key: cord-0046371-97u7bkcs authors: Bartlett, Jeanine H. title: Microorganisms date: 2020-06-22 journal: Theory and Practice of Histological Techniques DOI: 10.1016/b978-0-443-10279-0.50024-5 sha: ac8774a8ef74aecec3cffe8479676a242856cdbf doc_id: 46371 cord_uid: 97u7bkcs nan We have all heard the expression, 'The world is getting smaller.' Nowhere is that statement truer than in the world of microorganisms. Microorganisms (also called microbes) are organisms which share the property of being sub-microscopic. Most do not normally cause disease in humans, existing in a state of commensalism, where there is little or no benefi t to the person, or mutualism, where there is some benefi t to both parties. Pathogens are agents that cause disease. These fall into fi ve main groups (Microbiology at Leicester website): • Viruses • Bacteria • Fungi • Protozoa • Helminths. With the advent of new and more powerful antibiotics, improved environmental hygiene, and advances in microbiological technique, it was widely expected that the need for diagnosis of infectious agents in tissue would diminish in importance. This assumption underestimated the infi nite capacity of infectious agents for genomic variation, enabling them to exploit new opportunities to spread infections that are created when host defenses become diminished and inadequate. The following are currently the most important factors infl uencing the presentation of infectious diseases: • Increased mobility of the world's population through tourism, immigration, and international commerce has distorted natural geographic boundaries to infection, exposing weaknesses in host defenses, and in knowledge. Some, such as Ebola, have been around for many years but the fi rst human outbreaks were not recorded until 1976. Previous outbreaks would fl are up and then burn themselves out, undetected and confi ned before deforestation and the like altered this state. • Immunodefi ciency states occurring either as part of a natural disease, such as acquired immune defi ciency syndrome (AIDS), or as an iatrogenic disease. As treatment becomes more aggressive, depression of the host's immunity often occurs, enabling organisms of low virulence to become life-threatening, and allows latent infections accrued throughout life to reactivate and spread unchecked. • Emerging, re-emerging, and antibiotic-resistant organisms such as the tubercle bacillus and staphylococcus are a constant concern. • Adaptive mutation occurring in microorganisms, which allows them to jump barriers of species and explore new physical environments, evading host defenses, and resisting agents of treatment. • Bioterrorism has become a major concern since September 11, 2001. The world public health systems and primary healthcare providers must be prepared to address varied biological agents, including pathogens that are rarely seen in the developed countries. High-priority agents include organisms that pose a risk to national security because they: • Can be easily disseminated or transmitted from person to person • Cause high mortality, with potential for a major public health impact • Might cause public panic and social disruption, and require special action for public health preparedness. The following are listed by the Centers for Disease Control and Prevention (CDC) in the United States as high-risk biological agents: • Anthrax • Smallpox • Botulism • Tularemia • Viral hemorrhagic fever. These factors, acting singly or together, provide an everchanging picture of infectious disease where clinical presentation may involve multiple pathological processes, unfamiliar organisms, and modifi cation of the host response by a diminished immune status. The term 'microorganism' has been interpreted liberally in this chapter. Space limitation precludes a comprehensive approach to the subject; the reader is referred to additional texts such as that of von Lichtenberg (1991) for greater depth. The organisms in Table 17 .1 are discussed, with techniques for their demonstration described. Most infectious agents are rendered harmless by direct exposure to formal saline. Standard fi xation procedures should be suffi cient to kill microorganisms, one exception being material from patients with Creutzfeldt-Jakob disease (CJD). It has