In microbiology and parasitology, staining is a  Kinyoun Stain- also known as the ‘“cold

technique used to enhance the visibility of method” as it does not utilize heat after…
microorganisms and parasites under a microscope.
It involves applying dyes or stains to the
specimens, allowing scientists to observe their 3. Special Stains- are used to demonstrate
structure, morphology, and other characteristics specific structure ms in a bacterial cell. For
more clearly. instance, metachromatic granules can be
visualize using…
Purpose of Staining:
Visualization: Staining helps in highlighting
specific features of microorganisms and parasites
that may be difficult to see with standard light
microscopy.

Differentiation: It allows for the differentiation

between different types of organisms, such as
bacteria, fungi, and parasites.

Identification: Staining aids in the identification

and classification of microorganisms based on their
staining properties.

TYPES OF STAINING

1. Simple Stains - make use of a single dye

which can either be aqueous (water-based)
or alcohol based. This method of staining is
quick an easy way to visualize shape, suze
and arrangements of bacteria
It uses basic dyes as safranin, mythelyne bkue lr
crystal violet. Yhese stains gives up or accept
hydrogen ion leaving stain positively charged. Most
bacterial cells and cytoplasm are negatively
charged and since the sye is positively charged it
adgeres readily…

2. Differential Stains - are used to Common Staining Techniques:

differentiate one group of bacteria from
another.
1. Gram Staining:
Two Types of Differential Stains Used to classify bacteria into Gram-positive and
 Gram stain - distinguishes gram-positive Gram-negative based on their cell wall properties.
from a gram negative bacteria. Gram
positive bacteria stain a blue or purple, Gram-positive bacteria retain the crystal violet stain
while gram negative bacteria stain red or and appear purple.
pink.
As a general rule all cocci are gram-positive Gram-negative bacteria do not retain the crystal
except Neisseria Veilonella, and branhamella. On violet stain and appear pink/red after
the other hand, all bacteria gram-negative except counterstaining with safranin.
Cornybacterium, Clostridium, Bacillus and
Mycobacterium.

2. Acid-Fast Staining:
 Acid-fast stain - stain used for bacteria Used to identify acid-fast bacteria, such as
with high lipid content in their wall, hence Mycobacterium species (e.g., the causative agent
cannot be stained using Gram stain. of tuberculosis).

Two Methods used in Acid-fast stain Acid-fast bacteria retain the primary stain (carbol
 Ziehl-Neelsen stain - also known as the fuchsin) and appear red, while non-acid-fast
‘“hot-method” because it requires steam- bacteria take up the counterstain (methylene blue)
bathing the prepared smear adter addition and appear blue.
of primary dye. This is because the primary
stain is used….
 or less that allows thickining the media
without producing afirm substance. They
have a soft consistentcy similar to custard,
3. Wright-Giemsa Staining: and are best suited for culture of
Commonly used in parasitology to stain blood microaerophilic bacteria or for the the study
smears and identify parasites such as Plasmodium of bacterial motility.
species (malaria) and Trypanosoma species. c. Solid media - contain a solidifying agent
such as 1.5%-2% agar, giving them a firm
Provides a detailed view of cellular components, surface on which cells can form discrete
allowing for the identification of parasites within colonies. They are used for isolation of
blood cells. bacteria and fungi or for determining the
colony characteristics of the organism under
study. Solid media come in two forms: (a)
liquefiable (or reversible) solid media and
(b) non-liquefiable (or non-reversible) solid
4. Endospore Staining: media.
Used to detect the presence of endospores in
bacteria. d. Differential media - allow the growth of
several types of microorganisms. These are
Endospores retain the primary stain (malachite designed to show visible differences among
green) and appear green, while vegetative cells certain groups of microorganisms. These
take up the counterstain (safranin) and appear red. allow the growth of more than one target
microorganism that demonstrate
morphologic variations in colony
5. Negative Staining: morphology. e. Transport media - used for
Used to visualize capsules around bacteria. clinical specimens that need to be
transported to the laboratory immediately
The background is stained, leaving the capsules after collection. These media prevent the
clear and visible. drying of specimen and inhibit the
overgrowth of commensals & contaminating
organisms. Chail is added to neutralize
CULTURE MEDIA inhibitory factors.

