PRINCIPLES OF MICROBIOLOGICAL

DIAGNOSTICS
MICROSCOPIC OBSERVATION OF
MICROORGANISMS
SIMPLE STAINING, NEGATIVE STAINING,
HANGING-DROP PREPARATIONS,
CAPSULE STAIN,
GRAM-STAINING, ZIEHL-NEELSEN STAINING,
DARK-FIELD MICROSCOPY,
FLUORESCENCE MICROSCOPY

 Microbial etiologic agents of infections are

bacteria, viruses, fungi, and parasites
The pathogen may be exogenous - acquired from
environmental (soil, water) or animal sources or
from other persons
or endogenous - from the normal flora.
Endogenous infections can occur when the
microorganism is aspirated from the upper to the
lower respiratory tract or when it penetrates the
skin or mucosal barrier as a result of trauma or
surgery.

THE DIAGNOSIS OF MICROBIAL INFECTION

The diagnosis of microbial infection begins with
an
assessment
of
clinical
and
epidemiological features, leading to the
formulation of a diagnostic hypothesis
Anatomic localization of the disease with the aid
of physical and radiologic findings is usually
included
However, this clinical diagnosis is suggestive of
a number of possible etiologic agents on the
basis of knowledge of infectious syndromes and
their courses
Therefore, it is frequently necessary to use
microbiologic laboratory methods to identify
a specific etiologic agent

IN THE DIAGNOSIS OF INFECTIOUS DISEASE,

SEVERAL IMPORTANT STEPS PRECEDE THE
LABORATORY WORK:
Choosing the appropriate specimen to examine
Obtaining the specimen to avoid contamination
from the normal microbial flora: skin disinfection
before aspirating or incising a lesion; alternatively,
the contaminated area may be bypassed
altogether (transtracheal puncture with aspiration
of lower respiratory secretions, suprapubic bladder
puncture with aspiration of urine)
Transporting the specimen promptly to the
laboratory or storing it correctly

THE ACTUAL LABORATORY WORK INCLUDES:

Observing the microorganism under the microscope after
staining
Obtaining a pure culture of the microorganism by inoculating it
onto a bacteriologic medium
Identifying the etiologic agent of infection on the basis of
biochemical reactions/characteristics of the organism, growth
on selective media, detection of specific microbial antigens,
detection of antibodies in the patients serum (classic methods)
One of the newest approaches to the identification of
microorganisms are nucleic acid amplification assays that allow
direct detection of genomic components of pathogens ,
however, only few are practical for routine use.
Antimicrobial agents susceptibility testing of the cultured

Specimens for the diagnosis of

infection.
A.Direct specimen. The pathogen is
localized in an otherwise sterile site, and
a barrier such as the skin must be passed
to sample it. This may be done surgically
or by needle aspiration as shown. The
specimen collected contains only the
pathogen. Examples are deep abscess
and cerebrospinal fluid.
B.Indirect sample. The pathogen is
localized as in A, but must pass through a
site containing normal flora in order to be
collected. The specimen contains the
pathogen, but is contaminated with the
nonpathogenic flora. The degree of
contamination is often related to the skill
with which the normal floral site was
"bypassed"
in
specimen
collection.
Examples are expectorated sputum
and voided urine.
C.Sample from site with normal flora.
The pathogen and nonpathogenic flora
are mixed at the site of infection. Both are

Specimen collection and transport

The sterile swab is the most convenient and most commonly used tool for
specimen collection; however, it provides the poorest conditions for survival
and can only absorb a small volume of inflammatory exudate.

The worst possible specimen is a dried-out swab; the best is a collection of

5 to 10 mL or more of the infected fluid or tissue.

The volume is important because infecting organisms that are present in

small numbers may not be detected in a small sample.

Specimens should be transported to the laboratory as soon after

collection as possible because some microorganisms survive only briefly
outside the body. For example, unless special transport media are used,
isolation rates of the organism that causes gonorrhea (Neisseria
gonorrhoeae) are decreased when processing is delayed beyond a few
minutes. Likewise, many respiratory viruses survive poorly outside the
body. On the other hand, some bacteria survive well and may even multiply
after the specimen is collected. The growth of enteric Gram-negative rods
in specimens awaiting culture may in fact compromise specimen
interpretation and interfere with the isolation of more fastidious organisms.
Significant changes are associated with delays of more than 3 to 4 hours.

Specimen collection and transport

Various

transport media have been developed to

minimize the effects of the delay between specimen
collection and laboratory processing.

In general, they are buffered fluid or semisolid media

containing minimal nutrients and are designed to

prevent drying, maintain a neutral pH, and minimize
growth of bacterial contaminants
Other

features may be required to meet special

requirements, such as an oxygen-free atmosphere for
obligate anaerobes

MICROSCOPY

Microscopy is the most common method used both for

detection of microorganisms directly in clinical specimens
and for the characterization of organisms grown in culture
Three major types of microscopy can be used to visualize
bacteria, namely, BRIGHT-FIELD/LIGHT MICROSCOPY,
FLUORESCENCE
MICROSCOPY,
AND
DARK-FIELD
MICROSCOPY

APPLICATIONS OF MICROSCOPY IN DIAGNOSTIC

MICROBIOLOGY

Rapid preliminary identification of organism by direct visualization in

patient specimens
Rapid final identification of certain organisms by direct visualization in
patient specimens
Detection of different organisms present in the same specimen
Detection of organisms not easily cultivated in the laboratory
Evaluation of patient specimens for the presence of cells indicative of
inflammation (phagocytes) or contamination (squamous epithelial cells)
Determination of an organisms clinical significance; bacterial
contaminants usually are not present in patient specimens at
sufficiently high numbers (>10 cells/ml) to be seen by light microscopy
Provide preculture information about which organisms might be
expected to grow so that appropriate cultivation techniques are used
Determine which tests and methods should be used for identification
and characterization of cultivated organisms
Provide a method for invastigating unusual or unexpected laboratory
test results