been shown that well-fi xed tissue, paraffi n-processed blocks, and stained slides from CJD remain infectious when introduced into susceptible animals. Treatment of fi xed tissue or slides in 96% formic acid for 1 hour followed by copious washing inactivates this infectious agent without adversely affecting section quality (Brown et al 1990) . Laboratory safety protocols should cover infection containment in all laboratory areas, and the mortuary, or necropsy area, where handling unfi xed material is unavoidable. When available, unfi xed tissue samples should be sent for microbiological culture as this offers the best chance for rapid and specifi c identifi cation of etiological agents, even when heavy bacterial contamination may have occurred. The diagnosis of illness from infectious disease starts with clinical presentation of the patient, and in most cases a diagnosis is made without a tissue sample being taken. Specimens submitted to the laboratory range from autopsy specimens, where material is plentiful and sampling error presents little problem, to cervical smears where cellular material is often scarce and lesions may easily be missed. A full clinical history is important, especially details of the patient's ethnic origin, immune status, any recent history of foreign travel, and current medication. The macroscopic appearance of tissue, such as abscesses and pus formation, cavitations, hyperkeratosis, demyelination, pseudo-membrane or fi brin formation, focal necrosis, and granulomas can provide evidence of infection. These appearances are often nonspecifi c but occasionally in hydatid cyst disease or some helminth infestations the appearances are diagnostic. The microscopic appearance of routine stains at lowpower magnifi cation often reveals indirect evidence of the presence of infection, such as neutrophil or lymphocytic infi ltrates, granulomata, micro-abscesses, eosinophilic aggregates, Charcot-Leyden crystals, and caseous necrosis. Some of these appearances may be suffi ciently reliable to provide an initial, or provisional, diagnosis and allow treatment to be started even if the precise nature of the suspect organism is never identifi ed, particularly in the case of tuberculosis. At the cellular level the presence of giant cells, such as Warthin-Finkeldy, or Langhans' giant cells, likely indicates measles and tuberculosis, respectively. Other cellular changes include intracytoplasmic edema of koilocytes, acantholysis, spongiform degeneration of brain, margination of chromatin, syncytial nuclear appearance, 'ground-glass' changes in the nucleus or At some stage in these processes, suspect organisms may be visualized. A well-performed hematoxylin and eosin (H&E) method will stain many organisms. Papanicolaou stain and Romanowsky stains, such as Giemsa, will also stain many organisms together with their cellular environment. Other infectious agents are poorly visualized by routine stains and require special techniques to demonstrate their presence. This may be due to the small size of the organism, as in the case of viruses where electron microscopy is needed. Alternatively, the organism may be hydrophobic, or weakly charged, as with mycobacteria, spirochetes, and cryptococci, in which case the use of specifi c histochemical methods is required for their detection. When organisms are few in number, fl uorochromes may be used to increase microscopic sensitivity of a technique. Finally, there are two techniques that offer the possibility of specifi c identifi cation of microorganisms that extend to the appropriate strain level. There is a growing catalog of biotinylated antisera against organism-specifi c proteins that can be demonstrated immunohistochemically. To date, those developed for protozoan, chlamydial, and viral organisms have been most widely used diagnostically in histopathology; however, this will undoubtedly change in the future. In situ hybridization has even greater potential for microbial detection. The use of single-stranded nucleic acid probes offers even greater possibilities by identifying latent viral genomic footprints in cells, which may have relevance to extending our knowledge of disease, AIDS and HIV being good examples. The polymerase chain reaction technique, to increase