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 CHAPTER 4 MICROSCOPY AND IN VITRO CULTURE T he foundation of microbiology was established in 1676 when Anton van Leeuwenhoek, using one of his early microscopes, observed bacteria in water....

 CHAPTER 4 MICROSCOPY AND IN VITRO CULTURE T he foundation of microbiology was established in 1676 when Anton van Leeuwenhoek, using one of his early microscopes, observed bacteria in water. It was not until to magnify the image of the specimen. In brightfield micros- copy the specimen is visualized by transillumination, with light passing up through the condenser to the specimen. The almost 200 years later that Pasteur was able to grow bacteria image is then magnified, first by the objective lens and then in the laboratory in a culture medium consisting of yeast by the ocular lens. The total magnification of the image is the extract, sugar, and ammonium salts. In 1881, Hesse used product of the magnifications of the objective and ocular agar from his wife’s kitchen to solidify the medium that then lenses. Three different objective lenses are commonly used: permitted the growth of macroscopic colonies of bacteria. low power (10-fold magnification), which can be used to Over the years, microbiologists have returned to the kitchen scan a specimen; high dry (40-fold), which is used to look to create hundreds of culture media that are now routinely for large microbes such as parasites and filamentous fungi; used in all clinical microbiology laboratories. Although tests and oil immersion (100-fold), which is used to observe bac- that rapidly detect microbial antigens and nucleic acid–based teria, yeasts (single-cell stage of fungi), and the morphologic molecular assays have replaced microscopy and culture details of larger organisms and cells. Ocular lenses can methods for the detection of many organisms, the ability to further magnify the image (generally 10-fold to 15-fold). observe microbes by microscopy and grow microbes in the The limitation of brightfield microscopy is the resolution laboratory remains an important procedure in clinical labo- of the image (i.e., the ability to distinguish that two objects ratories. For many diseases, these techniques remain the are separate and not one). The resolving power of a micro- definitive methods to identify the cause of an infection. This scope is determined by the wavelength of light used to illu- chapter will provide an overview of the most commonly used minate the subject and the angle of light entering the objective techniques for microscopy and culture, and more specific lens (referred to as the numerical aperture). The resolving details will be presented in the chapters devoted to labora- power is greatest when oil is placed between the objective tory diagnosis in the individual organism sections. lens (typically the 100× lens) and the specimen, because oil reduces the dispersion of light. The best brightfield micro- scopes have a resolving power of approximately 0.2 μm, Microscopy which allows most bacteria, but not viruses, to be visualized. Although most bacteria and larger microorganisms can be In general, microscopy is used in microbiology for two basic seen with brightfield microscopy, the refractive indices of purposes: the initial detection of microbes and the prelimi- the organisms and background are similar. Thus organisms nary or definitive identification of microbes. The micro- must be stained with a dye so they can be observed, or an scopic examination of clinical specimens is used to detect alternative microscopic method must be used. bacterial cells, fungal elements, parasites (eggs, larvae, or adult forms), and viral inclusions present in infected cells. Darkfield Microscopy Characteristic morphologic properties can be used for the The same objective and ocular lenses used in brightfield preliminary identification of most bacteria and are used microscopes are used in darkfield microscopes; however, a for the definitive identification of many fungi and parasites. special condenser is used that prevents transmitted light The microscopic detection of organisms stained with anti- from directly illuminating the specimen. Only oblique scat- bodies labeled with fluorescent dyes or other markers has tered light reaches the specimen and passes into the lens proved to be very useful for the specific identification of systems, which causes the specimen to be brightly illumi- many organisms. Five general microscopic methods are nated against a black background. The advantage of this used (Box 4-1). method is that the resolving power of darkfield microscopy is significantly improved compared with that of brightfield Microscopic Methods microscopy (i.e., 0.02 μm versus 0.2 μm) and makes it pos- Brightfield (Light) Microscopy sible to detect extremely thin bacteria such as Treponema The basic components of light microscopes consist of a light pallidum (etiologic agent of syphilis) and Leptospira spp. source used to illuminate the specimen positioned on a (leptospirosis). The disadvantage of this method is that light stage, a condenser used to focus the light on the specimen, passes around rather than through organisms, making it dif- and two lens systems (objective lens and ocular lens) used ficult to study their internal structure. 16  CHAPTER 4 MICROSCOPY AND IN VITRO CULTURE 17 produced. Today, electron microscopy is used more as a Box 4-1 Microscopic Methods research tool than a diagnostic aid, with highly sensitive and Brightfield (light) microscopy specific nucleic acid amplification assays the primary diag- Darkfield microscopy nostic test in current use. Phase-contrast microscopy Fluorescent microscopy Examination Methods Electron microscopy Clinical specimens or suspensions of microorganisms can be placed on a glass slide and examined under the microscope (i.e., direct examination of a wet mount). Although large organisms (e.g., fungal elements, parasites) and cellular Phase-Contrast Microscopy material can be seen using this method, analysis of the inter- Phase-contrast microscopy enables the internal details of nal detail is often difficult. Phase-contrast microscopy can microbes to be examined. In this form of microscopy, as overcome some of these problems; alternatively, the speci- parallel beams of light are passed through objects of different men or organism can be stained by a variety of methods densities, the wavelength of one beam moves out of “phase” (Table 4-1). relative to the other beam of light (i.e., the beam moving through the more dense material is retarded more than the Direct Examination other beam). Through the use of annular rings in the con- Direct examination methods are the simplest for preparing denser and the objective lens, the differences in phase are samples for microscopic examination. The sample can be amplified so that in-phase light appears brighter than out- suspended in water or saline (wet mount), mixed with alkali of-phase light. This creates a three-dimensional image of the to dissolve background material (potassium hydroxide organism or specimen and permits more detailed analysis of [KOH] method), or mixed with a combination of alkali and the internal structures. a contrasting dye (e.g., lactophenol cotton blue, iodine). The dyes nonspecifically stain the cellular material, increas- Fluorescent Microscopy ing the contrast with the background, and permit examina- Some compounds called fluorochromes can absorb short- tion of the detailed structures. A variation is the India ink wavelength ultraviolet or ultrablue light and emit energy at method, in which the ink darkens the background rather a higher visible wavelength. Although some microorganisms than the cell. This method is used to detect capsules sur- show natural fluorescence (autofluorescence), fluorescent rounding organisms, such as the yeast Cryptococcus (the dye microscopy typically involves staining organisms with fluo- is excluded by the capsule, creating a clear halo around the rescent dyes and then examining them with a specially yeast cell) and encapsulated Bacillus anthracis. designed fluorescent microscope. The microscope uses a high-pressure mercury, halogen, or xenon vapor lamp that Differential Stains emits a shorter wavelength of light than that emitted by A variety of differential stains are used to stain specific organ- traditional brightfield microscopes. A series of filters are isms or components of cellular material. The Gram stain is used to block the heat generated from the lamp, eliminate the best known and most widely used stain and forms the infrared light, and select the appropriate wavelength for basis for the phenotypic classification of bacteria. Yeasts can exciting the fluorochrome. The light emitted from the fluo- also be stained with this method (yeasts are gram-positive). rochrome is then magnified through traditional objective The iron hematoxylin and trichrome stains are invaluable and ocular lenses. Organisms and specimens stained with for identifying protozoan parasites, and the Wright-Giemsa fluorochromes appear brightly illuminated against a dark stain is used to identify blood parasites and other selected background, although the colors vary depending on the fluo- organisms. Stains such as methenamine silver and toluidine rochrome selected. The contrast between the organism and blue O have largely been replaced by more sensitive or techni- background is great enough that the specimen can be cally easier differential or fluorescent stains. screened rapidly under low magnification, and then the material is examined under higher magnification once fluo- Acid-Fast Stains rescence is detected. At least three different acid-fast stains are used, each exploit- ing the fact that some organisms retain a primary stain even Electron Microscopy when exposed to strong decolorizing agents such as mixtures Unlike other forms of microscopy, magnetic coils (rather of acids and alcohols. The Ziehl-Neelsen is the oldest method than lenses) are used in electron microscopes to direct a beam used but requires heating the specimen during the staining of electrons from a tungsten filament through a specimen procedure. Many laboratories have replaced this method and onto a screen. Because a much shorter wavelength of with either the cold acid-fast stain (Kinyoun method) or the light is used, magnification and resolution are improved dra- fluorochrome stain (auramine-rhodamine method). The matically. Individual viral particles (as opposed to viral inclu- fluorochrome method is the stain of choice because a large sion bodies) can be seen with electron microscopy. Samples area of the specimen can be examined rapidly by simply are usually stained or coated with metal ions to create con- searching for fluorescing organisms against a black back- trast. There are two types of electron microscopes: transmis- ground. Some organisms are “partially acid-fast,” retaining sion electron microscopes, in which electrons such as light the primary stain only when they are decolorized with a pass directly through the specimen, and scanning electron weakly acidic solution. This property is characteristic of only microscopes, in which electrons bounce off the surface of a few organisms (see Table 4-1), making it quite valuable for the specimen at an angle and a three-dimensional picture is their preliminary identification. 18 MEDICAL MICROBIOLOGY Table 4-1 Microscopic Preparations and Stains Used in the Clinical Microbiology Laboratory Staining Method Principle and Applications Direct Examination Wet mount Unstained preparation is examined by brightfield, darkfield, or phase-contrast microscopy. 10% KOH KOH is used to dissolve proteinaceous material and facilitate detection of fungal elements that are not affected by strong alkali solution. Dyes such as lactophenol cotton blue can be added to increase contrast between fungal elements and background. India ink Modification of KOH procedure in which ink is added as contrast material. Dye primarily used to detect Cryptococcus spp. in cerebrospinal fluid and other body fluids. Polysaccharide capsule of Cryptococcus spp. excludes ink, creating halo around yeast cell. Lugol iodine Iodine is added to wet preparations of parasitology specimens to enhance contrast of internal structures. This facilitates differentiation of amebae and host white blood cells. Differential Stains Gram stain Most commonly used stain in microbiology laboratory, forming basis for separating major groups of bacteria (e.g., gram-positive, gram-negative). After fixation of specimen to glass slide (by heating or alcohol treatment), specimen is exposed to crystal violet and then iodine is added to form complex with primary dye. During decolorization with alcohol or acetone, complex is retained in gram-positive bacteria but lost in gram-negative organisms; counterstain safranin is retained by gram-negative