicate blue with a pale blue background.

Limitations
 It can only be used as a presumptive identification method of fungi
which should be followed up with other diagnostic tools such as
biochemical and cultural examination.
 The components of the solution should be used before expiry,
including the use of the solution before it expires.
 The solution may disrupt the original morphology of the fungi.
 The stain can only be used to identify mature fungi and its structures
and not the young vegetative forms of fungi.
 The stain can not be stored for a long period of time.