This document discusses procedures for preparing and staining microbial smears and slides. It describes how microbes are fixed to slides through air drying or heat, and then stained using simple, differential or special staining techniques. Key points covered include how gram staining classifies bacteria as either gram-positive or gram-negative based on cell wall structure and staining properties, and how acid-fast staining identifies mycobacteria by their lipid-rich cell walls. Negative staining can reveal capsules around cells.

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Preparing Smears for Staining
Most initial observations of microorganisms are made with stained preparations. Staining
simply means coloring the microorganisms with a dye that emphasizes certain structures.
Before the microorganisms can be stained, however, they must be fixed (attached) to the
microscope slide. Fixing simultaneously kills the microorganisms and fixes them to the slide. It
also preserves various parts of microbes in their natural state with only minimal distortion .
When a specimen is to be fixed, a thin film of material containing the microorganisms 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 fixed by passing it through the flame of a Bunsen burner several
times, smear side up, or by covering the slide with methyl alcohol for I minute. Stain is applied
and then washed off with water; then the slide is blotted with absorbent paper. Without fixing,
the stain might wash the microbes off the slide. The stained microorganisms are now ready for
microscopic examination.
Stains are salts composed of a positive and a negative ion, one of which is colored and is known
as the chromoplwre. The color of so-called basic dyes is in the positive ion; in acidic dyes. it is in
the negative ion. 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. Basic dyes, which include
crystal violet, methylene blue, malach ite green, and safran in, are more commonly used than
acidic dyes. Acidic dyes are not attracted to mosttypesof bacteriabecause the dye's negative ions
are repelled by the negatively charged bacterial surface, so the stain colors the background
instead. Preparing colorless bacteria against a colored background is called negative staining. It
is valuable for observing overall cell shapes, sizes, and capsules because the cells are made
highly visible against a contrasting dark background . Distortions of cell size and shape are
minimized because fixing is not necessary and the cells do not pick up the stain . Examples of
acidic dyes are eosin, acid fuchsin, and nigrosin.
To apply acidic or basic dyes, microbiologists use three kinds of staining techniques: simple,
differential, and special.
 Simple Stains
A simple stain is an aqueous or alcohol solution of a single basic dye. Although different dyes
bind specifically to different part s of cells, the primary purpose of a simple stain is to highlight
the entire microorganism so that cellular shapes and basic structures are visible. The stain is
applied 10 the fixed smear for a certain length of lime and then washed off, and the slide is

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dried and examined. Occasionally, a chemical is added to the solution to intensify the slain;
such an additive is called a mordant. One function of a mordant is 10 increase the affinity of a
stain for a biological specimen; another is to coal a structure (such as a flage llum) to make it
thicker and easier to see after it is stained with a dye. Some of the simple stains commonly used
in the laboralory are methylene blue, carbolfuchsin, crystal violet, and safranin.
DifferentialStains
Unlike simple stains, differential stains react differently with different kinds of bacteria and thus
can be used to distinguish them. The differential stains most frequently used for bacteria are
the Gram stain and the acid-fast stain.
 Gram Stain:
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is
one of the most useful staining procedures because it classifies bacteria into two large groups:
gram-positive and gram-negative.
1. A heat -fixed smear is covered with a basic purple dye, usually crystal violet. Because the
purple stain imparts its color to all cells, it is referred to as a primary stain.
2. 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.
3. Next, the slide is washed with alcohol or an alcohol-acetone solution. This solution is a
discoloring agent. Which removes the purple from the cells of some species but not
from others.
4. The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye. The
smear is washed again, blotted dry, and examined microscopically.
The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark
violet or purple. Bacteria that retain this color after the alcohol has attempted to decolorize
them are classified as gram-positive; bacteria that lose the dark violet or purple color after
depolarization are classified as gramnegative . Because gram-negative bacteria are colorless
after the alcohol wash, they are no longer visible. This is why the basic dye safranin is applied; it
turns the gram-negative bacteria pink. Stains such as safranin that have a contrasting color to
the primary stain arc called counterstains. Because grampositive bacteria retain the original
purple stain, they are not affected by the safranin counterstain .