organisms (hence their red color). The degree to which organism retains stain is function of organism, culture conditions, and staining skills of the microscopist. Iron hematoxylin Used for detection and identification of fecal protozoa. Helminth eggs and larvae retain too much stain and are more easily stain identified with wet-mount preparation. Methenamine silver In general, performed in histology laboratories rather than in microbiology laboratories. Used primarily for stain detection of fungal elements in tissue, although other organisms (e.g., bacteria) can be detected. Silver staining requires skill because nonspecific staining can render slides unable to be interpreted. Toluidine blue O Used primarily for detection of Pneumocystis organisms in respiratory specimens. Cysts stain reddish-blue to dark purple on stain light blue background. Background staining is removed by sulfation reagent. Yeast cells stain and are difficult to distinguish from Pneumocystis cells. Trophozoites do not stain. Many laboratories have replaced this stain with specific fluorescent stains. Trichrome stain Alternative to iron hematoxylin for staining protozoa. Protozoa have bluish-green to purple cytoplasms with red or purplish-red nuclei and inclusion bodies; specimen background is green. Wright-Giemsa stain Used to detect blood parasites, viral and chlamydial inclusion bodies, and Borrelia, Toxoplasma, Pneumocystis, and Rickettsia spp. This is a polychromatic stain that contains a mixture of methylene blue, azure B, and eosin Y. Giemsa stain combines methylene blue and eosin. Eosin ions are negatively charged and stain basic components of cells orange to pink, whereas other dyes stain acidic cell structures various shades of blue to purple. Protozoan trophozoites have a red nucleus and grayish-blue cytoplasm; intracellular yeasts and inclusion bodies typically stain blue; rickettsiae, chlamydiae, and Pneumocystis spp. stain purple. Acid-Fast Stains Ziehl-Neelsen stain Used to stain mycobacteria and other acid-fast organisms. Organisms are stained with basic carbolfuchsin and resist decolorization with acid-alkali solutions. Background is counterstained with methylene blue. Organisms appear red against light blue background. Uptake of carbolfuchsin requires heating specimen (hot acid-fast stain). Kinyoun stain Cold acid-fast stain (does not require heating). Same principle as Ziehl-Neelsen stain. Auramine-rhodamine Same principle as other acid-fast stains, except that fluorescent dyes (auramine and rhodamine) are used for primary stain, and potassium permanganate (strong oxidizing agent) is the counterstain and inactivates unbound fluorochrome dyes. Organisms fluoresce yellowish-green against a black background. Modified acid-fast Weak decolorizing agent is used with any of three acid-fast stains listed. Whereas mycobacteria are strongly acid-fast, other stain organisms stain weaker (e.g., Nocardia, Rhodococcus, Tsukamurella, Gordonia, Cryptosporidium, Isospora, Sarcocystis, and Cyclospora). These organisms can be stained more efficiently by using a weak decolorizing agent. Organisms that retain this stain are referred to as partially acid-fast. Fluorescent Stains Acridine orange Used for detection of bacteria and fungi in clinical specimens. Dye intercalates into nucleic acid (native and denatured). At neutral stain pH, bacteria, fungi, and cellular material stain reddish-orange. At acid pH (4.0), bacteria and fungi remain reddish-orange, but background material stains greenish-yellow. Auramine-rhodamine Same as acid-fast stains. stain Calcofluor white Used to detect fungal elements and Pneumocystis spp. Stain binds to cellulose and chitin in cell walls; microscopist can mix dye stain with KOH. (Many laboratories have replaced traditional KOH stain with this stain.)  CHAPTER 4 MICROSCOPY AND IN VITRO CULTURE 19 Table 4-1 Microscopic Preparations and Stains Used in the Clinical Microbiology Laboratory—cont’d Staining Method Principle and Applications Direct fluorescent Antibodies (monoclonal or polyclonal) are complexed with fluorescent molecules. Specific binding to an organism is detected by antibody stain presence of microbial fluorescence. Technique has proved useful for detecting or identifying many organisms (e.g., Streptococcus pyogenes, Bordetella, Francisella, Legionella, Chlamydia, Pneumocystis, Cryptosporidium, Giardia, influenza virus, herpes simplex virus). Sensitivity and specificity of test are determined by number of organisms present in test sample and quality of antibodies used in reagents. KOH, Potassium hydroxide. Fluorescent Stains Relatively few laboratories prepare their own media today. The auramine-rhodamine acid-fast stain is a specific example Most media are produced by large commercial companies of a fluorescent stain. Numerous other fluorescent dyes have with expertise in media production. Although this has also been used to stain specimens. For example, the acridine obvious advantages, it also means that media are not “freshly orange stain can be used to stain bacteria and fungi, and produced.” Although this is generally not a problem, it can calcofluor white stains the chitin in fungal cell walls. impact the recovery of some fastidious organisms (e.g., Bor- Although the acridine orange stain is rather limited in its detella pertussis). Thus laboratories that perform sophisti- applications, the calcofluor white stain has replaced the cated testing frequently have the ability to make a limited potassium hydroxide stains. Another procedure is the exam- amount of specialized media. Dehydrated formulations of ination of specimens with specific antibodies labeled with most media are available, so this can be accomplished with fluorescent dyes (fluorescent antibody stains). The presence minimal difficulty. Please refer to the references in the Bib- of fluorescing organisms is a rapid method for both detection liography for additional information about the preparation and identification of the organism. and quality control of media. Types of Culture Media In Vitro Culture Culture media can be subdivided into four general catego- ries: (1) enriched nonselective media, (2) selective media, (3) The success of culture methods is defined by the biology of differential media, and (4) specialized media (Table 4-2). the organism, the site of the infection, the patient’s immune Some examples of these media are summarized below. response to the