sensitivity and make use of stored blocks and slides to study evolutionary aspects of infectious disease, is being used increasingly in research. Future demonstration methods for infectious diseases may lie with these techniques. While modern advances in technique are important, emphasis is also placed upon the ability of the microscopist to interpret suspicious signs from a good H&E stain. The growing number of patients whose immune status is compromised, and who can mount only a minimal or inappropriate response to infection, further complicates the picture, justifying speculative use of special stains such as those for mycobacteria and fungi on tissue from AIDS patients. It should be remembered that, for a variety of reasons, negative results for the identifi cation of an infectious agent do not exclude its presence. For instance, administration of antibiotics to the patient before a biopsy might be the reason for failure to detect a causal microorganism in tissue. When bacteria are present in large numbers in an abscess or in vegetation on a heart valve, they appear as bluegray granular masses with an H&E stain; often organisms are invisible or obscured by cellular debris. The reaction of pyogenic bacteria to the Gram stain, together with their morphological appearance, i.e. cocci or bacilli, provides the basis for a simple classifi cation: see Table 17 .2. The use of known positive control sections with all special stain methods for demonstrating microorganisms is essential. Results are unsafe in the absence of positive controls, and should not be considered valid. The control section should be appropriate, where possible, for the suspected organism. A pneumocystiscontaining control, for instance, should be used for demonstrating Pneumocystis carinii. A Gram control should contain both Gram-positive and Gram-negative organisms. Postmortem tissues can often be a good source of control material or, as a last resort, a suspension of Gram-positive and Gram-negative organisms can be injected into the thigh muscle of a rat shortly before it is sacrifi ced for some other purpose. Gram-positive and Gram-negative organisms can also be harvested from microbiological plates, suspended in 10% neutral buffered formalin (NBF), centrifuged, and small amounts mixed with minced normal kidney, then chemically processed along with other tissue blocks (Swisher & Nicholson 1989) . In spite of more than a century having passed since Gram described his technique in 1884, its chemical rationale is still obscure. It is probably due to a mixture of factors, the most important being increased thickness, chemical composition, and the functional integrity of cell walls of Gram-positive bacteria. When these bacteria die, they become Gram negative. The following procedure is only suitable for the demonstration of bacteria in smears of pus and sputum. It may be of value to the pathologist in the necropsy room where a quick technique such as this may enable rapid identifi cation of the organism causing a lung abscess, wound infection, septicemic abscesses, or meningitis. Method 1. Fix dry fi lm by passing it three times through a fl ame or placing on a heat block. 2. Stain for 15 seconds in 1% crystal violet or methyl violet, then pour off excess. 3. Flood for 30 seconds with Lugol's iodine, pour off excess. 4. Flood with acetone for not more than 2-5 seconds; wash with water immediately. 5. Alternatively decolorize with alcohol until no more stain comes out. Wash with water. 6. Counterstain for 20 s with dilute carbol fuchsin, or freshly fi ltered neutral red for 1-2 min. 7. Wash with water and carefully blot section until it is dry. Gram-positive organisms blue-black Gram-negative organisms red Modifi ed Brown-Brenn method for Grampositive and Gram-negative bacteria in paraffi n sections (Churukian & Schenk 1982) Sections Formalin-fi xed, 4-5 micron, paraffi n-embedded sections. Crystal violet, 10% alcoholic 2 ml Distilled water 18 ml Ammonium oxalate, 1% 80 ml Mix and store; always fi lter before use. Iodine 2 g Potassium iodide 4 g Distilled water 400 ml Dissolve potassium iodide in a small amount of the distilled water, add iodine and dissolve; add remainder of distilled water. Ethyl alcohol, absolute 50 ml Acetone 50 ml Basic fuchsin or pararosaniline 0.5 g Distilled water 100 ml Dissolve with aid of heat and a magnetic stirrer. Basic fuchsin solution (stock) 10 ml Distilled water 40 ml Picric acid 0.1 g Acetone 100 ml With concerns over the explosiveness of dry picric acid in the lab, it is recommended that you purchase the picric acid-acetone solution pre-made. It is available through most histology vendors. Acetone 50 ml Xylene 50 ml 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Stain with fi ltered crystal violet solution, 1 min. 3. Rinse well in distilled water. 