Types of staining in bacteria

1. Lecture 7
Staining Techniques – Principle and Types of
Stains – Staining Techniques – Simple, Negative,
Differential and Structural Staining Methods

2. Why do we stain ?
Staining has for its first and primary purpose is rendering
outlines and structures more distinct by giving them a color
contrast with their surroundings (color image).
A second and more important use is for the differentiation of
particular structures or substances which by their selective
staining facilitate the histological analysis

3. But interchangable in microbiology
Dye Stain
Used on inanimate objects Used on animate objects
Has penetrate property Has surface property
Permanent and can be removed
only after cell wall destruction
Temporary
Coloring agent used for general
purposes
Stain is used for any
biological specimen staining
Dye is crude Stain is purified form
Staining in Microbiology
The process of adding a dye to a bacterial culture

4. 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 organisms
Staining
Staining is an auxiliary technique used in microscopy to enhance contrast in
the microscopic image. Stains and dyes are frequently used in biology and
medicine to highlight structures in biological tissues for viewing, often with
the aid of different microscopes.
Terms used in Staining

5. What can be used as stain
Substance to be used as a stain must be colored or it should react in the
system to give a colored product, because of which some portion of the
system becomes colored and the rest remains colorless.
Staining renders the organism more visible, it displays the structure and
finer details of bacteria and it helps to differentiate between organisms
Staining techniques
Direct staining - The organism is stained and background is left unstained
Negative staining - The background is stained and the organism is left
unaltered
Fixation
Fixation by itself consists of several steps–aims to preserve the shape of the
cells or tissue involved as much as possible. Sometimes heat fixation is used
to kill, adhere, and makes them permeable so it will accept stains

6. Classification of Stains
(a) According to their chemical composition as
(1) Organic; -- hematoxylin stains, carmine stains, anilin stains
(coal-tar dyes; benzene derivatives),
(2) Inorganic
(b) From Charge based
(1) Basic (+)
(2) Acid (-)
(3) Neutral
(c) Histological stains are:
(1) Nuclear (chromatin stains)
(2) Plasma or general stains
(3) Special stains – Flagella stain, Endospore stain, DNA stain etc.

7. BIOLOGICAL STAINING
DYES ARE CLASSIFIED INTO TWO GROUPES:
• 1. Natural dyes:
a. Haematoxylin -----from plant
b. Carmine -----------from female cochineal bug
• c. Orcein --------------a vegetables dye extract
• 2. Synthetic: these are derived from hydrocarbon benzene

8. Chemical Makeup of Stain
• Benzene = organic compound
• Chromophore = color
• Auxochrome = ionization properties
• Benzene + Chromophore = Chromogen
– Chromogen is a colored compound only
• Auxochrome with Chromogen allows the dye to form salt compounds
that adhere to cells.
• Stains consist of a positive and negative ion.
• In a basic dye, the chromophore is a cation (+).
• In an acidic dye, the chromophore is an anion (-).
• Bacteria are slightly negative at neutral pH

10. Examples
Methylene Blue
Acid Red
Giemsa

11. Principle of staining
Stains → combine chemically with the bacterial protoplasm.
Commonly used stains are salts
 Basic dyes: colored cation + colorless anion
e.g. methylene blue
Nuclei take the basic dye
 Acidic dyes: colored anion + colorless cation e.g. eosin
Cytoplasm takes the acid dye
Neutral dye: such as Leishmans stains are obtained by combining
aqueous solutions of basic and acid dyes.
The resultant precipitates are usually insoluble in water but they
are soluble in alcohol.

12. Stains
• All dyes are salts
– Ionize
• Cationic
• Anionic
• Techniques
– Single dyes
– Multiple dyes

13. Bacterial cells are slightly negatively
charged ( rich in nucleic acids bearing
negative charges as phosphate groups)
→ combine with positively charged
basic dyes
Acidic dyes do not stain the bacterial
cell → can stain the background
material with a contrasting color.

14. Basic Dyes
• Ionizes (Cl-, SO4-)
• Creates (+) Cationic
chromogen
• Attracted to (-) acidic cell
components [DNA, proteins]
• Examples
– Methylene Blue
– Crystal Violet
– Carbol Fuchsin (CF)
– Safranin
– Malachite Green
– Basic fuschin
CF
This dye have positive charge & bind to negatively charged molecules
(nucleic acid, -COOH -OH). Since, surface of bacterial cells are
negatively charged (due to Teichoic acid), basic dyes are most
commonly used in bacteriology.

15. Acidic Dyes
• Works best in acidic pH
• Ionizes (Na+, K+, Ca++)
• Creates Anionic (-) chromogen
• Attracted to (+) cell components
[Amino Acids]
• Examples
– Picric Acid
– Nigrosin
– India Ink
– Eosin
– Acid fuschin Nigrosin
It is dye which has negative charge so they bind to positively
charged cell structures like some proteins. Acidic dyes are not very
often used in Microbiology lab except to provide background
staining like Capsule staining.