infection, and the quality of the culture media. The bacterium Legionella is an important respiratory Enriched Nonselective Media pathogen; however, it was never grown in culture until it was These media are designed to support the growth of most recognized that recovery of the organism required using organisms without fastidious growth requirements. The fol- media supplemented with iron and l-cysteine. Campylo- lowing are some of the more commonly used media: bacter, an important enteric pathogen, was not recovered in stool specimens until highly selective media were incubated Blood agar. Many types of blood agar media are used in at 42° C in a microaerophilic atmosphere. Chlamydia, an clinical laboratories. The media contain two primary important bacterium responsible for sexually transmitted components—a basal medium (e.g., tryptic soy, brain diseases, is an obligate intracellular pathogen that must be heart infusion, Brucella base) and blood (e.g., sheep, grown in living cells. Staphylococcus aureus, the cause of horse, rabbit). Various other supplements can also be staphylococcal toxic shock syndrome, produces disease by added to extend the range of organisms that can grow on release of a toxin into the circulatory system. Culture of the media. blood will almost always be negative, but culture of the site Chocolate agar. This is a modified blood agar medium. where the organism is growing will detect the organism. In When blood or hemoglobin is added to the heated basal many infections (e.g., gastroenteritis, pharyngitis, urethri- media, it turns brown (hence the name). This medium tis), the organism responsible for the infection will be present supports the growth of most bacteria, including some that among many other organisms that are part of the normal do not grow on blood agar (i.e., Haemophilus, some microbial population at the site of infection. Many media pathogenic Neisseria strains). have been developed that suppress the normally present Mueller-Hinton agar. This is the recommended medium for microbes and allow easier detection of clinically important routine antibiotic susceptibility testing of bacteria. It has organisms. The patient’s innate and adaptive immunity may a well-defined composition of beef and casein extracts, suppress the pathogen, so highly sensitive culture techniques salts, divalent cations, and soluble starch that is necessary are frequently required. Likewise, some infections are char- for reproducible test results. acterized by the presence of relatively few organisms. For Thioglycolate broth. This is one of a variety of enrichment example, most septic patients have less than one organism broths used to recover low numbers of aerobic and anaer- per milliliter of blood, so recovery of these organisms in a obic bacteria. Various formulations are used, but most traditional blood culture requires inoculation of a large include casein digest, glucose, yeast extract, cysteine, volume of blood into enrichment broths. Finally, the quality and sodium thioglycolate. Supplementation with hemin of the media must be carefully monitored to demonstrate it and vitamin K will enhance the recovery of anaerobic will perform as designed. bacteria. 20 MEDICAL MICROBIOLOGY Table 4-2 Types of Culture Media Type Media (examples) Purpose Nonselective Blood agar Recovery of bacteria and fungi Chocolate agar Recovery of bacteria including Haemophilus and Neisseria gonorrhoeae Mueller-Hinton agar Bacterial susceptibility test medium Thioglycolate broth Enrichment broth for anaerobic bacteria Sabouraud dextrose agar Recovery of fungi Selective, MacConkey agar Selective for gram-negative bacteria; differential for lactose-fermenting species differential Mannitol salt agar Selective for staphylococci; differential for Staphylococcus aureus Xylose lysine deoxycholate agar Selective, differential agar for Salmonella and Shigella in enteric cultures Lowenstein-Jensen medium Selective for mycobacteria Middlebrook agar Selective for mycobacteria CHROMagar Selective, differential for selected bacteria and yeasts Inhibitory mold agar Selective for molds Specialized Buffered charcoal yeast extract (BCYE) agar Recovery of Legionella and Nocardia Cystine-tellurite agar Recovery of Corynebacterium diphtheriae Lim broth Recovery of Streptococcus agalactiae MacConkey sorbitol agar Recovery of Escherichia coli O157 Regan Lowe agar Recovery of Bordetella pertussis Thiosulfate citrate bile salts sucrose (TCBS) agar Recovery of Vibrio species Sabouraud dextrose agar. This is an enriched medium con- and phenol red. Staphylococci can grow in the presence sisting of digests of casein and animal tissue supplemented of a high salt concentration, and S. aureus can ferment with glucose that is used for the isolation of fungi. A mannitol, producing yellow-colored colonies on this agar. variety of formulations have been developed, but most Xylose-lysine deoxycholate (XLD) agar. This is a selective mycologists use the formulation with a low concentration agar used for detection of Salmonella and Shigella in of glucose and neutral pH. By reducing the pH and adding enteric cultures. This is an example of a very clever antibiotics to inhibit bacteria, this medium can be made approach to detecting important bacteria in a complex selective for fungi. mixture of insignificant bacteria. The medium consists of yeast extract with xylose, lysine, lactose, sucrose, sodium Selective Media and Differential Media deoxycholate, sodium thiosulfate, ferric ammonium Selective media are designed for the recovery of specific citrate, and phenol red. Sodium deoxycholate inhibits the organisms that may be present in a mixture of other organ- growth of the majority of nonpathogenic bacteria. Those isms (e.g., an enteric pathogen in stool). The media are sup- that do grow typically ferment lactose, sucrose, or xylose, plemented with inhibitors that suppress the growth of producing yellow colonies. Shigella does not ferment unwanted organisms. These media can be made differential these carbohydrates, so the colonies appear red. Salmo- by adding specific ingredients that allow identification of an nella ferments xylose but also decarboxylates lysine, pro- organism in a mixture (e.g., addition of lactose and a pH ducing the alkaline diamine product cadaverine. This indicator to detect lactose fermenting organisms). The fol- neutralizes the acid fermentation products, thus the colo- lowing are some examples of