CH-2 :Observation of Microbes

under Microscope

Staining
Purpose of Bacterial staining
Visualization of microbes in the living state is most
difficult, not only because they are minute, but also
because they are transparent and particularly
colorless when suspended in an aqueous medium.
To study their properties and to divide microbes
into specific groups for diagnostic purposes,
biological stains and staining procedures in
conjunction with light microscopy have become
major tools in microbiology.
 What is stain?
➢ Chemically, a stain (dye) may be defined as an
organic compound containing a benzene ring plus a
chromophore and auxochrome group.
Benzene (organic colorless solvent) + Chromophore
(chemical group that imparts color to benzene) =
Chromogen (colored compound, not a stain)
Chromogen + Auxochrome (chemical group that
conveys the property of ionization to the chromogen,
enabling it to form salts and bind to fibers or tissues)
= Stain
Basic rules of staining
 Basic rules of staining
The basic rules of staining are:
1. Preparation of glass slides:
– Slides are cleaned essential for the preparation of microbial
smears.
– Grease or oil from fingers on slides must be removed by
washing the slides with soap and water or scouring powders,
followed by a water rinse and a rinse of 95% alcohol.
– After cleaning, the slides are dried and placed on lab towels until
ready for use.
Basic rules of staining (contd…)
2. Preparation of smear:
– Avoidance of thick, dense smears is absolutely
essential.
– A good smear is one that, when dried, appears as a
thin whitish layer or film.

– Those made from broth cultures or cultures from a

solid medium require variations in technique.
3. Heat fixation:
– Unless fixed on the glass slide, the bacterial smear
will wash away during the staining procedure.
– This is avoided by heat fixation, during which
bacterial proteins are coagulated and fixed to
the surface.

– Heat fixation is performed by rapid passage of the

air-dried smear two or three times over the flame
of the Bunsen burner.
Preparing Smears for Staining

Most studies of the shapes and cellular arrangements

of microbes are made with stained preparations.
Staining simply means coloring the microbes with a dye
that emphasizes certain structures.
Before the microbes can be stained, they must be
attached (or fixed) to the microscopic slide; without
fixing, the stain might wash them off the slide.
Preparing Smears for Staining (contd…)
When a specimen is to be fixed, a thin film of material
containing the microbes is spread over the surface of
the slide. This film, called a smear, is allowed to air dry.
In most staining procedures the slide is then slowly passed
through the flame of a Bunsen burner several times,
smear side up.
Air drying and flaming fix the microbes to the slide and
usually kill them. Stain is applied and then washed off with
water, then the slide is blotted with absorbent paper.
The stained microbes are now ready for microscopic
examination.
 Microbiological Stains
A more practical classification for the
cytologist is on based on the chemical
behavior of the dye; namely,
a) Acidic dye
b) Basic dye
c) Neutral dye
 Acid dye (anionic dye)
Acidic dye is negatively charged which bind to positively
charged groups of the cell.
Acid dye generally stain basic cell components.Works best
at low pH.
Acid dye’s negative ions are repelled by the negatively
charged bacterial surface, so the stain colors the
background instead.
Proteins, positively charged cellular components readily
bind to and accept the color of the negatively charged,
anionic chromogen of an acidic stain.
E.g.eosin, acid fuchsin, and nigrosin, picric acid etc.
 Basic dye (Cationic dye)
Basic dye is positively charged and bind to negatively
charged groups.

Basic dye generally stain acidic cell components.

Bacteria are slightly negatively charged at pH 7. Thus,

the colored positive ion in a basic dye is attracted to the
negatively charged bacterial cell. Works best at high pH.

E.g.crystal violet, methylene blue, malachite green, and

safranin.
 Neutral dye
• Neutral dye is a complex salt of a dye acid
with a dye base
• E.g., eosinate of a methylene blue
 Process of staining
Stains are salts composed of a positive and a
negative ions, one of which is colored
The process of staining may involve ion-
exchange reactions between the stain and the
active sites at the surface of or within the cell.
Dyes contain chromophore groups and bind
to cells by ionic, covalent and hydrophobic
forces.
 Process of staining (contd…)
In a basic dye, like methylene blue, the colored ion is
positively charged (a cation), and if we represent this ion
by the symbol MB, the dye, which is actually methylene
blue chloride, may be represented as
MB+Cl-
The ion exchange which take place during the staining
can be represented by the following equation, in which
the MB+ cation replaces the Na+ cation in the cell:
(Bacterial cell-)(Na+) + (MB+)(Cl-) (Bacterial cell-
)(MB+)+ (Na+Cl-)
 Staining Techniques
a) Simple stain: use of single stain for
visualization of morphological shape and
arrangement.
b) Differential stain: Use of two contrasting
stain for separation into groups and
visualization of structure.
c) Special stain
 Simple Stain
A simple stain is an aqueous solution of a single basic dye. Although
different dye bind specifically to different parts of cells, the primary
purpose of simple stain is to highlight the entire microorganism so
that cellular shapes and basic structures are visible.
E.g. methylene blue, carbolfuchsin, crystal violet, and safranin.
The stain is applied to the fixed smear for a certain period of time and
then washed off, and the slide is dried and examined.
Occasionally, a chemical is added to the solution to intensify the
stain ; such an additive is called a mordant. One function of a
mordant is to increase the affinity of a stain for a biological specimen;
another is to coat a structure (such as flagellum) to make it thicker
and easier to see after it is stained with a dye.
 Differential Stain
Unlike simple stain differential staining
procedures that make visible the differences
between bacterial cells or parts of a bacterial
cell.
They are slightly more elaborate than the simple
staining technique in that the cells may be
exposed to more than one dye solution or
staining reagent.
1) Gram stain
2) Acid-fast stain
 Special stains
➢ are used to color and isolate specific parts of
microorganisms, such as endospores, flagella,
and capsule etc.
1) Endospore(Spore) stain
2) Negative staining for capsule
3) Flagella stain
4) Cytoplasmic inclusion stains
 Gram stain
❑One of the most important and widely used
differential staining.