4. Iodine solution, 1 min. 5. Rinse in distilled water, blot slide but NOT the tissue section. 6. Decolorize by dipping in alcohol-acetone solution until the blue color stops running. (One to two dips only!) 7. Counterstain in working basic fuchsin for 1 min. Be sure to agitate the slides well in the basic fuchsin before starting the timer. 8. Rinse in distilled water and blot slide but not section. 9. Dip in acetone, one dip. 10. Dip in picric acid-acetone until the sections have a yellowish-pink color. 11. Dip several times in acetone-xylene solution. At this point, check the control for proper differentiation. (Go back to picric acid-acetone if you need more differentiation.) 12. Clear in xylene and mount. Gram-positive organisms, fi brin, some blue fungi, Paneth cells granules, keratohyalin, and keratin Gram-negative organisms red Nuclei red Other tissue elements yellow Be sure you do not allow the tissue sections to dry at any point in the staining process. If this occurs after treatment with iodine, decolorization will be diffi cult and uneven. Gram-Twort stain (Twort 1924; Ollet 1947) Formalin fi xed, paraffi n. 1% neutral red in ethanol 9 ml 0.2% fast green in ethanol 1 ml Distilled water 30 ml Mix immediately before use. 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Stain in crystal violet solution, 3 min. 3. Rinse in gently running tap water. 4. Treat with Gram's iodine, 3 min. 5. Rinse in tap water, blot dry, and complete drying in a warm place. 6. Differentiate in preheated acetic alcohol until no more color washes out (2% acetic acid in absolute alcohol, pre-heated to 56°C). This may take 15-20 min; the section should be light brown or straw colored. 7. Rinse briefl y in distilled water. 8. Stain in Twort's, 5 min. 9. Wash in distilled water. 10. Rinse in acetic alcohol until no more red runs out of the section; this takes only a few seconds. 11. Rinse in fresh absolute alcohol, clear, and mount. Gram-positive organisms blue-black Gram-negative organisms pink-red Nuclei red Red blood cells and most green cytoplasmic structures Elastic fi bers black These organisms are diffi cult to demonstrate by the Gram technique because they possess a capsule containing a long-chain fatty acid (mycolic acid) that makes them hydrophobic. The fatty capsule infl uences the penetration and resistance to removal of the stain by acid and alcohol (acid-and alcohol-fastness), and is variably robust between the various species that make up this group. Phenolic acid, and frequently heat, are used to reduce surface tension and increase porosity, thus forcing dyes to penetrate this capsule. The speed with which the primary dye is removed by differentiation with acid alcohol is proportional to the extent of the fatty coat. The avoidance of defatting agents, or solvents, such as alcohol and xylene, in methods for Mycobacterium leprae, is an attempt to conserve this fragile fatty capsule. Mycobacteria are PAS positive due to the carbohydrate content of their cell walls; however, this positivity is evident only when large concentrations of the microorganisms are present. When these organisms die, they lose their fatty capsule and consequently their carbol fuchsin positivity. The carbohydrate can still be demonstrated by Grocott's methenamine silver reaction, which may prove useful when acid-fast procedures fail, particularly if the patient is already receiving therapy for tuberculosis. A possible source of acid-fast contamination may be found growing in viscous material sometimes lining water taps and any rubber tubing connected to them. These organisms are acid-and alcohol-fast but are usually easily identifi ed as contaminants by their appearance as clumps, or fl oaters, above the