selective and differential media: nies appear red. Because most Salmonella produce hydro- gen sulfide from sodium thiosulfate, the colonies will turn MacConkey agar. This is a selective agar for gram-negative black in the presence of ferric ammonium citrate, thus bacteria and differential for differentiation of lactose- differentiating Salmonella from Shigella. fermenting and lactose-nonfermenting bacteria. The Lowenstein-Jensen (LJ) medium. This medium, used for medium consists of digests of peptones, bile salts, lactose, the isolation of mycobacteria, contains glycerol, potato neutral red, and crystal violet. The bile salts and crystal flour, salts, and coagulated whole eggs (to solidify the violet inhibit gram-positive bacteria. Bacteria that ferment medium). Malachite green is added to inhibit gram- lactose produce acid that precipitates the bile salts and positive bacteria. causes a red color in the neutral red indicator. Middlebrook agar. This agar medium is also used for the Mannitol salt agar. This is a selective medium used for the isolation of mycobacteria. It contains nutrients required isolation of staphylococci. The medium consists of digests for the growth of mycobacteria (i.e., salts, vitamins, oleic of casein and animal tissue, beef extract, mannitol, salts, acid, albumin, catalase, glycerol, glucose) and malachite  CHAPTER 4 MICROSCOPY AND IN VITRO CULTURE 21 green for the inhibition of gram-positive bacteria. In con- John Franklin Enders described a technique for cultivating trast with LJ medium, it is solidified with agar. mammalian cells for the isolation of poliovirus. This tech- CHROMagar. These selective differential agars are used for nique has been expanded for the growth of most strict intra- the isolation and identification of a variety of bacteria cellular organisms. The cell cultures can either be cells that (e.g., Staphylococcus aureus, enteric bacteria) and yeasts. grow and divide on a surface (i.e., cell monolayer) or grow An example of design of these media is the one developed suspended in broth. Some cell cultures are well established for Candida species. This medium has chloramphenicol and can be maintained indefinitely. These cultures are com- to inhibit bacteria and a mixture of proprietary chromo- monly commercially available. Other cell cultures must be genic substrates. The different species of Candida have prepared immediately before they are infected with the bac- enzymes that can use one or more of the substrates, teria or viruses and cannot be maintained in the laboratory releasing the color compound and producing colored for more than a few cycles of division (primary cell cul- colonies. Thus Candida albicans forms green colonies, tures). Entry into cells is frequently regulated by the pres- Candida tropicalis forms purple colonies, and Candida ence of specific receptors, so the differential ability to infect krusei forms pink colonies. specific cell lines can be used to predict the identity of the Inhibitory mold agar. This medium is an enriched selective bacterium or virus. Additional information about the use of formulation used for the isolation of pathogenic fungi cell cultures is described in the following chapters. other than dermatophytes. Chloramphenicol is added to suppress the growth of contaminating bacteria. Bibliography Specialized Media Chapin K: Principles of stains and media. In Murray P, et al, editors: Manual A large variety of specialized media have been created for of clinical microbiology, ed 9, Washington, DC, 2007, American Society for Microbiology Press. the detection of specific organisms that may be fastidious or Murray P, Shea Y: ASM pocket guide to clinical microbiology, ed 3, typically present in large mixtures of organisms. The more Washington, DC, 2004, American Society for Microbiology Press. commonly used media are described in the specific organism Snyder J, Atlas R: Handbook of media for clinical microbiology, ed 2, Boca chapters in this textbook. Raton, Fla, 2006, CRC Press. Wiedbrauk D: Microscopy. In Versalovic J, et al, editors: Manual of clinical Cell Culture microbiology, ed 10, Washington, DC, 2011, American Society for Microbiology. Some bacteria and all viruses are strict intracellular Zimbro M, Power D: Difco and BBL manual: manual of microbiological microbes; that is, they can only grow in living cells. In 1949, culture media, Sparks, Md, 2003, Becton Dickinson and Company.  CHAPTER 5 MOLECULAR DIAGNOSIS L ike the evidence left at the scene of a crime, the DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or pro- teins of an infectious agent in a clinical sample can be used a specific organism produced on cleavage with one or more restriction endonucleases is termed restriction fragment length polymorphism (RFLP). to help identify the agent. In many cases, the agent can be DNA or RNA fragments of different sizes or structures detected and identified in this way even if it cannot be iso- can be distinguished by their electrophoretic mobility in an lated or detected by immunologic means. New techniques agarose or polyacrylamide gel. Different forms of the same and adaptations of older techniques are being developed for DNA sequence and different lengths of DNA move through the analysis of infectious agents. the mazelike structure of an agarose gel at different speeds, The advantages of molecular techniques are their sensitiv- allowing their separation. The DNA can be visualized by ity, specificity, and safety. From the standpoint of safety, these staining with ethidium bromide. Smaller fragments ( 11 amino acids) pro- cells to arrive at the site in response to infection; they are duced from phagocytosed proteins (exogenous route) are followed later by monocytes and macrophages. Recruitment bound to class II major histocompatibility complex (MHC) of immature band forms of neutrophils from the bone molecules and presented by these antigen-presenting cells marrow during infection is indicated by a “left shift” in the (APCs) to naïve CD4 TH0 T cells. TH0 provides the first complete blood cell count. Neutrophils are recruited and stage, a generic expansion of the immune cells needed to activated by the TH17 response and macrophages, and DCs respond to the infection. The CD4 T cells are activated by a are activated by IFN-γ produced by NK and NKT cells and combination of (1) antigenic peptide in the cleft of the MHC the TH1 response. II molecule with the T-cell antigen