16. Stains used for demonstrating the general relationship of
tissue to each other:
Simple stain- staining solution contains one dye.
Compound stain- staining solution is composed of more than one
or more dye
Direct stains- the stains work without adding a mordant.
Indirect staining- stained needs a mordant to work.
Specific stain- when the stain acts only on a certain
constituents of the cell or tissue and has a little or no effect upon
other elements.

17. Types of staining techniques
Simple staining
(use of a single stain)
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
For visualization of
morphological
shape & arrangement.
Identification Visualization
of structure
Gram
stain
Acid fast
stain Spore
stain
Capsule
stain

18. Staining Methods
• Negative Stain
• Simple Stain
• Differential Stains
– Group
• Gram Stain
• Acid Fast Stain
– Special Structures
• Capsule Stain
• Endospore Stain
• Flagellar Stain

19. Negative Stain
• Acid Dye
• (-) chromogen
• Repelled by (-) cell
wall
• Cells
– Colorless
– Seen against dark
background

21. Definition:
It is the use of single basic dye to color the bacterial
organism.
e.g. methylene blue,
crystal violet,
safranin.
All bacteria take the color of the dye.
Objective:-
To show the morphological shapes and arrangement of
bacterial cells.
Simple Stain

22. Simple Staining
Type of staining: Simple Stain
Name of dye:- Crystal violet.
Shape of cells:- cocci
Arrangement of cells: clusters
Color:- Purple
Name of m.o:- Staphylococci

23. Simple Stain (shapes and arrangements)
• One reagent used
• Soak smear 30-60 seconds
• Rinse with H20
• Background stained
• Bacteria stained
• Basic dye
– Shows morphology
• Size
• Shape
• Arrangement
• Examples
– Methylene Blue
– Carbol Fuschin
– Crystal Violet

24. • Two or more reagents
• Distinguish
– Bacterial groups
– Specific Structures
• Example
– Gram stain
– Acid Fast Stain
– Capsule stain
– Endospore stain
Differential Staining (Gram reactions)

25. Gram Stain:
It is the most important
differential stain used in
bacteriology because
it classified bacteria
into two major groups:
a)Gram positive:
Appears violet after
Gram’s stain
b) Gram negative:
Appears red after Gram’s
stain

26. Crystal violet
↓
Iodine
↓
Alcohol
↓
Safranin

27. A basic dye, ie., the primary stain is one, which has its chromogen in its
positive ionic part (cation). Eg.Crystal violet.
Mordant is a substance that increases the affinity between the cell and
the dye thus helps in fixing the dye on the cell in some way. Eg., Acids,
bases, metallic salts and iodine.
Under the action of a mordant, a cell is more intensely stained; it is also
much more difficult to wash out the stain after the application of a mordant.
A decolorizing agent, is one that removes the dye from a stained cell.
Some stained cells decolorize more easily than others, and it is this variation
in the rate of decolorization that differentiates diverse types of bacteria when
the gram stain and other differential stains are used. Eg. Alcohol
The counter stain is a basic dye of a different color from the primary stain
and it is used to give the decolorized cells a color different from that of the
initial stain. Those organisms that are not easily decolorized retain the color
of the initial stain, and those that are easily decolorized take the color of the
counter stain. Eg. Safranin, Basic Fuchsin

28. Time Frame
1) 1 minute
2) 1 minute
3) 15 seconds
4) 1 minute
Rinse with water between each step

30. Gram-positive bacteria
Have a thick peptidoglycan layer surrounds the cell.
The stain gets trapped into this layer and the bacteria
turned purple.
Retain the color of the primary stain (crystal violet)
after decolorization with alcohol
Gram-negative bacteria
have a thin peptidoglycan layer that does not retain
crystal violet stain.
Instead, it has a thick lipid layer which dissolved
easily upon decolorization with Acetone-Alcohol.
Therefore, cells counterstained with safranin turn red.

31. Principle
Gram staining is based on the differences in the cell wall composition of
gram positive and negative organisms.
Gram -ve bacterial cell wall is thin, complex, multilayered with relative high
lipid content.
Lipid is readily dissolvable by alcohol, forming large pores in the cell wall
resulting in the easy decolorization of crystal violet - Iodine (CVI) complex.
Later they take up the counter stain and appear red.
In contrast, the Gram +ve cell walls are thick with less of lipids, so will not
loose CVI and the cells remain blue.
It requires four solutions; a basic dye, a mordant, a decolorizing agent and
a counter stain.