microscopic focal plane of the section. Formalin or fi xative other than Carnoy's, paraffi n. Basic fuchsin 0.5 g Absolute alcohol 5 ml 5% aqueous phenol 100 ml Mix well and fi lter before use. Hydrochloric acid 10 ml 70% alcohol 1000 ml Methylene blue 1.4 g 95% alcohol 100 ml Methylene blue (stock) 10 ml Tap Mycobacteria golden yellow (using blue light fl uorescence below 530 nm) Background dark green The advantage of increased sensitivity of this technique is offset by the inconvenience of setting up the fl uorescence microscope. Preparations fade over time, as a result of their exposure to UV light. Fixation 10% neutral buffered formalin (NBF). Paraffi n sections at 4-5 μm. Carbol fuchsin solution commercially available, or 0.5 g basic fuchsin dissolved in 5 ml of absolute alcohol; add 100 ml of 5% aqueous phenol. Mix well and fi lter before use. Filter before each use with #1 fi lter paper. 25% ethanol 95 ml Sulfuric acid, concentrated 5 ml Methylene blue 1.4 g 95% alcohol 100 ml Stock methylene blue 5 ml Tap water 45 ml Xylene-peanut oil 1 part oil : 2 parts xylene Method 1. Deparaffi nize in two changes of xylene-peanut oil, 6 minutes each. 2. Drain slides vertically on paper towel and wash in warm, running tap water for 3 minutes. (The residual oil preserves the sections and helps accentuate the acid fastness of the bacilli.) 3. Stain in carbol fuchsin at room temperature for 25 minutes. (Solution may be poured back into bottle and reused.) 4. Wash in warm, running tap water for 3 minutes. 5. Drain excess water from slides vertically on paper towel. 6. Decolorize with 5% sulfuric acid in 25 % alcohol, two changes of 1.5 minutes each. (Sections should be pale pink.) 7. Wash in warm, running tap water for 5 minutes. 8. Counterstain in working methylene blue, one quick dip. (Sections should be pale blue.) 9. Wash in warm, running tap water for 5 minutes. 10. Blot sections and dry in 50-55°C oven for 5 minutes. 11. Once dry, one quick dip in xylene. 12. Mount with permanent mountant. bright red Nuclei and other tissue elements pale blue Be careful not to over-stain with methylene blue and do not allow sections to dry between carbol fuchsin and acid alcohol. Cresyl violet acetate method for Helicobacter sp. Formalin fi xed, paraffi n. 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Filter 0.1% cresyl violet acetate onto slide or into Coplin jar, 5 min. 3. Rinse in distilled water. 4. Blot, dehydrate rapidly in alcohol, clear, and mount. Helicobacter and nuclei blue-violet Background shades of blue-violet This simple method allows for good differentiation of Helicobacter sp. from other organisms. Gimenez method for Helicobacter pylori (Gimenez 1964; McMullen et al 1987) Sections Formalin fi xed, paraffi n. Buffer solution (phosphate buffer at pH 7.5, or 0.1 M) 0.1 M sodium dihydrogen orthophosphate 3.5 ml 0.1 M disodium hydrogen orthophosphate 15.5 ml Commercial cold acid-fast bacilli stain, or Basic fuchsin 1 g Absolute alcohol 10 ml 5% aqueous phenol 10 ml Filter before use. Phosphate buffer 10 ml Stock carbol fuchsin 4 ml Filter before use. Malachite green 0.8 g Distilled water 100 ml Method 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Stain in working carbol fuchsin solution, 2 min. 3. Wash well in tap water. 4. Stain in malachite green, 15-20 seconds. 5. Wash thoroughly in distilled water. 6. Repeat steps 4 and 5 until section is blue-green to the naked eye. 7. Blot sections dry, and complete drying in air. 8. Clear and mount. Helicobacter red-magenta Background blue-green The greatest problem with this method is over staining, or irregularity of staining, with Malachite green. It is valuable in demonstrating the Legionella bacillus in postmortem lung smears. Formalin fi xed, paraffi n. Sorenson's phosphate buffer pH 6.8 50 ml 1% aqueous toluidine blue 1 ml Method 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Stain in buffered toluidine blue, 20 min. 3. Wash well in distilled water. 4. Dehydrate, clear, and mount. Warthin-Starry method for spirochetes (Warthin & Starry 1920) Sections Formalin fi xed, paraffi n. Sodium acetate 4.1 g Acetic acid 6.25 ml Distilled water 500 ml Dissolve 3 g of hydroquinone in 10 ml pH 3.6 buffer, and mix 1 ml of this solution and 15 ml of warmed 5% Scotch glue or gelatin; keep at 40°C. Take 3 ml of 2% silver nitrate in pH 3.6 buffer solution and keep at 55°C. Mix these two solutions immediately before use. 