receptor (TCR) and with Bacteria are bound to the neutrophils and macrophages CD4, (2) co-stimulatory signals provided by sufficient with receptors for bacterial carbohydrates (lectins [specific numbers of interactions of B7 molecules on the DC with sugar-binding proteins]), fibronectin receptors (especially CD28 molecules on the T cells, and (3) IL-6 and other cyto- for Staphylococcus aureus), and receptors for opsonins, kines produced by the DCs. The TH0 cells produce IL-2, including complement (C3b), C-reactive protein, mannose- IFN-γ, and IL-4. Simultaneously, bacterial molecules with binding protein, and the Fc portion of antibody. The microbes repetitive structures (e.g., capsular polysaccharide) interact are internalized in a phagocytic vacuole that fuses with with B cells expressing surface IgM and IgD specific for the primary lysosomes (macrophages) or granules (PMNs) to antigen and activate the cell to grow and produce IgM. LPS allow inactivation and digestion of the vacuole contents (see and also the C3d component of complement activate B cells Figure 8-7 and Box 8-4). and promote the specific IgM antibody responses. Swollen The neutrophil kills the phagocytosed microbes by oxygen- lymph nodes are an indication of lymphocyte growth in dependent killing with hydrogen peroxide, superoxide ion, response to antigenic challenge. and hypochlorous ions and with oxygen-independent killing Early responses are also provided by γ/δ T cells, NKT upon fusion of the phagosome with azurophilic granules cells, and innate lymphoid cells (including NK cells). γ/δ T containing cationic proteins (e.g., cathepsin G) and specific cells in tissue and blood sense phosphorylated amine metab- granules containing lysozyme and lactoferrin. These proteins olites from some bacteria (Escherichia coli, mycobacteria) kill gram-negative bacteria by disrupting their cell membrane but not others (streptococci, staphylococci). DCs can present integrity, but they are far less effective against gram-positive bacterial glycolipids to activate NKT cells. These T cells and bacteria, which are killed principally through the oxygen- innate lymphoid cells produce IFN-γ, which activates mac- dependent mechanism. Nitric oxide produced by neutro- rophages and DCs to enforce local cellular inflammatory phils and activated macrophages has antimicrobial activity reactions. and is also a major second messenger molecule that enhances The conversion of TH0 cells to TH17 and TH1 cells initi- the inflammatory and other responses. ates expansion of the host response. Acute-phase cytokines Neutrophils contribute to the inflammation in several IL-1 and TNF-α together with the omnipresent transform- ways. Prostaglandins and leukotrienes are released and ing growth factor (TGF)-β promote the development of CD4 increase vascular permeability, cause swelling (edema), and TH17 T cells (see Animation 9-5). The acute phase cytokines stimulate pain receptors. During phagocytosis, the granules provide a cry for help despite the calming influence of TGF-β may leak their contents to cause tissue damage. The neutro- to elicit a quick inflammatory cytokine yell by the CD4 TH17 phils have short lives, and upon death, neutrophils release T cells to the epithelial cells and neutrophils to activate inflam- a sticky DNA net (neutrophil extracellular trap) and matory responses. Memory TH17 cells are activated by IL-23. become pus. TH17 cells produce IL-17 and TNF-α to activate epithelial In contrast to neutrophils, macrophages have long lives, cells and neutrophils and also promote production of anti- but the cells must be activated (made angry) with IFN-γ microbial peptides. TH17 responses are important for early (best) in order to kill phagocytized microbes. Granulocyte- antibacterial responses and antimycobacterial responses. A macrophage colony-stimulating factor (GM-CSF), TNF-α, balance of TH17 and Treg responses are also important to and lymphotoxin (TNF-β) maintain the antimicrobial action regulate the populations of intestinal flora. (keep them aggravated). Early in the infection, IFN-γ is pro- DCs producing IL-12 promote TH1 responses. CD4 TH1 duced by NK and NKT cells and later by CD4 T cells. Splenic T cells (1) promote and reinforce inflammatory responses macrophages are important for clearing bacteria, especially (e.g., IFN-γ activation of macrophage) and growth of T and encapsulated bacteria, from blood. Asplenic (congenitally or B cells (IL-2) to expand the immune response, and (2) surgically) individuals are highly susceptible to pneumonia, promote B cells to produce complement-binding antibodies meningitis, and other manifestations of Streptococcus pneu- (IgM and then IgG upon class switching) and mature into  CHAPTER 10 IMMUNE RESPONSES TO INFECTIOUS AGENTS 81 Mθ B cell DC IL-2 Memory CD8 T T TH1 TH1 IL-2 IL-2, IFN-γ TLR IL-12 IFN-γ Naïve TH0 iDC DC Antigen IL-4 No IL-12 IL-4 Cytokines TH2 IL-4 TH2 Memory T IL-10 Periphery Lymph node B cell DC Mθ FIGURE 10-2 Initiation and expansion of specific immune responses. Immature dendritic cells (iDCs) at the site of infection acquire microbial debris, Toll-like receptors (TLRs) and other pathogen pattern receptors bind their ligands and activate dendritic cells (DCs) that produce cytokines, mature, and move to the lymph node. DCs present antigen to naïve T cells to initiate the antigen-specific and cytokine- directed response. During a secondary or memory response, B cells, macrophages, and DCs can present antigen to initiate the response. IL, Interleukin; IFN-γ, interferon-γ; Mθ, macrophage; TH, T helper (cell). plasma cells and memory cells. These responses are impor- sewage inspectors who promote a response to clear out tant for the early phases of an antibacterial defense. TH1 excess and damaged protein. This is the same type of response responses are also essential for combating intracellular bac- that occurs to injection of a bolus of antigen for an inacti- terial infections, including mycobacteria, which are hidden vated vaccine. Binding of antigen to the cell surface antibody from antibody. IFN-γ activates macrophage to kill phagocy- on B cells activates the B cells and also promotes uptake, tized microbes. Upon chronic stimulation by macrophages processing of the antigen, and presentation of antigenic pep- expressing microbial (e.g., mycobacterial or histoplasma) tides on class II MHC molecules to the CD4 TH2 cell. The antigen, CD4 TH1 T cells will