THE LIGHT MICROSCOPY

For light microscopy, visible light is passed through the specimen

and then through a series of lenses that reflect the light in a
manner that results in magnification of the organisms present in
the specimen
The objective lens, which is closest to the specimen, magnifies
objects 100x, whereas the ocular lens, which is nearest the eye,
magnifies 10x
Using these two lenses in combination, organisms in the specimen
are magnified 1000x their actual size when viewed through ocular
lens

THE LIGHT MICROSCOPY

RESOLUTION the extent to which detail in the magnified object is

maintained (without it everything would be an indistinguishable
blur)
It refers to the ability of the lenses to distinguish between two
points a specified distance apart.
CONTRAST needed to make objects stand out from the
background
Because microorganisms are essentially transparent, owing to their
microscopic dimensions and high water content, they cannot be
easily detected among the background materials and debris in
patient specimens
Contrast is most commonly achieved by staining techniques that
highlight organisms and allow them to be differentiated from one
another and from background material and debris

To achieve the level of resolution desired with 1000x magnification, oil

immersion must be used in conjunction with light microscopy.
It is used to fill the space between the objective lens and the glass slide
onto which the specimen has been affixed.
The oil enhances resolution by preventing light rays from dispersing and
changing wavelength after passing through the specimen.
It must be remembered that a special objective lens the oil immersion
lens is used with oil. This lens provides 100x magnification.

WET MOUNT PREPARATION

Sometimes assay results are compromised because a

contaminating organism grows in the medium instead of
the intended bacterial isolate.
For a quick check to verify that cell morphology is
consistent with the culture from which the inoculum was
taken, a WET MOUNT can be prepared and examined in
dark field and/or phase contrast
Wet mounts (unstained preparations of fluid material) are
widely used in looking at cells in urine, cerebrospinal fluid,
faeces and vaginal secretions

HANGING DROP METHOD

Identification of an unknown organism frequently requires
knowledge of its motility
A good quick check for motility is to examine a very young
culture using the HANGING DROP METHOD
A young culture would be a broth culture inoculated the night
before, or a broth culture that was diluted 10 fold or so in the
morning, incubated and examined in the afternoon
A hanging drop culture is prepared by placing a very small drop
of medium on a coverslip, then inverting the coverslip over a
depression slide so that the bottom of the drop does not make
contact with the slide itself. Vaseline can be used if necessary,
to make a sealed chamber.
Hanging drops can be examined using all objective lenses. The
curved depression slide will distort the effects of phase
contrast, but dark field may work and will be sufficient to detect
movement. All live bacteria move by Brownian (molecular)
motion, at a vibration rate inversely proportional to the size of
the cell.
Rapid Brownian movement is a common characteristic of nonmotile cocci
such
as
Staphylococcus, Streptococcus,
Micrococcus. However, some bacteria are flagellated and
exhibit translational movement as well. Truly motile organisms

STAINS:
Stains are chemicals containing chromophores, groups
that impart color
Stains are generally salts in which one of the ions is
colored; a salt is a compound composed of a positively
charged ion and a negatively charged ion
Stains fall into basic stains and acidic stains (India Ink is a
neutral stain)
A basic dye (crystal violet, safranin, basic fuchsin,
methylene blue, malachite green) is a stain that is cationic
(positively charged) and will therefore react with material
that is negatively charged. The cytoplasm of all bacterial
cells have a slight negative charge when growing in a
medium of near neutral pH and will therefore attract and
bind with basic dyes.
Acidic dyes (nigrosine, congo red, eosin, acid fuchsin)
have negatively charged chromophores and are repelled
by the bacteria surface forming a deposit around the
organism. They stain the background and leave the
microbe transparent.

SIMPLE (POSITIVE) STAINING

SIMPLE STAIN is an aqueous or alcohol solution of a
single basic dye
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 to the fixed smear for a certain
length of time and then washed off, and the slide is
dried and examined
Simple stains: methylene blue, carbolfuchsin, crystal
violet, and safranin

Cocci which has been stained with crystal violet

SIMPLE (NEGATIVE) STAINING

PROCEDURE:
After preparing a clean, greaseless slide, a small drop of
nigrosine is mixed with a small drop from a broth culture
or with a quantity of dry material
The drop is spread across the slide using the edge of
another slide as a spreader. This same procedure is used
for blood smears.
After air drying, the smear is observed using the high dry
lens (40x), or oil immersion if desired
The background should be blue-grey
Bacteria will be evidenced by the absence of any color

Negatively Stained Cocci

Negatively stained Bacillus: (A) Vegetative Cell

INDIA INK PREPARATION

It is useful to detect Cryptococcus neoformans in
cerebrospinal fluid (CSF)
A drop of centrifuged CSF is mixed with a drop of India ink on
a microscope slide beneath a glass cover slip
Cryptococci are identified by their large transparent capsules
that displace the India ink particles

CAPSULES are highly ordered polymers of sugars and proteins that surround
some bacterial cells.
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. Accordingly, capsule stains are not heatfixed, and water is never used to rinse.
To demonstrate the presence of capsules, we can mix the bacteria in a
solution containing a fine colloidal suspension of colored particles (usually
India ink, nigrosin) to provide a dark 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.

Capsules have a role in adherence,

protection (they inhibit ingestion and killing
by phagocytes), securing nutrients, and
cell-to-cell recognition)

CAPSULE STAINING (2)

The primary stain applied is crystal violet, which stains both the bacterial
cell and the surrounding capsule.
A 20% copper sulfate solution is then applied, which serves a dual function
as both decolorizer and counterstain. It removes and replaces the crystal
violet in the capsule only.
At the end of the staining procedure, the capsule appears as a faint blue or
white halo around a purple cell.