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Celloidinize in 0.5% celloidin, drain, and harden in distilled water, 1 min. 3. Impregnate in preheated 55-60°C silver solution (b), 90-105 minutes. 4. Prepare and preheat developer in a water bath. 5. Treat with developer (solution c) for 3 1 / 2 minutes at 55°C. Sections should be golden-brown at this point. 6. Remove from developer and rinse in tap water for several minutes at 55-60°C, then in buffer at room temperature. 7. Tone in 0.2% gold chloride. 8. Dehydrate, clear, and mount. It is wise to take a few slides through at various incubation times to insure optimum impregnation. Modifi ed Steiner for fi lamentous and nonfi lamentous bacteria (Steiner & Steiner 1944 ; modifi ed Swisher 1987) Formalin fi xed, paraffi n. Uranyl nitrate 1 g Distilled water 100 ml Silver nitrate 1 g Distilled water 100 ml Make fresh each time and fi lter with #1 or #2 fi lter paper before use. Silver nitrate 0.04 g Distilled water 100 ml Refrigerate and use for only 1 month. Gum mastic 2.5 g Absolute alcohol 100 ml Allow to dissolve for 24 hours then fi lter until clear yellow before use. Refrigerate unused portion. Hydroquinone 1 g Distilled water 25 ml Make fresh solution for each use. Techniques for mycobacteria SOME IMPORTANT BACTERIA Staphylococcus aureus is perhaps the most important pathogen of this group. It causes boils, wound and burn infections, and a form of cavitating pneumonia in children and adults. Septicemic states and the formation of multiple scattered abscesses sometimes occur. Staphylococci tend to form clusters (cf. streptococci). Multiresistance to antibiotics is sometimes encountered. Mix 10 ml of 2.5% gum mastic, 25 ml of 2.0% hydroquinone, and 5 ml absolute alcohol. Make just prior to use and fi lter with #4 fi lter paper; add 2.5 ml of 0.04% silver nitrate. Do not fi lter this solution. When gum mastic is added, solution will have a milky appearance. Do not boil. Remove from oven, loosely cover jar, and allow to stand in hot silver nitrate, 6-7 min; alternatively, preheat silver nitrate for 20-30 min in a 60°C water bath, add slides, and allow to impregnate for 1 1 / 2 hours. 5. Rinse in three changes of distilled water. 6. Dehydrate in two changes each of 95% alcohol and absolute alcohol. 7. Treat with 2.5% gum mastic, 5 min. 8. Allow to air dry, 5 min. 9. Rinse in two changes of distilled water. Slides may stand here while reducing solution is being prepared. 10. Reduce in preheated reducing solution at 45°C in a water bath for 10-25 min, or until sections have developed satisfactorily with black microorganisms against a light yellow background. Avoid intensely stained background. 11. Rinse in distilled water to stop reaction. 12. Dehydrate, clear, and mount. (Fig. 17.2) Spirochetes, cat-scratch dark brown-black organisms, Donovan bodies, non-fi lamentous bacteria of L. pneumophila Bring all solutions to room temperature before using. All glassware making contact with silver nitrate should be chemically cleaned. Avoid the use of metal forceps in silver solutions. When doing a bacterial screen, Gram controls should be run along with diagnostic slides. As spirochetes take longer to develop, Gram controls should be used in addition to spirochete controls. When Gram controls have a yellow appearance, remove them to distilled water, and check on microscope for microorganisms. Return to silver solution if they are not ready, and repeat, realizing that spirochetes will take longer. Most solutions can be made in large quantities and kept in the refrigerator. Neisseria meningitidis (meningococcus) is a common cause of meningitis, and may produce a fulminating septicemia. Organisms can be seen in histological sections of meningococcal meningitis, but are diffi cult to see because they are usually within neutrophil cytoplasm. Neisseria gonorrheae (gonococcus) is the cause of gonorrhea. Organisms may be seen within polymorphs in sections of cervix, endometrium, or fallopian tubes in cases of gonorrhea, but, again, are diffi cult to fi nd. Members of the Neisseria family are generally diffi cult to see in histological