produce IFN-γ and TNF-α TH2 cell produces IL-4, IL-5, IL-6, IL-10, and IL-13, which and cause transformation of other macrophages into epithe- enhance IgG production and, depending on other factors, lioid cells and giant cells, which can surround the infection the production of IgE or IgA. CD4TFH cells are a conduit and produce a granuloma. Granulomas wall off intracellular for the TH1 or TH2 responses to promote memory cell pro- infections arising either because the microbe can evade anti- duction and terminal differentiation of B cells to plasma-cell microbial responses (e.g., Mycobacterium tuberculosis), the antibody factories. macrophages are not activated and cannot kill them (normal CD4+CD25+ regulatory T cells (Treg) prevent spurious alveolar macrophages), or a genetic defect prevents genera- activation of naïve T cells, curtail both TH1 and TH2 tion of antimicrobial reactive oxygen substances, as in responses, and promote development of some of the antigen- chronic granulomatous disease. CD8 T cells facilitate clear- specific cells into memory T cells. Only DCs can override ance of intracellular infections by producing cytokines but are the Treg block to activate naïve T cells. not essential for antibacterial immunity. Antibodies are the primary protection against extracel- CD4 TH2 T-cell responses occur in the absence of IL-12 lular bacteria and toxins and promote the clearance and at more distant lymph nodes. These responses are also initi- prevent the spread of bacteria in the blood (bacteremia). ated by DCs and are sustained by the B-cell presentation of Antibody promotes complement activation, opsonizes antigen. TH2 responses can occur at the same time as TH1 bacteria for phagocytosis, blocks bacterial adhesion, and responses when antigen is delivered in lymph fluid to lymph neutralizes (inactivates) exotoxins (e.g., tetanospasmin, nodes other than the draining lymph node. The DCs act as botulinum toxin) and other cytotoxic proteins produced by 82 MEDICAL MICROBIOLOGY bacteria (e.g., degradative enzymes). Vaccine immunization mutation in the IL-23 receptor or NOD2 receptor for pepti- with inactivated exotoxins (toxoids) is the primary means doglycan enhances chances for certain types of Crohn of protection against the potentially lethal effects of disease. exotoxins. In the skin, Langerhans cells are sentinel DCs responsive IgM antibodies are produced early in the antibacterial to trauma and infection. Memory CD4 and CD8 T cells response (see Animation 10-1). IgM bound to bacteria constantly cycle into the skin from the blood. In the respira- activates the classical complement cascade, promoting both tory tract, antimicrobial peptides and secreted IgA control the direct killing of gram-negative bacteria and the inflam- bacteria, mucus traps, and cilia move the mucus and bacteria matory responses. IgM is usually the only antibody pro- out of the lungs. Inflammatory responses are controlled by duced against capsular polysaccharides and promotes alveolar macrophages (M2 macrophages) to prevent tissue opsonization of the bacteria with complement. Splenic damage to normal flora. Like in the gastrointestinal tract, macrophages depend upon IgM bound to capsular poly- DCs monitor the epithelium for normal and abnormal saccharides to activate complement and opsonize the microbes. encapsulated bacteria so they can be recognized, phagocy- tized, and eliminated. The large size and limited transport Bacterial Immunopathogenesis mechanisms for IgM limits its ability to spread into tissue. Activation of the inflammatory and acute-phase responses IgM produced in response to polysaccharide vaccines (as can initiate significant tissue and systemic damage. Activa- for Streptococcus pneumonia) can prevent bacteremia but tion of macrophages and DCs in the liver, spleen, and blood not infection of the interstitium of the lung. Approximately by endotoxin can promote release of acute-phase cytokines a week later, T-cell help promotes differentiation of the B into the blood, causing many of the symptoms of sepsis, cell and immunoglobulin class switching to produce IgG. including hemodynamic failure, shock, and death (see IgG antibodies are the predominant serum antibody, espe- Cytokine Storm section and Chapter 14). Although IL-1, cially on rechallenge. IgG antibodies fix complement and IL-6, and TNF-α promote protective responses to a local promote phagocytic uptake of the bacteria through Fc infection, these same responses can be life threatening when receptors on macrophages. IgA is the primary secretory activated by systemic infection. Increased blood flow and antibody and is important for protecting mucosal mem- fluid leakage can lead to shock when it occurs throughout branes. Large amounts of secretory IgA are released to reg- the body. Antibodies produced against bacterial antigens ulate the normal flora population, prevent adhesion of that share determinants with human proteins can initiate bacteria, and neutralize toxins at epithelial cell surfaces. autoimmune tissue destruction (e.g., antibodies produced A primary antigen-specific response to bacterial infection in poststreptococcal rheumatic fever). Nonspecific activa- takes at least 5 to 7 days. Movement of the DC to the lymph tion of CD4 T cells by superantigens (e.g., toxic shock syn- node may take 1 to 3 days, followed by activation, expansion, drome toxin of S. aureus) promotes production of large and maturation of the response. On rechallenge to infection, amounts of cytokines and, eventually, the death of large long-lived plasma cells may still be producing antibody. numbers of T cells. The sudden massive release of cytokines Memory T cells can respond quickly to antigen presentation (“cytokine storm”) can cause shock and severe tissue damage by DCs, macrophages, or B cells, not just DCs; memory B (e.g., toxic shock syndrome) (see Cytokine Storm section cells are present to respond quickly to antigen, and the sec- and Chapter 14). ondary antibody response occurs within 2 to 3 days. Bacterial Evasion of Protective Responses Skin, Intestinal, and Mucosal Immunity The mechanisms used by bacteria to evade host-protective The skin, intestine, and mucous membranes are populated responses are discussed in Chapter 14 as virulence factors. with bacteria upon traversing the birth canal and soon there- These mechanisms include (1) inhibition of phagocytosis after. The immune response matures, and a balance