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 patient’s 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 organism performed in vitro in order to determine whether it is susceptible to antibiotics

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 collected and the non-pathogen is either inhibited by the use of selective culture methods or discounted in interpretation of culture results. Examples are throat and stool.

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 organism’s clinical significance; bacterial

contaminants usually are not present in patient specimens at sufficiently high numbers (>10⁵ 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 non-motile cocci such as Staphylococcus, Streptococcus, Micrococcus. However, some bacteria are flagellated and exhibit translational movement as well. Truly motile organisms will zip across the microscope field.

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  (B)  Endospore

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 heat-fixed, 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.

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.

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.

ENDOSPORES

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.

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; B-Lophotrichous; C-Amphitrichous; 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 component common to all bacteria (except Mycoplasma species which are bounded by a cell membrane, not a cell wall)

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 Gram-positive and Gram-negative bacteria have been established; they consist of a glycan backbone of repeating groups of N-acetylglucosamine and N-acetylmuramic acid connected by a beta-1,4-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 peptide-bridge 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 cross-linking is lysine or diaminopimelic acid, the latter being uniformly present in Gram-negative peptidoglycans.

The ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine 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

PEPTIDOGLYCANPEPTIDOGLYCAN

MURAMIC ACID

GLUCOSAMINE

L-ALANINED-GLUTAMIC ACIDL-LYSINE/DIAMINOPIMELIC ACIDD-ALANINED-ALANINE

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, and the individual monosaccharide constituents confer serologic specificity on these components.

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, IL-1 ("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 COAGULATION, HEMORRHAGE, AND SHOCK.

LPS also acts as a B cell mitogen, stimulating the polyclonal differentiation and multiplication of B-cells and the secretion of immunoglobulins, especially IgG and IgM.

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 Gram-negative 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 aminoglycoside-modifying 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)

43

CytoplasmCytoplasm

Inner (cytoplasmic) membrane

Ou

ter M

emb

rane

LipopolysaccharidePorin

Braun lipoprotein

GRAM NEGATIVE CELL ENVELOPEGRAM NEGATIVE CELL ENVELOPE

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).

45

r r rrr

r

GRAM POSITIVE CELL ENVELOPEGRAM POSITIVE CELL ENVELOPE

CytoplasmCytoplasm

rrrr

Lipoteichoic acid

Peptidoglycan-teichoic acid

Cytoplasmic membrane

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 VIOLET-IODINE COMPLEX: ALL CELLS CONTINUE TO APPEAR BLUE

3. THE ORGANIC SOLVENT: ACETONE/ETHANOL EXTRACTS THE BLUE DYE COMPLEX FROM THE LIPID-RICH, THIN-WALLED GRAM-NEGATIVE 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 GRAM-NEGATIVE 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 CV-I.

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 cocciClusters: 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 bacilliThick : 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. meningitidisIn 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

Urine smear, Escherichia coli/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)

Acinetobacter- A major characteristic of this species is its gram stain morphology: they appear as gram-negative 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, gram-negative, 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 gram-negative 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 PHYSICIANSEXAMINE GRAM-STAINED SMEARS?

Determine the adequacy of the specimen for cultureThe 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 decisionsImmediate 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 Gram-stained 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 "poly-pharmacy."

WHY SHOULD PHYSICIANSEXAMINE GRAM-STAINED SMEARS?

Suggest a need for non-routine laboratory procedureThe 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 resultsThe 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 infectionIn 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 PHYSICIANSEXAMINE 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 antibiotic-treated bacteria that are normally Gram-positive may appear Gram-variable or even Gram-negative, 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.

Don't overinterpret what is seen. The natural tendency is to leap to more specific interpretations than the Gram-stained smear warrant. To cite one pitfall, all Gram-positive cocci, including staphylococci, can occur in pairs and short chains. Thus, the impulse to conclude, for example, that all paired cocci are pneumococci can lead to clinical errors. The description should be limited to what is actually seen -- Gram-positive cocci in pairs. Then, with the recognition that morphology doesn't necessarily establish a specific etiology, the observation can be interpreted in light of the other clinical findings. This is why the physician's judgment is vital in evaluating Gram-stained smears.

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 acid-alcohol 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.

67

r r rrr

r

Acid Fast Cell EnvelopeAcid Fast Cell Envelope

CytoplasmCytoplasm

rrrr

Peptidoglycan-arabinogalactan-mycolic acid

Cytoplasmic membrane

Mycolic acid lipids

Mycobacterial cell wall: 1-outer lipids, 2-mycolic acid, 3-polysaccharides (arabinogalactan), 4-peptidoglycan, 5-plasma membrane, 6-lipoarabinomannan (LAM), 7-phosphatidylinositol mannoside, 8-cell 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 observer’s ability to detect stained cells 10-fold greater than would be observed by light microscopy. The most common fluorochroming methods include acridine orange stain, auramine-rhodamine 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 mycobacterial cells appear bright yellow or orange against a greenish background. This method can be used to enhance detection of mycobacteria directly in patient specimens and for initial characterization of cells grown in culture.

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 specimensRespiratory 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 microscopy under UV light.

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 μm versus 0.2 μm)

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.