❑The widely used technique in microbiology is

Gram staining because it classifies bacteria into
two large groups: gram-positive and gram-
negative. This technique is introduced by Hans
Christian Gram of Denmark in 1884.
 Procedure of Gram Staining
❑ A heat-fixed smear is covered with basic purple dye, usually crystal
violet. Because the purple stain imparts its color to all the cells, it is
referred to as primary stain.
❑ After a short time, the purple dye is washed off, and the smear is
covered with iodine, a mordant. When the iodine is washed off, both
gram-positive and gram-negative bacteria appear dark violet or purple.
❑ Next the slide is washed with alcohol or an alcohol-acetone solution.
This solution is called decolorizing agent, which removes the purple
from the cells of some species. But not from others,
❑ The alcohol is rinsed off, and the slide is then stained with safranin, a
basic red dye. The smear is then washed again, blotted dry, and
examine microscopically.
Procedure of Gram Staining (contd..)

Primary Stain: Crystal violet

Mordant: Gram’s Iodine
Decolorizing agent: 95% ethyl alcohol
Counter stain: Safranin
Procedure of Gram Staining
Differential Stains: Gram Stain
 Differential Stains: Gram Stain
Color of Color of
Gram + cells Gram – cells
Primary stain: Purple Purple
Crystal violet
Mordant: Purple Purple
Iodine
Decolorizing agent: Purple Colorless
Alcohol-acetone
Counterstain: Purple Red
Safranin
 Differential Stains: Gram Stain
⧫Gram positive bacteria have thicker peptidoglycan
(disaccharides and amino acids) cell wall than gram
negative bacteria.

⧫In addition gram negative bacteria contain a layer of

lipopolysaccharides (lipids and polysaccharides)
as part of their cell wall.

⧫When applied to both gram positive and gram

negative cells, crystal violet and then iodine readily
enter the cells.
 Differential Stains: Gram Stain (contd….)
⧫ Inside the cells, the crystal violet and iodine combine
to form CV-I complex. This CV-I complex binds to the
magnesium-ribonucleic acid components of the
cell wall.

⧫ The resultant magnesium-ribonucleic acid-crystal

violet-iodine (Mg-RNA-CV-I) complex is more
difficult to remove than the smaller CV-I complex. This
complex is larger than the crystal violet molecule that
entered the cells, and, because of its size, it cannot be
washed out of the intact peptidoglycan layer of gram
positive cells by alcohol.
 Differential Stains: Gram Stain (contd….)
❑ Ethyl Alcohol, 95%: This reagent serves as a duel
function as a lipid solvent and as a protein
dehydrating agent.

❑ Its action is determined by the lipid concentration of

the microbial cell walls.