ENDOSPORES
These are a dormant form of a bacterial cell produced by certain bacteria when
starved; the actively growing form of the cell is referred to as vegetative. The spore is
resistant to adverse conditions (including high temperatures and organic solvents).
The spore cytoplasm is dehydrated and contains calcium dipicolinate (dipicolinic acid)
which is involved in the heat resistance of the spore. Spores are commonly found in
the genera Bacillus and Clostridium.

The Schaeffer-Fulton stain is a technique

designed to isolate endospores by staining
any present endospores green, and any
other bacterial bodies red. The green stain
is malachite green, and the counterstain is
safranin, which dyes any other bacterial
bodies red.

Electron micrograph of a bacterial endospore. The spore has a core wall of unique
peptidoglycan surrounded by several layers, including the cortex, the spore coat and the
exosporium. The dehydrated core contains the bacterial chromosome and a few ribosomes and
enzymes to jump-start protein synthesis and metabolism during germination.

THE FLAGELLA STAIN

Flagella are too fine (12-30 nm in diameter) to be visible in

the light microscope

Their presence and arrangement can be observed by treating

the cells with an unstable colloidal suspension of tannic acid
salts, causing a heavy precipitate to form on the cell walls
and flagella

In this manner, the apparent diameter of flagella is increased

to such an extent that subsequent staining with basic
fuchsin makes the flagella visible in the light microscope

Examples of bacterial flagella

arrangement schemes.
A-Monotrichous;
BLophotrichous;
CAmphitrichous; D-Peritrichous;

THE GRAM STAIN AS A DIFFERENTIAL STAINING

PROCEDURE
The principal stain used for microscopic examination of
bacteria
The Gram stain broadly differentiates bacteria into
Gram-positive and Gram-negative groups; a few
organisms are consistently Gram-variable
Gram-positive and Gram-negative organisms differ drastically
in the organization of the structures outside the plasma
membrane but below the capsule: in Gram-negative
organisms these structures constitute the cell envelope,
whereas in Gram-positive organisms they are called a cell
wall
The

cell

wall

is

the

outermost,

multilayered

THE BACTERIAL CELL WALL

It is composed of an inner layer of PEPTIDOGLYCAN (also known
as mucopeptide or murein) surrounded by an outer membrane
that varies in thickness and chemical composition depending on
the bacterial type
The peptidoglycan provides structural support, protects against
osmotic pressure and maintains the characteristic shape of the
bacterial cells
Additionally, it is the site of action beta-lactam antibiotics including
penicillins and cephalosporins
The primary chemical structures of peptidoglycans of both Grampositive and Gram-negative bacteria have been established; they
consist of a glycan backbone of repeating groups of Nacetylglucosamine and N-acetylmuramic acid connected by a beta1,4-glycoside bond

 Tetrapeptides
of
L-alanine-D-isoglutamic
acid-L-lysine
(or
diaminopimelic acid)-n-alanine are linked through the carboxyl
group by amide linkage of muramic acid residues of the glycan
chains;
the D-alanine residues are directly cross-linked to the e-amino
group of lysine or diaminopimelic acid on a neighboring
tetrapeptide, or they are linked by a peptide bridge
In S aureus peptidoglycan, a glycine pentapeptide bridge links the
two adjacent peptide structures. The extent of direct or peptidebridge cross-linking varies from one peptidoglycan to another.
The staphylococcal peptidoglycan is highly cross-linked, whereas
that of E coli is much less so, and has a more open peptidoglycan
mesh. The diamino acid providing the e-amino group for crosslinking is lysine or diaminopimelic acid, the latter being uniformly
present in Gram-negative peptidoglycans.

 The -1,4 glycosidic bond between N-acetylmuramic acid and Nacetylglucosamine is specifically cleaved by the bacteriolytic
enzyme lysozyme.
Widely distributed in nature, this enzyme is present in human
tissues and secretions (tears, mucus, saliva) and can cause
complete digestion of the peptidoglycan walls of sensitive
organisms. This enzyme contributes to the natural resistance of the
human host to bacterial infection.
Lysozyme-treated bacteria may swell and rupture as a result of the
entrance of water into the cells, which have a high internal osmotic
pressure
When lysozyme is allowed to digest the cell wall of Gram-positive
bacteria suspended in an osmotic stabilizer (such as sucrose),
protoplasts are formed. These protoplasts are able to survive and
continue to grow on suitable media in the wall-less state.
Gram-negative bacteria treated similarly produce spheroplasts,
which retain much of the outer membrane structure

L-ALANINE
D-GLUTAMIC ACID
L-LYSINE/DIAMINOPIMELIC ACID
D-ALANINE
D-ALANINE

PEPTIDOGLYCAN

MURAMIC ACID
GLUCOSAMINE

THE CELL WALLS OF GRAM-POSITIVE

AND GRAM-NEGATIVE BACTERIA
The peptidoglycan layer is much thicker in Gram-positive
than in Gram-negative bacteria
Some Gram-positive bacteria also have a layer of teichoic acid
outside the peptydoglikan, whereas Gram-negative do not
In contrast, the Gram-negative microorganisms have a complex
outer
layer/outer
membrane
(OM)
consisting
of
lipopolysaccharide, lipoprotein, and phospholipid. Lying between
the outer membrane layer and the cytoplasmic membrane in
Gram-negative bacteria is the periplasmic space (in some
species it is the site of enzymes, called beta-lactamases that
degrade beta-lactam antibiotics).
The OM has several specialized functions. Its strong
negative charge is an important factor in evading
phagocytosis and the actions of complement
The OM also provides a barrier to certain antibiotics (for
example, penicillin), digestive enzymes such as lysozyme,
detergents, heavy metals, bile salts, and certain dyes

