MICROBIOLOGY QUICK LEARNSTAINING TECHNIQUESPART I GRAM STAINING, SIMPLE STAINING Ms Saajida Sultaana Mahusook

1. MICROBIOLOGY QUICK LEARN
Ms Saajida Sultaana Mahusook

2.  Staining of microorganisms is essential to study their properties and to divide them
into specific groups for diagnostic purposes.
 Chemically, a stain (dye) may be defined as an organic compound containing a
benzene ring plus a chromophore and an auxochrome group.
 The ability of a stain to bind to macromolecular cellular components such as
proteins or nucleic acids depends on the electrical charge found on the chromogen
portion, as well as on the cellular component to be stained.
 Acidic stains are anionic- on ionization of the stain, the chromogen portion exhibits
a negative charge and has a strong affinity for the positive constituents of the cell.
Proteins, positively charged cellular components, will readily bind to and accept the
color of the negatively charged, anionic chromogen of an acidic stain.
Example picric acid.
 Basic stains are cationic- on ionization the chromogen portion exhibits a positive
charge and therefore has a strong affinity for the negative constituents of the cell.
Nucleic acids, negatively charged cellular components, will readily bind to and
accept the color of the positively charged, cationic chromogen of a basic stain.
Example methylene blue

3. Types of
staining techniques
Simple staining:
Use of single stain
For visualization of morphological
shape (cocci, bacilli, and spirilli) and
arrangement (chains, clusters, pairs,
and tetrads)
Differential staining:
Use of two contrasting stains
Separation into groups
Gram stain
Acid-fast stain
Visualization of structures
Flagella stain
Capsule stain
Spore stain
Nuclear stain

4. Simple staining
Principle
In simple staining, the bacterial smear is stained with a single reagent, which
produces a distinctive contrast between the organism and its background. Basic
stains with a positively charged chromogen are preferred because bacterial nucleic
acids and certain cell wall components carry a negative charge that strongly
attracts and binds to the cationic chromogen.
Commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin.
Application
 To elucidate the morphology(shape) and arrangement of bacterial cells.
 To determine the numbers of bacteria present in a sample.
 Simple staining methods are not considered differential or diagnostic and will
have limited uses.
 It is a quick procedure for determining whether a clinical sample has the
presence of a foreign bacterial pathogen.

5. B. cereus
S. aureus
Shapes and arrangement of bacterial cells
S. pyogenes

6. Gram staining
It is a differential staining requires the use of at least four chemical reagents named
after Dr. Hans Christian Gram.
It divides bacterial cells into two major groups, gram-positive and gram-negative,
which makes it an essential tool for classification and differentiation of
microorganisms.
Primary Stain
Crystal Violet (Hucker’s)
Impart its color to all cells.
Mordant
Gram’s Iodine
Killing agent, intensify the
color of the primary stain.
Decolorizing Agent
Ethyl Alcohol, 95%
protein-dehydrating agent and
as a lipid solvent.
Counterstain
Safranin
used to stain pink those cells
that have been previously
decolorized.

7. Principle
The Gram stain reaction is based on the difference in the chemical composition of
bacterial cell walls.
Crystal violet stains all cells purple.
Gram’s Iodine increases the cells’ affinity for a stain by binding to the primary stain,
forming an insoluble complex. The resultant crystal-violet–iodine (CV-I) complex
serves to intensify the color of the stain. All cells will appear purple-black.
Ethyl Alcohol, 95% Its action is determined by the concentration of lipids and
thickness of the peptidoglycan layer in bacterial cell walls. In gram-negative cells, the
alcohol increases the porosity of the cell wall by dissolving the lipids in the outer
layers. Thus, the CV-I complex can be more easily removed from the thinner
peptidoglycan layer. Therefore, the washing-out effect of the alcohol facilitates the
release of the unbound CV-I complex, leaving the cells colorless or unstained. The
thicker peptidoglycan layer in gram-positive cells is responsible for the stringent
retention of the CV-I complex, as the pores are made smaller due to the dehydrating
effect of the alcohol. The tightly bound primary stain complex is difficult to remove,
and the cells remain purple.
Safranin is used to stain pink those cells that have been previously decolorized. Since
only gram-negative cells undergo decolorization, they may now absorb the
counterstain. Gram-positive cells retain the purple color of the primary stain.

8. Clinical Application :
 The Gram stain is a diagnostic staining procedure that can be done on body
fluids, tissue biopsies, throat cultures, samples from abscesses when infection
is suspected, and more.
 Clinically important results are obtained much more rapidly from staining than
from culturing the specimen. Once the bacterial gram type, shape, and
orientation are determined, it expedites the appropriate choice of antibiotic
needed to treat the patient.
E. coli (Gram negative) S. aureus (Gram positive)

9. Reference :
Microbiology: A Laboratory Manual (9th Edition)
by James Cappuccino , Natalie Sherman, Pearson Education (2010).
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