Gram staining  Gram-positive cells have a thick layer of
Staining
 Stains, or dyes, contain salts made up of a positive
ion and a negative ion.
 Depending on the type of dye, the positive or the
negative ion may be the chromophore (the colored
ion); the other, uncolored ion is called the
counterion.
 If the chromophore is the positively charged ion, the
stain is classified as a basic dye
 if the negative ion is the chromophore, the stain is
considered an acidic dye.
 Staining is almost always applied to color certain
features of a specimen before examining it under a
light microscope
Simple Staining
 Direct staining with a positively charged dye in order
to see bacterial detail
 Emphasize particular structures in the specimen.
 Make all of the organisms in a sample appear to be
the same color, even if the sample contains more
than one type of organism.
Differential Staining
 Uses more than one chemical stain.
 differentiate between microorganisms or
structures/cellular components of a single organism.
 Two organisms in a differentially stained sample may
appear to be different colors.
Gram Stain
 The Gram staining method, named after the Danish
bacteriologist who originally devised it in 1882
(published 1884), Hans Christian Gram, is one of the
most important staining techniques in microbiology.
 It is almost always the first test performed for the
identification of bacteria.
 Distinguish and classify the bacterial species into the
Gram-positive bacteria and Gram-negative bacteria
peptidoglycan in the cell wall that retains the primary
stain
 Gram-negative cells have a thinner peptidoglycan
layer that the Gram- positive, it allows the crystal
violet to wash out during the decolorization process
instead it will be stained with safranin, a red dye.
Acid-Fast Stain
 Ziehl-Neelsen stain is also known as the acid-fast
stain.
 This method is used for Mycobacterium, which can
only be visualized by acid-fast staining. Special Staining
 Special stains are processes that generally employ a
Principle dye or chemical that has an affinity for the particular
 Organisms such as Mycobacteria are extremely tissue component that is to be demonstrated.
difficult to stain by ordinary methods like Gram Stain  They allow the presence/or absence of certain cell
because of the high lipid content of the cell wall. types, structures and/or microorganisms to be
 The phenolic compound carbol fuchsin is used as the viewed microscopically.
primary stain because it is lipid soluble and
penetrates through lipoidal wall and enters into Negative Stain
cytoplasm, Acid is used to decolorize nonacid-fast  Negative staining requires the use of an acidic stain
cells; acid-fas such as India ink or nigrosin.
 t cells resist this decolorization. The ability of the  The practical application of negative staining is
bacteria to resist decolorization with acid confers a twofold.
acid-fastness to the bacterium.  First, since heat fixation is not required and the cells
 Following decolorization, the smear is counterstained are not subjected to the distorting effects of
with methylene blue which stains the background chemicals and heat, their natural size and shape can
material, providing a contrast colour against which be seen.
the red AFB can be seen.  Second, it is possible to observe bacteria that are
difficult to stain, such as some spirilla. Because heat
Expected Result fixation is not done during the staining process, keep
 Mycobacterium spp. will appear red or have a red- in mind that the organisms are not killed and slides
blue, bended appearance, whereas nonmycobacteria should be handled with care.
will appear blue.
Z.N Reagents:
 Primary stain : 0.3% Carbol Fuchsin ( Consist of
phenol in ethanol or methanol and basic fuchsin ).
 Decolorization solution : 25 % Sulphuric acid
 Counter stain : 0.3% methylene blue or malachite
green.

 Nigrosin is a simple and indirect stain used for
determining bacterial morphology.
 The shapes and sizes of the organisms are seen as
color-free outlines against the dark background.
 An advantage of using this method is that prior
fixation by heat is not needed, so the organisms are
seen in more lifelike shapes.
 Nigrosin is an acidic stain which becomes negatively
charged. Since the surface of most bacterial cells is
negatively charged, the cell surface repels the stain.
 The glass of the slide will stain, but the bacterial cells
will not.
OBSERVATIONS & RESULTS ON NEGATIVE STAINING
 In Negative staining Preparations, the Bacterial cells
observed as the Clear transparent bodies or objects,
may be of variable size and shape if you are using a
mixture of bacteria, against a dark background.
 Vegetative Cells and Spores of Bacilli are clearly
visible in negative stain preparation
Endospore Stain
 AKA: Schaeffer-Fulton stain
 Stains bacterial endospores.
 The main purpose is to differentiate bacterial spores
from other vegetative cells and to differentiate spore
formers from non-spore formers.
Endospore Stain
 The endospore stain is a differential stain used to
visualize bacterial endospores. Endospores are
formed by a few genera of bacteria, such as Bacillus.  During the staining procedure, the alcoholic solution
By forming spores, bacteria can survive in hostile evaporates and leaves a precipitate around the
conditions. Spores are resistant to heat, desiccation, flagella, increasing its apparent size to the human eye.
chemicals, and radiation.
Flagella Stain
 Flagella Staining is recommended to use in detecting
the presence and arrangement of flagella on the
bacterial cell. Bacterial flagella, due to their narrow
diameter, cannot be seen with a light microscope.
 Crystal violet in an alcoholic solution as the primary
stain.
 Tannic acid and aluminum potassium phosphate as
mordants
 Phenol as an antifungal agent.
 Flagella Stains appear opaque, and blue-violet in
color.
 During the staining procedure, the alcoholic solution
evaporates and leaves a precipitate around the
flagella, increasing its apparent size to the human eye.