 In Gram-negative bacteria, the cell wall contains endotoxin, a

lipopolysaccharide (LPS)
The LPS of all Gram-negative species are also called endotoxins,
thereby distinguishing these cell-bound, heat-stable toxins from
heat-labile, protein exotoxins secreted into culture media
Endotoxins possess an array of powerful biologic activities
and play an important role in the pathogenesis of many
Gram-negative bacterial infections
The endotoxic properties of LPS reside largely in the lipid A
components
Usually, the LPS molecules have three regions:
the LIPID A structure required for insertion in the outer leaflet of
the outer membrane bilayer; Lipid A consists of a phosphorylated
N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA)
attached.
a covalently attached CORE composed of 2-keto-3 deoxyoctonic
acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose,
and galactose;
polysaccharide chains linked to the core. The polysaccharide
chains constitute the O-ANTIGENS of the Gram-negative bacteria,

THE O POLYSACCHARIDE AND VIRULENCE

O-specific antigens could allow organisms to adhere specifically to
certain tissues, especially epithelial tissues
The O antigens probably allow resistance to phagocytes
The hydrophilic O polysaccharides could act as water-solubilizing
carriers for toxic Lipid A
The O antigens could provide protection from damaging
reactions with antibody and complement
The O-polysaccharide or O antigen is the basis of antigenic
variation among many important Gram-negative pathogens
including E. coli, Salmonella and Vibrio cholerae. Antigenic
variation guarantees the existence of multiple serotypes of the
bacterium, so that it is afforded multiple opportunities to infect its
host if it can bypass the immune response against a different
serotype.
Virulence, and the property of "smoothness", is associated with an intact O
polysaccharide. The involvement of the polysaccharide chain in virulence is
shown by the fact that small changes in the sugar sequences in the side
chains of LPS result in major changes in virulence.

 In humans, LPS binds to a lipid binding protein (LBP) in the serum which
transfers it to CD14 on the cell membrane, which in turn transfers it to
another non-anchored protein, MD2, which associates with Toll-like
receptor-4
(TLR4).
This
triggers
the
signaling
cascade
for
macrophage/endothelial cells to secrete pro-inflammatory cytokines and
nitric oxide that lead to characteristic "endotoxic shock". CD14 and TLR4
are present on several cells of the immunological system cells, including
macrophages and dendritic cells. In monocytes and macrophages, three
types of events are triggered during their interaction with LPS:
1. Production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor
(TNF) and platelet-activating factor. These, in turn, stimulate production of
prostaglandins and leukotrienes. These are powerful mediators of
inflammation and septic shock that accompanies endotoxin toxemia. LPS
activates macrophages to enhanced phagocytosis and cytotoxicity.
Macrophages are stimulated to produce and release lysosomal enzymes, IL1 ("endogenous pyrogen"), and tumor necrosis factor (TNFalpha), as well as
other cytokines and mediators.
2. Activation of the complement cascade. C3a and C5a cause
histamine release (leading to vasodilation) and affect neutrophil chemotaxis
and accumulation. The result is inflammation.
3. Activation of the coagulation cascade. Initial activation of Hageman
factor (blood-clotting Factor XII) can activate several humoral systems
resulting in:
a. coagulation: a blood clotting cascade that leads to coagulation,
thrombosis, acute disseminated intravascular coagulation, which depletes
platelets and various clotting factors resulting in internal bleeding.
b. activation of the complement alternative pathway (as above, which leads
to inflammation)
c. plasmin activation which leads to fibrinolysis and hemorrhaging.
d. kinin activation releases bradykinins and other vasoactive peptides
which causes hypotension.
THE NET EFFECT IS TO INDUCE INFLAMMATION, INTRAVASCULAR

 The outer membrane possesses several major outer membrane

proteins; the most abundant is called porin
The assembled subunits of porin form a channel that limits
the passage of hydrophilic molecules across the outer membrane
barrier to those having molecular weights that are usually less than
600 to 700
Evidence also suggests that hydrophobic pathways exist across the
outer membrane and are partly responsible for the differential
penetration and effectiveness of certain beta-lactam antibiotics
(ampicillin, cephalosporins) that are active against various Gramnegative bacteria
Several outer membrane proteins are also involved in the
specific uptake of metabolites (maltose, vitamin B12,
nucleosides) and iron from the medium
Thus, outer membranes of the Gram-negative bacteria provide a
selective barrier to external molecules and thereby prevent the
loss of metabolite-binding proteins and hydrolytic enzymes
(nucleases, alkaline phosphatase) found in the periplasmic space

 The periplasmic space is the region between the outer surface of

the inner (plasma) membrane and the inner surface of the outer
membrane
Thus, Gram-negative bacteria have a cellular compartment that
has no equivalent in Gram-positive organisms
In addition to the hydrolytic enzymes, the periplasmic space holds
binding proteins (proteins that specifically bind sugars, amino
acids, and inorganic ions) involved in membrane transport and
chemotactic receptor activities
Moreover, plasmid-encoded beta-lactamases and aminoglycosidemodifying enzymes (phosphorylation or adenylation) in the
periplasmic space produce antibiotic resistance by degrading or
modifying an antibiotic in transit to its target sites on the
membrane (penicillin-binding proteins) or on the ribosomes
(aminoglycosides)