32. Gram Stain General Theory
Dr. Hans Christian Gram, a
Danish physician in 1884,
developed this technique.

33. Negative staining
(Indirect staining with acidic
dye)
The negative staining technique does not
stain the bacteria due ionic repulsion.
but stain the background.
The bacteria will appear colorless against
a dark background.
No heat fixation or strong chemicals are
used→ the bacteria are less distorted
than in other staining procedure.
Example: Nigrosine

34. Negative staining
Candida

35. Endospore Stain
Difficult to stain but once stained they resist decolorizing
Intense heating causes the Endospores to be penetrated by the
malachite green
Safranin counter stain stains all material other than the endospores
Spore stains are typically performed on older cultures
Bacterial endospores are metabolically inactive, highly resistant structures
produced by some bacteria as a defensive strategy against unfavorable
environmental conditions.
Primary stain - Malachite green, which stains both vegetative cells and
endospores and heat is applied to help the primary stain penetrate the
endospore.
Decolorized with water - removes the malachite green from vegetative cell
but not the endospore
Safranin – counter stain any cells which have been decolorized
Vegetative cells will be pink
Endospores will be dark green
Eg. Gram-positive organisms, Clostridium and Bacillus

36. Ziehl–Neelsen stain, also known as the acid-fast stain, widely used
differential staining procedure.
Described by two German doctors; Franz Ziehl (1859 to 1926), a
bacteriologist and Friedrich Neelsen (1854 to 1894) a pathologist.
Some bacteria resist decolourization by both acid and alcohol and hence
they are referred as acid fast organisms.
Two groups namely acid-fast and non acid-fast.
Extensively used in the diagnosis of tuberculosis and leprosy.
Mycobacterium tuberculosis and M. leprae is the most important of this
group, as it is responsible for the disease called tuberculosis (TB) along
with some others of this genus
Acid-fast stain

37. Principle
Mycobacterial cell walls
contain a waxy substance
composed of mycolic
acids.
These are β-hydroxy
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

38. Capsule staining
To reveal the presence of the bacterial capsule
Capsule may appear as clear halo when a fresh sample is stained
by Leishman stain or Negative staining- using - India ink, Nigrosin
Procedure
Place a loop full of India ink on the slide
A small portion of the culture is emulsified in the drop of ink
Place a clean cover slip over the preparation without bubbles.
Press down gently
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

39. Silver staining
Silver staining is the use of silver to stain histologic sections. This
kind of staining is important especially to show proteins and DNA.
It is used to show both substances inside and outside cells. Silver
staining is also used in temperature gradient gel electrophoresis.
Sudan staining
Sudan staining is the use of Sudan dyes to stain sudanophilic
substances, usually lipids.
Sudan III, Sudan IV, Oil Red O, and Sudan Black B are often used.

40. Properties of Some important staining agents
Acridine orange (AO) is a nucleic acid selective fluorescent cationic dye
useful for cell cycle determination.
It is cell-permeable, and interacts with DNA and RNA by intercalation or
electrostatic attractions.
When bound to DNA, it is very similar spectrally to fluorescein.
Coomassie blue - nonspecifically stains proteins strong blue colour.
It is often used in gel electrophoresis.
Crystal violet, when combined with a suitable mordant, stains cell walls purple.
Important component in Gram staining.
Eosin is most often used as a counterstain to haematoxylin, imparting a pink
or red colour to cytoplasmic material, cell membranes,
It also imparts a strong red colour to red blood cells.
Used as a counterstain in some variants of Gram staining
Eosin Y - slightly yellowish cast. Eosin B - bluish or imperial red
It produces red nuclei, and is used primarily as a counterstain.

41. Acid fuchsine may be used to stain collagen or mitochondria or nuclear
and cytoplasmic stain
Haematoxylin is a nuclear stain. Used with a mordant, haematoxylin stains
nuclei blue-violet or brown. It is most often used with eosin in H&E
(haematoxylin and eosin) staining—one of the most common procedures
in histology.
Iodine is one component in the staining technique known as Gram
staining, used in microbiology. Lugol's solution or Lugol's iodine (IKI) is a
brown solution that turns black in the presence of starches and can be
used as a cell stain, making the cell nuclei more visible. Iodine is also
used as a mordant in Gram's staining, it enhances dye to enter through
the pore present in the cell wall/membrane.
•Malachite green can be used as a blue-green counterstain to safranin in
the Gimenez staining technique for bacteria. It also can be used to directly
stain spores.
•Methyl green is used commonly with bright-field microscopes to dye the
chromatin of cells so that they are more easily viewed.

42. •Methylene blue is used to stain animal cells, such as human cheek
cells, to make their nuclei more observable.
•Nile blue (or Nile blue A) stains nuclei blue. It may be used with
living cells.
•Osmium tetraoxide is used in optical microscopy to stain lipids. It
dissolves in fats, and is reduced by organic materials to elemental
osmium, an easily visible black substance.
•Rhodamine is a protein specific fluorescent stain commonly used in
fluorescence microscopy.

43. Stains used in Electron microscopy
As in light microscopy, stains can be used to enhance contrast in transmission
electron microscopy. Electron-dense compounds of heavy metals are typically
used.
Phosphotungstic acid is a common negative stain for viruses, polysaccharides,
and other biological tissue materials.
Osmium tetroxide is used in optical microscopy to stain lipids. It dissolves in
fats, and is reduced by organic materials to elemental osmium, an easily visible
black substance. Because it is a heavy metal that absorbs electrons, it is
perhaps the most common stain used for morphology in biological electron
microscopy.
It is also used for the staining of various polymers for the study of their
morphology by TEM.
OsO4 is very volatile and extremely toxic..
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. 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.