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As you will see in Chapter 4, different kinds of bacteria react differently to the Gram stain
because structural differences in their cell walls affect the retention or escape of a combination
of crystal violet and iodine, called the crystal violet- iodine (CV-I) complex. Among other
differences, gram-positive bacteria have a thicker peptidoglycan (disaccharides and amino
acids) cell wall than gram-negative bacteria. In addition, gram- negative bacteria contain a layer
of lipopolysaccharide (lipids and polysaccha - rides) 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. Inside the cells, the crystal violet and iodine combine to form CV- 1. This complex is larger
than the crystal violet molecule that entered the cells, and, because of its size, it can not be
washed out of the intact pept idoglycan layer of gram-positive cells by alcohol. Consequently,
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 . As a result, gramnegative cells are colorless until
counterstained with safranin, after which they arc pink.
In summary, gram-positive cells retain the dye and remain purple. Gram-negative cells do not
retain the dye; they are colorless until counterstained with a red dye.
The Gram method is one of the most important staining techniques in medical microbiology.
But Gram staining results arc not universally applicable, because some bacterial cells stain
poorly or not at all. The Gram reaction is most consistent when it is used on young, growing
bacteria.
In summary, gram-positive cells retain the dye and remain purple. Gram-negative cells do not
retain the dye; they are colorless until counterstained with a red dye.
The Gram method is one of the most important staining techniques in medical microbiology.
But Gram staining results arc not universally applicable, because some bacterial cells stain
poorly or not at all. The Gram reaction is most consistent when it is used on young, growing
bacteria.
 Acid-Fast Stain:
Another important differential stain (one that differentiates bacteria into distinctive groups) is
the acid-fast stain, which 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 .
In the acid-fast staining procedure, the red dye car- bolfuchsin is applied to a fixed smear, and
the slide is gently heated for several minutes. (Heating enhances penetration and retention of

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the dye.) 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 carbolfuchsin is more soluble in the
cell wall lipids than in the acid-alcohol. In non- acid-fast bacteria, whose cell walls lack the lipid
components, the carbolfuchsin is rapidly removed during 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.
Special Stains
Special stains are used to color and isolate specific parts of microorganisms, such as endospores
and flagella, and to reveal the presence of capsules.
Negative Staining for Capsules
Many microorganisms contain a gelatinous covering called a capsule.. In medical microbiology,
demonstrating the presence of a capsule is a means of determining the organism's virulence.
the degree to which a pathogen can cause disease.
Capsule staining is more difficult than other types of staining procedures because capsular
materials are soluble in water and may be dislodged or removed during rigorous washing. To
demonstrate the presence of capsules, a microbiologist can mix the bacteria in a solution
containing a fi ne colloidal suspension of colored particles (usually India ink or nigrosin) to
provide a contrasting background and then stain the bacteria with a simple stain, such as
safranin. Because of their chemical composition, capsules do not accept most biological dyes,
such as safranin, and thus appear as halos surrounding each stained bacterial cell.
 Endospore (Spore)Staining:
An endospore is a special resistant, dormant structure formed within a cell that protects a
bacterium from adverse environmental conditions. Although endospores arc relatively
uncommon in bacterial cells, they can be formed by a few genera of bacteria.
Endospores cannot be stained by ord inary methods, such as simple staining and Gram sta
ining, because the dyes do not penetrate the wall of the endospore.
The most commonly used endospore stain is the Schaeffer- Fulton endospore stain. Malachite
green, the primary stain, is applied to a heat-fixed smear and heated to steaming for about 5
minutes. The heat helps the stain penetrate the endospore wall. Then the prepa ration is
washed for about 30 seconds with water to remove the malachite green from all of the cells'

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parts except the endospores. Next, safranin, a counterstain, is applied to the smear to sta in
portions of the cell other than endospores. In a properly prepared smear, the endospores
appear green within red or pink cells. Because endospores are highly refractive, they can be
detected under the light microscope when unstained, but they cannot be differentiated from
inclusions of stored material without a special stain .
 Flagella Staining
Bacterial flagella (singular: flagllum) are structures of loco - motion too small to be seen with a
light microscope without staining. A tedious and delicate staining procedure uses a mordant
and the stain carbolfuchsin to build up the diameters of the flagella until they become visible
under the light micro - scope. Microbiologists use the number and arrangement of flagella as
diagnostic aids. Animation Staining .
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