GRAM NEGATIVE CELL ENVELOPE

Outer Membrane

Porin

Braun lipoprotein

Inner (cytoplasmic)
membrane

Cytoplasm
43

Lipopolysaccharide

Peptidoglycan

 Wall teichoic acids are found only in certain Gram-positive bacteria

(such as staphylococci, streptococci, lactobacilli, and Bacillus spp); so
far, they have not been found in gram-negative organisms
Teichoic acids are polyol phosphate polymers, with either ribitol or
glycerol linked by phosphodiester bonds, bearing a strong negative
charge; substituent groups on the polyol chains can include D-alanine
(ester linked), N-acetylglucosamine, N-acetylgalactosamine, and
glucose; the substituent is characteristic for the teichoic acid from a
particular bacterial species and can act as a specific antigenic
determinant.
These acids are covalently linked to the peptidoglycan. Teichoic
acids are antigenic and induce species-specific antibodies. In
staphylococci, the acids mediate adherence of the
microorganism to mucosal cells. They may bind and regulate the
movement of cations (positive ions) into and out of the cell.
Some polymers of glycerol teichoic acid penetrate the peptidoglycan
layer and are covalently linked to the lipid in the cytoplasmic
membrane, in which case they are called lipoteichoic acid; others
anchor to the muramic acid of the peptidoglycan. These highly
negatively charged polymers of the bacterial wall can serve as a
cation-sequestering mechanism. They are antigenic, cytotoxic and
adhesins (S. pyogenes).

GRAM POSITIVE CELL ENVELOPE

Lipoteichoic
acid
r

Peptidoglycan-teichoic acid
r

r
r

Cytoplasmic membrane

Cytoplasm

45

r
r

r
r

THE GRAM STAINING PROCEDURE INVOLVES

FOUR STEPS
1. THE CRYSTAL VIOLET DYE STAINS ALL CELLS BLUE
2. THE IODINE SOLUTION IS ADDED TO FORM A CRYSTAL VIOLETIODINE COMPLEX: ALL CELLS CONTINUE TO APPEAR BLUE
3. THE ORGANIC SOLVENT: ACETONE/ETHANOL EXTRACTS THE
BLUE DYE COMPLEX FROM THE LIPID-RICH, THIN-WALLED GRAMNEGATIVE BACTERIA TO A GREATER DEGREE THAN FROM THE
LIPID-POOR, THICK WALLED GRAM-POSITIVE BACTERIA. THE
GRAM-NEGATIVE MICROORGANISMS APPEAR COLORLESS;
THE GRAM-POSITIVE BACTERIA REMAIN BLUE
4. THE RED DYE SAFRANIN STAINS THE DECOLORIZED GRAMNEGATIVE CELLS RED; THE GRAM-POSITIVE BACTERIA REMAIN
BLUE

 Both Gram-positive and Gram-negative bacteria take up the

same amounts of crystal violet (CV) and iodine (I).
Inside the cells, the CV and I combine to form CV-I complex.
This complex is larger than the CV 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.
The application of alcohol dehydrates the peptidoglycan of
Gram-positive cells to make it more impermeable to the CVI.
Consequently, Gram-positive cells retain the color of the
crystal violet dye.
In Gram-negative cells, however, the alcohol wash disrupts
the outer LPS layer, dissolves the OM and even leaves small
holes in the thin peptidoglycan layer through which CV-I
diffuse.
The CV-I complex is washed out through the thin layer of
peptidoglycan.
As a result, Gram-negative cells are colorless, until
counterstained with a red dye, after which they are pink.

VIRTUALLY ALL OF THE EUBACTERIA WITH

WALLS
CAN BE ASSIGNED A GRAM RESPONSE
THE FEW EXCEPTIONS INCLUDE SOME MEDICALLY
IMPORTANT ORGANISMS:

PREPARING SMEARS FOR STAINING

Because most microorganisms appear almost colorless when viewed
through a standard light microscope, we often must prepare them for
observation. One of the ways this can be done is by staining.
Staining simply means coloring the microorganisms with a dye that
emphasizes certain structures
DIFFERENTIAL STAINS REACT DIFFERENTLY WITH DIFFERENT KINDS
OF BACTERIA AND THUS CAN BE USED TO DISTINGUISH AMONG
THEM
THE DIFFERENTIAL STAINS MOST FREQUENTLY USED FOR BACTERIA
ARE THE GRAM STAIN AND THE ACID-FAST STAIN
Before the microorganisms can be stained, however, they must be fixed
(attached) to the microscope slide. Fixing simultaneously kills the bacteria
and attaches them to the slide.
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 1 minute.
Air drying and flaming fix the microorganisms to the slide
Stain is applied and then washed off with water; then the slide is blotted
with absorbent paper
The stained microorganisms are now ready for microscopic examination

If the specimen is received on a swab, gently roll the swab on a

clean glass slide to avoid rupturing host cells. Allow to air dry.

If the specimen is a fluid, place a drop of fluid on a clean glass slide

and allow to air dry.
*In both cases, the specimen is fixed to the glass slide by passing it
a few times over a flame.

STAINING PROCEDURE
Step 1. Flood the slide with crystal violet for 1 minute. Rinse with water.
Step 2. Flood the slide with Gram's iodine for 1 minute. Rinse with water.
Step 3. Decolorize the slides by gently rinsing with an acetone - alcohol solution for 1 to 10 seconds dependent
on content of acetone in solution. Rinse with water.
Step 4. Flood the slide with saffranin, the counterstain, for 1 minute. Rinse with water and dry air.

THEORY: CELL WALL CONSTRUCTION

Gram positive and gram negative bacteria stain differently because of the structure of their cell walls.
Gram - positive bacteria and yeasts stain purple.
Gram negative bacteria and host cells stain pink.

TYPICAL GRAM STAINS (GRAM POSITIVE BACTERIA)

Gram-positive cocci
Clusters: usually characteristic of Staphylococcus spp., such as
S. aureus

Chains: usually characteristic of Streptococcus spp., such as

S. pneumoniae, B group streptococci

Tetrads: usually characteristic of Micrococcus spp.