sections, although easily detectable in smears of fresh pus or cerebrospinal fl uid (CSF), characteristically in pairs. They are easier to detect using the Gram-Twort method. Lactobacillus acidophilus (Doderlein's bacillus) is a normal inhabitant of the human vagina and is seen in cervical smears taken in the secretory phase of the cycle. Corynebacterium vaginale is a short Gram-negative bacillus which may cause cervicitis, and is present in about 6% of women of childbearing age. It may be seen in cervical smears where it accumulates as bluestained masses on the surface of squamous cells stained by Papanicoloau's method; these cells are known as 'clue cells'. Helicobacter pylori is frequently seen in endoscopic biopsies. A spiral vibrio organism is heavily implicated as the organism causing many cases of chronic gastritis. It is seen as small, weakly hematoxyphilic organisms (usually in clumps) in the lumina of gastric glands, often adherent to the luminal surface of the epithelial cells. With practice, these can be identifi ed from an H&E stain, but Warthin-Starry, Steiner, Gimenez, toluidine blue, or cresyl violet acetate methods demonstrate them more clearly. A commercial specifi c antiserum has recently become available for their demonstration. Clostridium diffi cile causes pseudomembranous colitis, an infl ammation of the large bowel. This arises following the administration of broad-spectrum antibiotics; the balance of the normal anerobic gut microfl ora is disturbed, allowing the organism to proliferate unchecked. C. diffi cile is diffi cult to stain but the 'explosive lesions' of purulent necrosis of the epithelium and lamina propria of the gut, giving rise to a 'mini volcano' effect, are a good indicator. Listeria monocytogenes is the cause of a rare form of meningitis and may cause septicemia in humans. Focal necrosis with macrophages that contain tiny intracellular rods arranged in a 'Chinese letter' formation, and staining variably with the Gram stain, are the hallmark of this disease. Mycobacterium tuberculosis remains a signifi cant pathogen in developed countries where the familiar caseating granulomatous lesion and its associated 1-2-μm, blunt-ended, acid and alcohol-fast bacilli can still be seen. In Africa and other countries, this organism has developed an opportunistic relationship with AIDS, where it is a major cause of death. Mycobacterium avium/intracellulare are representatives of a group of intracellular opportunistic mycobacteria that are frequently present in the later stages of immunosuppression, particularly that associated with AIDS. They frequently persist in spite of treatment, and are often lethal. The lesions produced are non-caseating and consist of collections of vacuolated macrophages that often contain vast numbers of organisms. On occasion, there is little evidence of a cellular reaction on an H&E-stained section, and the organism is detected only by routinely performing an acid-fast stain, such as the ZN, on all tissue from AIDS patients. This group also includes M. kansasii. Mycobacterium leprae is an obligate intracellular, neurotrophic mycobacterium that attacks and destroys nerves, especially in the skin. Tissue reaction to leprosy depends on the immune status of the host; it can be minimal with a few macrophages packed with crescentic, pointed, intracytoplasmic bacilli (lepromatous leprosy), or may contain scanty organisms and show fl orid granulomatous response (tuberculoid leprosy). M. leprae is only acid-fast and can often be demonstrated with a standard Ziehl-Neelsen technique. Legionella pneumophila was fi rst identifi ed in 1977 as the cause of a sporadic type of pneumonia of high mortality. The small Gram-positive coccobacillus is generally spread in aerosols from stagnant water reservoirs, usually in air-conditioning units. The bacterium may be diffi cult to stain except with the Dieterle and modifi ed Steiner silver stains, and specifi c antiserum. Treponema pallidum is the organism causing syphilis, and is infrequently seen in biopsy specimens as the primary lesion or 'chancre' is diagnosed clinically. The spirochete is quite obvious using dark-ground microscopy, as an 8-13-μm corkscrew-shaped microorganism that often kinks in the center. Dieterle, Warthin-Starry, or