STAINING - procedure only give clues as to the

probable organisms being studied. To identify a
specific organisms, culture using specific, culture e. f. Anaerobic media - media used
media is the most ideal specifically for organisms that cannot
survive in the presence of oxygen and
Media (sing, medium) are used to grow require reduced oxidation-reduction
microorganisms. potential and other nutrients. These are
Culture Medium - is basically an aqueous solution supplemented with nutrients such as vitamin
to which all the necessary nutrients essential for the K and hemin. They undergo boiling to
growth of organisms are added. remove dissolved oxygen. To reduce the
oxidation-reduction potential substances
such as 1% glucose, 0.1% ascorbic acid,
0.1% thioglycolate, or 0.05% cysteine are
added. Methylene blue or resazurin is
THREE PRIMARY LEVELS OF CULTURE
added as an indicator of the oxidation-
MEDIA
reduction potential.

2. According to Chemical Composition

1. According to Physical State
a. Synthetic media - contain a chemically-defined
a. Liquid Media. - commonly called broths, substances which are pure organic and/or inorganic
milk or infusion, these are water-based compounds. The precise chemical composition of
solutions ghat do not solidify at temperature synthetic medium is known. They may be simple or
above the freezing point. These contains complex, depending on what supplement is added
specific amounts of nutrients but do not to it.
contain gelling agents such as gelatin or
agar. These are suited for gelling agents
b. Non-synthetic media - complex media that
such as gelatin or agar. These are suited for
contain at least one ingredient that is not chemically
the propagation of a large number of
defined, which means that it is neither a simple or
organisms, fermentation studies and other
pure compound. It is not representable by an exact
test.
chemical formula.
b. Semi-solid media - exhibit a clot like
consistency in ordinary room temperature 3. According to Functional Type
and contain agar at concentration of 0.5%
a. General Purpose media are designed for 5. Saboraud's dextrose agar- used for the
primary o- isolation of fungi.
isolation of a broad spectrum of microbes and
contain a mixture of nutrients that support the
growth of both pathogenic and non-pathogenic
organisms.
Examples: peptone water, nutrient broth, and
nutrient agar

b. Enrichment media - contain complex organic

substances such as blood, serum or special growth
factors, and are designed to increase the number of
desired microorganisms without stimulatirte rest of
the bacterial population.

Two commonly used Enrichment Media

1. Blood agar - contains general nutrients with 5%-

10% (by volume) blood added to a blood agar
base. Certain gram- positive bacteria produce
exotoxins that cause hemolysis of red blood cells
contained in the blood agar. Their hemolytic
reaction is categorized into three, which is useful in
the classification of these bacteria.

a. Beta hemolysis - shows complete lysis of red

blood cells resulting in complete clearing around
the colonies.
b. Alpha hemolysis - shows incomplete lysis of
red blood cells, producing a greenish discoloration
of the blood agar around the colonies.
c. Gamma hemolysis - shows no hemolysis,
resulting in no change in the medium.

2. Chocolate agar - a type of nutrient medium that

is used for the culture of fastidious om to turn
brown.

c. Selective media - contain one or more

substances that encourage the growth of only a
specific target microorganisms and inhibit the
growth of others. It is designed to prevent the
growth of unwanted contaminating bacteria or
commensals so only the target bacteria will grow.

Types of Selective Media

1. Thayer- Martin Agar - contains the antibiotics

trimethroprim, nystatin, vancomysin, and colistin. It
is used for the isolatior of Neisseria

2. Mannitol Salt Agar - contains 10% NaCl and

used for the isolation of Staphylococcus aureus.

3. MacConkey's agar- promotes the growth of

gram- negative bacteria, primarily those belonging
to the family Enterobacteriacea, and inhibits the
growth of gram-positive bacteria through the
addition of bile salts. It is both selective and
differential.

4. Lowenstein-Jensen medium - a selective

medium used to recover Mycobacterium
tuberculosis. It is made selective by the
incorporation of malachite green.