Gram positive bacilli

Thick : usually characteristic of Clostridium spp., such as C. perfringens,
C. septicum, C. tetanomorphum
Thin: usually characteristic of Listeria spp.

Branched: usually characteristic of Actinomycetes and Nocardia , such as

A. israelii Note: Mycobacteria are not branched, and often stain poorly with
Gram stain. Some mycobacteria do appear as Gram-positive rods, not unlike
diptheroids, sometimes with Gram-positive beading.

TYPICAL GRAM STAINS (GRAM NEGATIVE BACTERIA)

Diplococci: usually characteristic of Neiseria spp., such as N. meningitidis
In addition, Moraxella spp. and Acinetobacter spp.are often diplococcal in
morphology. Acinetobacter can be pleomorphic, and sometimes appear as
Gram-positive cocci.

Coccobacilli: usually characteristic of Acinetobacter spp., which can be either

Gram-positive or Gram-negative, and is often Gram-variable.

Thin rods: usually characteristic of enterobacteriaceae, such as E. coli

Coccobacilli: usually characteristic of Haemophilus spp., such as

H. influenzae

Curved:usually characteristic of Vibrio spp.; Campylobacter spp., such as V.

cholerae, C. jejuni;

Thin needle shape:usually characteristic of Fusobacterium spp.

Urethral smear, Neisseria gonorrhoeae

Pus smear (wound), Staphylococcus aureus

Tissue smear, Clostridium perfringens

Chains and pairs of Gram-positive cocci, characteristic

of streptococci. Enterococci can also look like this.
(Source: Susan D. Caston, Clinical Microbiology Laboratory of the Hospital of the
University of Pennsylvania)

Sputum smear, Staphylococcus & Streptococcus

pneumoniae

Sputum smear, Streptococcus pneumoniae/PMN

Streptococcus pneumoniae appear as gram-positive

diplococci, although some very short chains may be
seen. The cells are somewhat tapered at the ends giving
the "lancet" shaped appearance. (Source: Susan D. Caston, Clinical
Microbiology Laboratory of the Hospital of the University of Pennsylvania )

Urine smear,Escherichia coli/PMN

Acinetobacter- A major characteristic of this species

is its gram stain morphology: they appear as gramnegative coccobacilli but are frequently confused
with gram-negative diplococci characteristic of
Neisseria sp. Acinetobacter sp. are nonfermentative,
non-motile and oxidase-negative. The most common
species causing human infection is Acinetobacter
baumanii (previous called A. calcoaceticus var
anitratus).
Campylobacter
sp.
are
microaerophillic,
gramnegative, spiral bacteria and usually have the
appearance of "seagulls". A major characteristic of this
species is the requirement for microaerobic conditions
for growth on laboratory media (5% O2). The most
common species isolated from human infections is
Campylobacter jejuni subsp. jejuni.

Pseudomonas aeruginosa is a strictly aerobic, oxidase

positive, gram-negative nonfermentative bacterium. The
Gram-stain appearance is not particularly characteristic
although rods are somewhat thinner than those seen for
the enteric-like bacteria. Mucoid strains that produce an
extracellular polysaccharide are frequently isolated from
patients with cystic fibrosis and this capsular material can
be seen in the photo.
(Source: Susan D. Caston, Clinical Microbiology Laboratory of the Hospital of the University of
Pennsylvania)

Gram Stain from Neisseria gonorrheae infection Urethral

discharge from a male patient. Stain shows gramnegative diplococci both intracellular and extracellular to
a polymorphonuclear leukocyte or puss cell. In a
symptomatic male patient, this Gram stain finding is
considered diagnostic of the sexually transmitted
disease caused by Neisseria gonorrheae. In female
patients, one cannot use this type of finding as
diagnostic of N. gonorrheae infection because the female
genital tract may contain commensal Neisseria species.

Bacillus anthracis in cerebrospinal fluid)

WHY SHOULD PHYSICIANS

EXAMINE GRAM-STAINED SMEARS?
Determine
the
adequacy
of
the
specimen
for
culture
The Gram-stained smear is useful in judging the adequacy of the specimen
obtained. In sputum and urine specimens, for example, a poorly collected or
contaminated specimen can be recognized by the presence of many
epithelial cells in the smear. Instead of spending laboratory effort and the
patient's money on a culture that may yield worthless or misleading
information, a better specimen should be obtained.
Make a presumptive etiologic diagnosis and early clinical decisions
Immediate examination of a Gram-stained smear of material from the
infection site can often provide important data on which to base early
clinical decisions, prior to the availability of culture results. In certain rapidly
progressive infections such as gas gangrene or acute meningitis, the Gramstained smear may allow a presumptive etiologic diagnosis to be made
within minutes, whereas culture results usually are not available for one to
two days. Information gleaned from the Gram-stained preparation rarely
permits definitive identification of organisms, but usually narrows the
possibilities in diseases such as gas gangrene, pneumonia or meningitis,
that have a variety of causative agents. Early diagnostic information
obtained from Gram-stained smears often allows the physician to prescribe
narrow-spectrum antimicrobial therapy, thereby reducing the risk of
toxicity, superinfection, and the expense of broad-spectrum "polypharmacy."

