The document briefs about the four commonly used staining techniques in the laboratory. It states the principle and identifies the color of the staining.

1. Principles of different staining
techniques
1. Gram staining: (purple-gram positive)
 The basic principle of Gram staining is the properties of
certain bacteria cell walls to retain the crystal violet dye. The
cell walls for Gram-positive microorganisms have a higher
peptidoglycan and lower lipid content than Gram-negative
bacteria.
 The Gram stain procedure distinguishes between Gram
positive and Gram-negative groups by coloring these cells red
or violet. Gram positive bacteria stain violet due to the
presence of a thick layer of peptidoglycan in their cell walls,
which retains the crystal violet these cells are stained with.
 The Gram stain is the most important staining procedure in
microbiology. It is used to differentiate between gram positive
organisms and gram negative organisms. Hence, it is a
differential stain
 Safranin is used as a counter stain in some staining protocols,
coloring all cell nuclei red. This is the classic counter stain in
both Gram stains, and endospore-staining. It can also be used
for the detection of cartilage, mucin and mast cell granules.
 In gram-positive bacteria, the S-layer is attached to the
peptidoglycan layer (in gram-negative bacteria, the S-layer is
attached directly to the outer membrane). Specific to gram-
positive bacteria is the presence of teichoic acids in the cell
wall.
 Although all bacteria have an inner cell membrane, gram-
negative bacteria have a unique outer membrane. This outer
membrane excludes certain drugs and antibiotics from
penetrating the cell, partially accounting for why gram-negative
bacteria are generally more resistant to antibiotics than
are gram-positive bacteria.

2. 2. Acid fast (red)
 Some bacteria contain a waxy lipid, mycolicacid, in their cell
wall. This lipid makes the cells more durable and is commonly
associated with pathogens. Acid fast cell walls are so durable
that the stain (carbol fuschin) must be driven into the cells with
heat.
 The acid-fast stain is a differential stain used to identify acid-
fast organisms such as members of the genus
Mycobacterium. Acid-fast organisms are characterized by wax-
like, nearly impermeable cell walls; they contain mycolic acid
and large amounts of fatty acids, waxes, and complex lipids.
 It is commonly used in the staining of mycobacterium as it has
an affinity for the mycolic acids found in their cell membranes.
It is a component of Ziehl–Neelsen stain. Carbol fuchsin is
used as a dye to detect acid fast bacteria because it is more
soluble in the cells wall lipids than in the acid alcohol.
 Sputum, or phlegm, is often used to test for Mycobacterium
tuberculosis, to find out if a patient has TB. This bacterium is
completely acid-fast, which means the entire cell holds onto the
dye. A positive test result from the acid-fast stain confirms the
patient has TB
 Acid-fast bacteria are gram-positive, but in addition to
peptidoglycan, the outer membrane or envelope of the acid-
fast cell wall of contains large amounts of glycolipids,
especially mycolic acids that in the genus Mycobacterium make
up approximately 60% of the acid-fast cell wall.
3. Capsular staining :
 Capsules are formed by organisms such as Klebsiella
pneumoniae. Most capsules are composed of polysaccharides,
but some are composed of polypeptides. The capsule differs
from the slime layer that most bacterial cells produce in that it is
a thick, detectable, discrete layer outside the cell wall.
 The main purpose of capsule stain is to
distinguish capsular material from the bacterial cell.
A capsule is a gelatinous outer layer secreted by bacterial cell
and that surrounds and adheres to the cell wall.

3. Most capsules are composed of polysaccharides, but some are
composed of polypeptides
4. Flagellar staining:
 Because bacterial flagella are very thin and fragile a
special stain (flagella stain) is prepared that contains a mordant.
This mordant allows piling of the stain on the flagella,
increasing the thickness until they become visible. Various
arrangements of flagella are seen on different cells.
 The flagella stain allows observation of bacterial flagella under
the light microscope. Bacterial flagella are normally too thin to
be seen under such conditions. The flagella stains employs a
mordant to coat the flagella with stain until they are thick
enough to be seen.