❑ In gram-positive cells, the low lipid concentration is

important to retention of the Mg-RNA-CV-I complex.
Therefore the small amount of lipid content is readily
dissolved by the action of the alcohol causing formation
of minute cell wall pores. These are then closed by
alcohol’s dehydrating effect.
 Differential Stains: Gram Stain (contd….)
❑ As a consequence, the tightly bound primary stain is difficult to
remove , and the cells remain purple. In the gram-negative cells,
the high lipid concentration found in outer layers of the cell wall is
dissolved by the alcohol, creating large pores in the cell wall that
do not close appreciably on dehydration of cell wall proteins. This
facilitates release of the unbound CV-I complex, leaving these
cells colorless or unstained.
❑ Therefore gram positive cells retain the color of the crystal violet
dye. In gram negative cells, however, the alcohol wash disrupts
the outer lipopolysaccharide layer and the CV-I complex is
washed out through the thin layer of peptidoglycan.
 Differential Stains: Gram Stain (contd….)
❑As a result, gram negative cells are colorless until
counterstain with safranin, after which they are
pink.
❑In summary, gram positive cells retain the dye and
remain purple. Gram negative cells do not retain
the dye, they are colorless until counterstain with a
red dye after which they appear pink.
❑The gram reaction is the most consistent when it is
used on young growing bacteria. The old culture
may show gram variable results.
 Acid fast staining
➢ The acid fast stain binds strongly only to bacteria that have a waxy
material in their cell walls.
➢ Microbiologists use this stain to identify all bacteria in the genus
Mycobacterium, including the two important pathogens,
Mycobacterium tuberculosis (tuberculosis)and Mycobacterium
leprae (leprosy).
➢ This stain is also used to identify the pathogenic strains of the
genus Nocardia.
➢ In AFB staining procedure, the red dye, Carbol fuchsin, is applied
to a fixed smear and the slide is gently heated for several
minutes (heating enhances penetration and retention of the dye.
 Acid fast staining (contd…)
➢ Then the slide is cooled and washed with water.
➢ The smear is next treated with acid alcohol, a decolorizer, which
removes the red stain from bacteria that are not acid fast.
➢ The acid fast microorganisms retain the red color because the
carbol fuchsin is more soluble in the cell wall lipids than in acid
alcohol.
➢ In non acid fast bacteria, whose cell walls lack the lipid
compounds, the carbon fuchsin is rapidly removed during the
decolorization, leaving the cells colorless.
➢ The smear is then stained with a methylene blue counterstain.
➢ Non acid fast cells appear blue after application of the
counterstain.
 Differential Stains: Acid-Fast Stain
❑ Cells that retain a
basic stain in the
presence of acid-
alcohol are called
acid-fast.
❑ Non–acid-fast cells
lose the basic stain
when rinsed with acid-
alcohol, and are
usually counterstained
(with a different color
basic stain) to see
them. Figure 3.11
 Ziehl-Neelson Method (modified
acid fast staining)
Purpose: To stain acid-fast mycobacteria.

Reagents: Ziehl-Neelson Carbol fucshin

Acid Alcohol
Methylene blue
Beaker of boiling water
 Ziehl-Neelson Method (modified acid fast
staining)
Procedure:
– Boil one inch of water in a beaker.
– Prepare smears, air dry and heat fix.
– Place the slide on a staining rack over the boiling water.
– Flood the slide with Ziehl-Neelson Carbol fucshin for five minutes while
applying steam. Continue to refresh the stain, not allowing it to dry.
– Pour off the excess stain and gently wash with a stream of water while
allowing the slide to cool.
– Decolorize with acid alcohol until no fuchsia drips off the slide.
– Allow the acid alcohol to stay on the slide for no longer than 30 seconds.
– Immediately wash the slide in water.
– Counterstain with Methylene blue for one to two minutes.
– Pour off the excess stain and gently wash with a stream of water.
– Gently blot dry and view under a microscope.
 Capsule staining
❖ microorganisms contain a gelatinous covering called a capsule.
In medical microbiology, demonstrating the presence of a
capsule is a means of determining the organisms virulence, the
degree to which a pathogen can cause disease.
❖ 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
❖ Bacterial capsules are non-ionic, so neither acidic nor basic
stains will adhere to their surfaces. Therefore, the best way to
visualize them is to stain the background using an acidic stain
and to stain the cell itself using a basic stain. We use India ink
and Gram crystal violet. This leaves the capsule as a clear halo
surrounding a purple cell in a field of black.
Capsule staining .
 Endospore (Spore) staining/Schaeffer-
Fulton method.
❖ An Endospore is a special resistant, dormant structure formed
within a cell that protects a bacteria from adverse environmental
conditions.
❖ Although endospores are relatively uncommon in bacterial cells,
they can be formed by a few genera of bacteria.
❖ Endospores cannot be stained by ordinary method such as simple
staining and gram staining, because the dyes do not penetrate the
wall of the endospore.
❖ The most commonly used endospore stain used is Schaeffer-
Fulton endospore stain. Malachite green, the primary stain, is
applied to a heat fixed smear and heated to steaming for about five
minutes.
 Endospore (Spore) staining (contd…)
➢ The heat helps the stain to penetrate the endospore wall.
Then the preparation is washed for about 30 seconds
with water to remove the malachite green from all of the
cells’ parts except the endospore.
➢ Next safranin, a counterstain, is applied to the smear to
stain portions of the cell for 2 minutes other than
endospores.
➢ In a properly prepared smear, the endospore appears
green within red or pink cells.
•Heat is required to drive a stain into endospores.
 Flagella staining

• Bacterial flagella (singular: flagellum) are

structures of locomotion too small to be
seen with a light microscope without
staining
• The flagella stains employs a mordant to
coat the flagella with stain until they are
thick enough to be seen.
 Staining procedure
➢ carefully remove a large loopful of the suspension and
place it at one end of the slide
➢ Air dry the film. Do not heat.
➢ Add about 1 ml. of the Flagella Stain solution(Carbol
fuchsin) (one dropper full) to the smear and allow to
stain for 10-15 minutes
➢ Flood off the stain by adding tap water to the slide
➢ Drain and flood the slide with carbol fuchsin for one
minute again . Rinse by flooding. Drain and air dry.
Flagella staining requires a mordant to make the
flagella wide enough to see
Thanks