WHY SHOULD PHYSICIANS

EXAMINE GRAM-STAINED SMEARS?
Suggest
a
need
for
non-routine
laboratory
procedure
The Gram-stained smear may indicate a need for laboratory procedures not
routinely employed, such as anaerobic and fungal cultures or special
staining techniques, without which the organism might be missed.
Help
make
accurate
interpretation
of
culture
results
The Gram-stained smear may provide clues that are important in
interpreting culture results. In patients who have already received
antibiotics, the direct smear may show organisms that will not grow in
culture. Moreover, in certain infections, such as Vincent's angina
(associated with fusobacteria and spirochetes), the organisms are not
identifiable by the culture techniques employed in most diagnostic
microbiology laboratories, and the Gram-stained smear together with the
clinical findings form the basis for diagnosis.
Provide a better insight into the nature of the current infection
In most cases, the Gram-stained smear may reflect what is happening in
the patient better than a culture. In mixed infections, due to several types
of aerobic and anaerobic bacteria, the smear may indicate the relative
abundance of different bacteria, whereas in culture, the bacteria may grow
at different rates, giving a false quantitative picture. Estimates made
regarding the total quantity of organisms present can sometimes be made
from the Gram-stained smear.

WHY SHOULD PHYSICIANS

EXAMINE GRAM-STAINED SMEARS?
For all these reasons, in the diagnosis of patients with acute
infections, the decision to send specimens of SPUTUM, URINE,
CEREBROSPINAL FLUID, OR MATERIAL FROM WOUNDS OR
ABSCESSES for culture should automatically trigger a response to
first examine a Gram-stained smear. At times when Gram-staining
cannot be done immediately, as in the operating room, a smear
can be made on a clean glass slide and saved for later staining.
THERE ARE SOME SPECIMENS THAT ARE NOT SUITABLE
FOR ROUTINE GRAM STAINING
Such examples are routine THROAT AND STOOL SPECIMENS in
which the pathogen usually cannot be distinguished from the
plethora of normal flora.
Blood specimens are rarely Gram-stained (although in acutely septic
patients a Gram-stained smear of the buffy coat may be useful).

TO OBTAIN USEFUL INFORMATION FROM GRAM STAINED PREPARATIONS, YOU SHOULD:

Look for one or two types of bacteria near inflammatory cells: In acute infections,
keep in mind that diagnostic fields usually contain only one or two types of bacteria near
inflammatory cells. Exceptions to this include infections caused by leakage from heavily
colonized areas, e.g., peritonitis secondary to perforated diverticulum. The temptation to
interpret minor morphologic variations of a single organism as multiple types of organisms
should be resisted.
Ignore contaminating flora. Varied types of bacteria in great numbers are often found near
epithelial cells. These almost always are organisms that constitute the normal flora of the
contaminating cell source - for instance, mouth bacteria in sputum or vaginal flora in poorly
collected urine. Knowledge on the normal microbial flora in various anatomical sites may be
helpful.
Beware of artifacts. Bits of irregularly shaped Gram-positive material or precipitated stain
are easy to misconstrue as Gram-positive cocci. In areas with these artifacts, nearby
inflammatory cells are often underdecolorized. Organisms can, however, acquire a bizarre
appearance after exposure to antibiotics. Keep in mind that old, damaged, or antibiotictreated bacteria that are normally Gram-positive may appear Gram-variable or even Gramnegative, presumably because their cell walls are more permeable to the decolorizing agent.
The opposite does not hold true -- Gram-negative bacteria do not become Gram-positive in
appearance with age or damage. When repeated Gram stains suggest the presence of
organisms (often yeasts) that do not fit the clinical picture, consider the possibility that the
Gram-stain reagents have become contaminated.
Examine the background. Small, pleomorphic Gram-negative organisms, such as
Haemophilus or Bacteroides, are easily overlooked especially when Gram-positive bacteria
are present. Nocardia and Actinomyces often appear as weakly Gram-positive, small, frail,
branching rods that blend easily into the background. Since the Actinomyces species are
strict anaerobes and the Nocardia species can be further identified by their relatively acid-fast
properties, recognition of these forms on Gram-stained smear is most helpful to further
identification.
Look at more than one area of the smear. Although the first bacteria seen may fit one's
clinical expectation, they may not be typical of the infectious material a whole. Check that
they are indeed representative. Find more than one or two organisms before drawing any
conclusion. Even when they are scarce, more than one or two examples of a bacterial type
can usually be discovered in a thorough search. Making another smear is often more
productive than exhaustively going over and over the original one. When no additional
organisms can be detected, the significance of the limited observation should be assessed in
the context of the other clinical data. In a number of difficult clinical situations, such as early
or partially treated meningitis, low concentrations of organisms are not uncommon.

Crystal violet precipitate on epithelial cell:

May be confused with Gram positive cocci.

Crystal violet precipitate crystal on gram stain.

UNDERDECOLORIZED NEUTROPHILS

Part of this slide (pink area) has been overdecolorized

with the acetone alcohol giving the false impression of
gram negative rods being present.

ACID-FAST STAIN
This important differential stain binds strongly only to bacteria that have a waxy
material in their cell walls.
This method is used to identify all bacteria in the genus Mycobacterium, including two
important pathogens: Mycobacterium tuberculosis the causative agent of
tuberculosis, and Mycobacterium leprae - the causative agent of leprosy. This stain
is also used to identify the pathogenic strains of the genus Nocardia.
These microorganisms are "acid-fast", since they resist decolorization with acidalcohol after being stained with carbolfuchsin. This property is related to the high
concentration in the cell wall of lipids called mycolic acids.
The classic acid-fast procedure is the ZIEHL-NEELSEN STAIN
In this procedure, the red dye carbolfuchsin is applied to a fixed smear, and the
slide is gently heated for several minutes / heating enhances penetration and
retention of the dye /
Then the slide is cooled and washed with water.
The smear is next treated /decolorized/ with a 3% solution of hydrochloric acid in
alcohol, 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 counterstained with methylene blue; acid-fast microorganisms
appear red against a blue background.
PONDER-KINYOUN (COLD) ACID-FAST STAIN is a variant of this method, in which
a more concentrated fuchsin is used and heating is omitted.
Another variant of the acid-fast stain is the FLUOROCHROME STAIN, which uses a
fluorescent dye, auramine, or an auramine-rhodamine mixture followed by
decolorization with acid-alcohol. Acid-fast organisms retain the fluorescent stain,
which allows their visualization by fluorescence microscopy.