develops and intracellular killing in the phagocyte, (2) inactivation of between regulatory and inflammatory cells in response to complement function, (3) binding of the Fc portion of IgG this normal flora. and cleavage of IgA, (4) intracellular growth (avoidance of The intestinal flora is constantly interacting with and antibody), and (5) change in bacterial antigenic appearance. being regulated by the innate and immune systems of the Some microorganisms, including but not limited to myco- gut-associated lymphoid tissue (see Figure 7-5). Similarly, bacteria (also Listeria and Brucella species), survive and mul- the immune response is shaped by its interaction with intes- tiply within macrophages and use the macrophages as a tinal flora as regulatory cells limit the development of auto- protective reservoir or transport system to help spread the immune responses and inflammation. DCs, innate lymphoid organisms throughout the body. However, cytokine-activated cells, Treg, TH17, TH1, and other T cells and B cells in the macrophages can often kill the intracellular pathogens. lamina propria, Peyer patches, and intestinal lymphoid fol- licles monitor and control the bacteria within the gut. These cells and epithelial and other cells lining the gut produce Antiviral Responses antimicrobial peptides, and plasma cells secrete IgA into the gut to maintain a healthy mixture of bacteria. At the same Host Defenses against Viral Infection time, regulatory cells prevent the development of detrimen- The immune response is the best and, in most cases, the only tal or excessive immune responses to the contents of the gut. means of controlling a viral infection (Figure 10-3 and Box Alterations in the microbial flora and its interaction with the 10-3). Unfortunately, it is also the source of pathogenesis for innate and immune cells can disrupt the system and result many viral diseases. Both humoral and cellular immune in inflammatory bowel diseases. For example, absence or a responses are important for antiviral immunity. The  CHAPTER 10 IMMUNE RESPONSES TO INFECTIOUS AGENTS 83 0-4 hr 4-96 hr 6 days + Innate responses Antigen-specific responses Early Later IFN-α NK cell IgM IFN-β Influenza CD4 IFN-γ IL-2 B cell TH2 IL-4 B cells IL-4 IL-5 IFN IFN-γ IL-12 IgA IL-6 IgG IL-10 IgE DC CD4 IL-4 TH0 IL-2 IFN-γ CD4 IL-2, IFN-γ B cell IL-1 DC TH1 IL-12 IgG IFN-γ IL-2 IFN-γ CD4 TNF Memory Inhibitory IL-6 Activating Activated DC CD8 Effector Mθ CTL IL-1 Mθ, DC T cell Lung Lymph node B cell FIGURE 10-3 Antiviral responses. The response to a virus (e.g., influenza virus) initiates with interferon production and action and natural killer (NK) cells. Activation of antigen-specific immunity resembles the antibacterial response, except that CD8 cytotoxic T lymphocytes (CTLs) are important antiviral responses. The time course of events is indicated at the top of the figure. IFN, Interferon, IL, interleukin; Mθ, macrophage; TH, T helper (cell); TNF, tumor necrosis factor. Box 10-3 Summary of Antiviral Responses Interferon Antigenic viral peptides (linear epitopes) can come from any viral protein Interferon is induced by double-stranded RNA, inhibition of cellular protein (e.g., glycoproteins, nucleoproteins) synthesis, or enveloped virus CD8 cytotoxic T cells respond to viral peptide: class I MHC protein complexes Interferon initiates the antiviral state in surrounding cells on the infected cell surface Antiviral state blocks viral replication CD4 TH2 responses may be detrimental if they prematurely limit the TH1 Interferon activates NK cells and systemic antiviral responses inflammatory and cytolytic responses NK Cells Antibody NK cells are activated by IFN-α and interleukin-12, which activate macro- Antibody neutralizes extracellular virus: phages with IFN-γ It blocks viral attachment proteins (e.g., glycoproteins, capsid NK cells target and kill virus-infected cells (especially enveloped viruses) proteins) It destabilizes viral structure Macrophages and DCs Antibody opsonizes virus for phagocytosis Macrophages filter viral particles from blood Antibody promotes killing of target cell by the complement cascade and Macrophages inactivate opsonized virus particles antibody-dependent cellular cytotoxicity Immature and plasmacytoid DCs produce IFN-α and other cytokines Antibody resolves lytic viral infections DCs initiate and determine the nature of the CD4 and CD8 T-cell response Antibody blocks viremic spread to target tissue DCs and macrophages present antigen to CD4 T cells IgM is an indicator of recent or current infection T Cells IgG is a more effective antiviral than IgM T cells are essential for controlling enveloped and noncytolytic viral Secretory IgA is important for protecting mucosal surfaces infections Resolution requires elimination of free virus (antibody) and the T cells recognize viral peptides presented by MHC molecules on cell virus-producing cell (viral or immune cell–mediated lysis) surfaces DC, Dendritic cell; IFN, interferon; Ig, immunoglobulin; MHC, major histocompatibility complex; NK, natural killer. 84 MEDICAL MICROBIOLOGY ultimate goal of the immune response in a viral infection Table 10-2 Basic Properties of Human Interferons (IFNs) is to eliminate both the virus and the host cells harboring or replicating the virus. Failure to resolve the infection may Property IFN-α IFN-β IFN-γ lead to persistent or chronic infection or death. Previous Leukocyte IFN Fibroblast IFN Immune IFN Interferons, NK cells, CD4 TH1 responses, and CD8 cyto- designations type I type I type II toxic killer T cells are more important for viral infections than for bacterial infections. Complement has a limited role Genes >20 1 1 in antiviral defense. Molecular 16,000-23,000 23,000 20,000-25,000 The course of the immune response and the nature of the mass (Da)* immunopathogenesis of bacterial and viral infections are Acid stability Stable† Stable Labile different. For bacteria, complement and the recruitment of neutrophils and macrophages are the initial response, and Primary Viruses Viruses Immune they rapidly drive the disease-associated inflammation. activator response Antibody can control extracellular bacteria and their toxins. Principal Epithelium, Fibroblast NK or T cell For viruses, type I interferons and other cytokines initiate source leukocytes the response, prodrome symptoms are driven by interferon and cytokines, but protection, inflammatory responses and Homology with 100% 30%-50%