Name: Hammed Aminat

Matric number: 21D/7HMB/186

Department: Microbiology
Course code: MCB 204

Question
Write on stain and staining techniques for differentiation of micro
organisms

Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic

image. Stains or dyes are used in biology and medicine to highlight structures in biological
tissues for viewing with microscope.

Purposes of Staining
— 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 or 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.
— Stains may be used to define and examine bulk tissues (e.g. muscle fibers or connective
tissues), cell populations (different blood cells) or organelles within individual cells.
— Biological staining is also used to mark cells in flow cytometry, flag proteins or nucleic
acids on gel electrophoresis
— Staining is not limited to biological materials, it can also be used to study the morphology
(form) of other materials e.g. the lamellar structure of semi crystalline polymers or domain
structures of block co-polymers

— Staining can be:

— In vivo: also known as vital staining which involves dyeing living tissues. Certain cells or
structures take up contrasting colors, the morphology or position within a cell or tissue can be
readily seen and studied. In in vivo staining, the cytological details that are not apparent are
revealed. It also reveals where certain chemicals or specific chemical reactions are taking place
within cells or tissues.
— In vitro staining: involves coloring of cells or structures that have been removed from their
biological context. Here certain stains are often combined to reveal more details and features
than a single stain alone. This staining can be used by scientists and physicians for consistent
and repeatable diagnostic tools.

Types of Staining Techniques Used in

Microbiology
Structural details of organisms cannot be seen under a light microscope due to a lack of
contrast. Hence, we use dyes to stain cells. Dyes bind with cellular constituents
producing color contrast and increasing their visibility. Positively charged (cationic) dyes
such as methylene blue, crystal violet, safranin, etc bind with negatively charged cellular
constituents such as nucleic acids and acidic polysaccharides and the cell surface of
bacteria.

Based on the types and number of dyes used, staining can be categorized into different
types.

Types of Microbiological Stains

Simple stain
Basic dyes, such as methylene blue or basic fuchsin are used as simple stains. They
produce color contrast but impart the same color to all the bacteria in the smear.

Negative staining
A drop of bacterial suspension is mixed with dyes, such as India ink or nigrosin. The
background gets stained black whereas the unstained bacterial or yeast capsule stands
out in contrast. This is very useful in the demonstration of capsules that do not take up
simple stains.

India ink preparation

Negative stains are used when a specimen or a part of it, such as the capsule resists
taking up the stain. India Ink preparation is recommended for use in the identification
of Cryptococcus neoformans.

Impregnation methods
Bacterial cells and structures that are too thin to be seen under the light microscope are
thickened by impregnation of silver salts on their surface to make them visible, e.g., for
demonstration of bacterial flagella and spirochetes.

Flagella stain
Demonstrate the presence and arrangement of flagella. Flagellar stains are
painstakingly prepared to coat the surface of the flagella with dye or a metal such as
silver. The number and arrangements of flagella are critical in identifying species of
motile bacteria.

Differential staining

Staphylococcus in Gram Stain

Here, two stains are used which impart different colors to different bacteria or bacterial
structures, which help in differentiating bacteria. The most commonly used differential
stains are:

Gram staining
Gram stain is a very important differential staining technique used in the initial
characterization and classification of bacteria in microbiology. Gram staining helps to
identify bacterial pathogens in specimens and cultures by their Gram reaction (Gram-
positive and Gram-negative) and morphology (cocci/rod).

Acid-fast stain (Ziehl-Neelsen technique)

It distinguishes acid-fast bacteria such as Mycobacterium spp from non-acid fast bacteria; which
do not stain well by the Gram staining. It is used to stain Mycobacterium species

Endospore stain
It demonstrates spore structure in bacteria as well as free spores. Relatively few
species of bacteria produce endospores, so a positive result from endospore staining
methods is an important clue in bacterial identification. Bacillus spp and Clostridium spp
are the main endospores producing bacterial genera.
Capsule stain
It helps to demonstrate the presence of capsulesin bacteria or yeasts. Streptococcus
pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Klebsiella
pneumoniae are common capsulated bacteria.

Giemsa stain
Giemsa stain is a Romanowsky stain. It is widely used in the microbiology laboratory for
the staining of:

1. Malaria and other blood parasites

2. Chlamydia trachomatis inclusion bodies
3. Borrelia species
4. Yersinia pestis
5. Histoplasma species
6. Pneumocystis jiroveci cysts (formerly Pneumocystis carinii)
7. Acridine orange Stain
8. This staining method is used to confirm the presence of bacteria in blood cultures
when Gram stain results are difficult to interpret or when the presence of bacteria
is highly suspected but none are detected using light microscopy. Acridine
orange binds to nucleic acid and stains them. It is also used for the detection of
mycoplasmas (cell wall deficient bacteria).
9. Cytoplasmic inclusion stains
10. Identifies intracellular deposits of starch, glycogen, polyphosphates,
hydroxybutyrate, and other substances. E.g. Albert staining is used to stain the
volutin or metachromatic granules of C. diphtheriae.
11. Other staining methods
12. Auramine-Rhodamine technique
13. This fluorochrome staining method is used to enhance the detection of
mycobacteria directly in patient specimens and the initial characterization of cells
grown in culture.
14. Calcofluor White Staining
It is commonly used to directly detect fungal elements and to observe the subtle
characteristics of fungi grown in culture. The cell walls of fungi will bind the stain
calcofluor white, which greatly enhances the visibility of fungal elements in tissue or
other specimens.

Lactophenol cotton blue (LPCB) wet mount

LPCB mount is the most widely used method of staining and observing fungi.