Acid Fast Cell Envelope

Mycolic acid lipids
Peptidoglycan-arabinogalactan-mycolic acid
r

r
r

r
r

Cytoplasmic membrane

Cytoplasm

67

r
r

r
r

Mycobacterial cell wall: 1-outer lipids, 2-mycolic acid, 3polysaccharides (arabinogalactan), 4-peptidoglycan, 5-plasma
membrane,
6-lipoarabinomannan (LAM), 7-phosphatidylinositol mannoside, 8cell wall skeleton

Pink: acid-fast bacteria (Mycobacterium bovis BCG)

Blue: non-acid-fast bacteria (Streptococcus mutans, for counter
stained)

FLUORESCENT MICROSCOPY
Certain dyes, called fluorochromes can be raised to a
higher energy level after absorbing ultraviolet (excitation)
light
When the dye molecules return to their normal, lower
energy state, they release excess energy in the form of
visible (fluorescent) light. This process is called
fluorescence.
In fluorescent microscopy the excitation light is emitted. An
excitation filter passes light of the desired wavelength to
excite the fluorochrome that has been used to stain the
specimen.
When observed through the ocular lens, fluorescing objects
appear brightly lit against a dark background.
The color of the fluorescent light depends on the dye and
light filters used
Which dye is used often depends on which organism is
being sought and the fluorescent method used

FLUORESCENT MICROSCOPY
Fluorescent staining techniques may be divided into two
general categories: fluorochroming (a fluorescent dye is
used alone) and immunofluorescence (fluorescent dyes
have been linked/conjugated to specific antibodies)
In FLUOROCHROMING a direct chemical interaction
occurs between the fluorescent dye and a component of
the bacterial cell. The use of a fluorescent dye enhances
contrast and amplifies the observers ability to detect
stained cells 10-fold greater than would be observed by
light microscopy. The most common fluorochroming
methods include acridine orange stain, auraminerhodamine stain, and calcofluor white stain.
The acridine orange binds to nucleic acid. This staining
method is used to confirm the presence of bacteria in
blood cultures when Gram stain results are difficult to
interpret or when the presence of bacteria is suspected but
not detected using light microscopy. Moreover, the stain
can be used to detect cell wall-deficient bacteria
(mycoplasmas) grown in culture. All microorganisms and
host cells will stain by this method and give a bright
orange fluorescence.
The waxy mycolic acids in the mycobacterial cell walls
have an affinity for auramine and rhodamine. The

More sensitive than an acid fast stain, this

auramine stain requires fluorescence microscopy.
Note the yellow-orange rod-like mycobacteria.

FLUORESCENT MICROSCOPY

Immunofluorescence is used to directly examine

patient specimens for bacteria that are difficult or slow
to grow (Legionella spp., Bordetella pertussis,
Chlamydia trachomatis) or to identify organisms
already grown in culture
Fluorescein isothiocyanate (FITC), which emits an
intense, apple green fluorescence, is most commonly
used for conjugation to antibodies

DFA technique to detect the Legionella antigen directly in patient specimens

Respiratory tract specimens are spread on a glass slide. A monoclonal antibody to
Legionella that is tagged with a fluorescein dye is added to the slide. If the antigen is
present, the antibody will bind and the outline of the bacilli can be detected by

Streptococcus pneumoniae in
spinal fluid

IFA reaction of a positive human serum

on Rickettsia rickettsii grown in chicken
yolk sacs, 400X

DARK-FIELD MICROSCOPY
By this method, the condenser does not allow light to pass
directly through the specimen but directs the light to hit the
specimen at an oblique angle
Only light that hits objects, such as microorganisms in the
specimen, will be deflected upward into the objective lens for
visualization. All other light that passes through the specimen
will miss the objective, thus making the background a dark
field.

The advantage of this method is that the resolving power of

darkfield microscopy is significantly improved compared
with that of brightfield microscopy (i.e., 0.02 m versus
0.2 m)

This method is useful for detecting certain bacteria directly in

patient specimens that, because of their thin dimensions,
cannot be seen by light microscopy and, because of their
physiology, are difficult to grow in culture
Dark-field microscopy is used to detect spirochetes
(Treponema pallidum the causative agent of syphilis), which
will appear extremely bright against a black field

Electron Microscopy

Unlike other forms of microscopy, magnetic coils (rather than

lenses) are used in electron microscopes to direct a beam of
electrons from a tungsten filament through a specimen and onto
a screen

Because a much shorter wavelength of light is used,

magnification and resolution are improved dramatically

Individual viral particles (as opposed to viral inclusion bodies) can

be seen with electron microscopy

Samples are usually stained or coated with metal ions to

create contrast

There are two types of electron microscopes: transmission

electron microscopes, in which electrons such as light pass
directly through the specimen, and scanning electron
microscopes, in which electrons bounce off the surface of the
specimen at an angle, and a three-dimensional picture is
produced.

Fungal and parasitic stains

The smallest fungi are the size of large bacteria and all parasitic
forms are larger. This allows detection in simple wet mount
preparations often without staining. Fungi in sputum or body
fluids can be seen by mixing the specimen with a potassium
hydroxide solution (to dissolve debris) and viewing with a
medium power lens. The use of simple stains or the fluorescent
calcofluor white improves the sensitivity of detection. Another
technique is to mix the specimen with India ink, which outlines
the fungal cells

Detection of the cysts and eggs of parasites requires a

concentration procedure if the specimen is stool, but once done
they can be visualized with a simple iodine stain

Iodine-stained parasite eggs. Two eggs of the intestinal fluke Clonorchis

sinensis are present in this stool specimen.