modifi ed Steiner methods demonstrate the organism; specifi c antiserum is also available. Leptospira interrogans is the organism causing leptospirosis or Weil's disease. It is a disease characterized by spirochetes, and is spread in the urine of rats and dogs, causing fever, profound jaundice, and sometimes death. Spirochetes can be seen in the acute stages of the disease where they appear in Warthin-Starry and modifi ed Steiner techniques as tightly wound 13-μm microorganisms with curled ends resembling a shepherd's crook. Intestinal spirochetosis appears as a massive infestation on the luminal border of the colon by spirochete Brachyspira aalborgi (Tomkins et al 1986) . It measures 2-6 μm long, is tightly coiled, and arranged perpendicularly to the luminal surface of the gut, giving it a fuzzy hematoxyphilic coat in an H&E stain. There is no cellular response to the presence of this spirochete. It is seen well with the Warthin-Starry and the modifi ed Steiner techniques. Cat-scratch disease presents as a self-limiting, local, single lymphadenopathy appearing about 2 weeks after a cat scratch or bite. Histologically the node shows focal necrosis or micro-abscesses. Two Gram-negative bacteria (Afi pia felis and Bartonella henselae) have been implicated. Because of the timing or maturation factor of the bacterium, it is diffi cult to demonstrate on paraffi n sections, but the modifi ed Steiner and the Warthin-Starry methods are valuable techniques for demonstrating this organism. Fungi are widespread in nature, and humans are regularly exposed to the spores from many species, yet the most commonly encountered diseases are the superfi cial mycoses that affect the subcutaneous or horny layers of the skin or hair shafts, and cause conditions such as athlete's foot or ringworm. These dermatophytic fungi belong to the Microsporum and Trichophyton groups and may appear as yeasts or mycelial forms within the keratin. They are seen fairly well in the H&E stain, but are demonstrated well with the Grocott and PAS stains. As with other infections, the increase in the number of patients with diminished or compromised immune systems has increased the incidence of systemic mycoses, allowing opportunistic attacks by fungi, often of low virulence, but sometimes resulting in death. When fungi grow in tissue they may display primitive asexual (imperfect) forms that appear as either spherical yeast or spore forms. Some may produce vegetative growth that appears as tubular hyphae that may be septate and branching; these features are important morphologically for identifying different types of fungi. A mass of interwoven hyphae is called a fungal mycelium. Only rarely, when the fungus reaches an open cavity, the body surface, or a luminal surface such as the bronchus, are the spore-forming fruiting bodies called sporangia, or conidia, produced. Some fungi may elicit a range of host reactions from exudative, necrotizing, to granulomatous; other fungi produce little cellular response to indicate their presence. Fortunately, most fungi are relatively large and their cell walls are rich in polysaccharides, which can be converted by oxidation to dialdehydes and thus detected with Schiff's reagent or hexamine-silver solutions. Fungi are often weakly hematoxyphilic. Some fungi, such as sporothrix, may be surrounded by a stellate, strongly eosinophilic, refractile Splendore-Hoeppli precipitates of host immunoglobulin and degraded eosinophils. Fluorochrome-labeled specifi c antibodies to many fungi are available, and are in use in mycology laboratories for the identifi cation of fungi on fresh and paraffi n sections. These antibodies have not found widespread use, however, on fi xed tissue where identifi cation still relies primarily on traditional staining methods. An H&E stain, a Grocott methenamine (hexamine)silver (GMS), a mounted unstained section to look for pigmentation, and a good color atlas (Chandler et al 1980) when experience fails, permit most fungal infections to be identifi ed to levels suffi cient for diagnoses. However, there is no substitute for microbiological culture. Grocott methenamine (hexamine)-silver for fungi and Pneumocystis spp. organisms (Gomori 1946; Grocott 1955; Swisher & Chandler 1982) Sections Formalin fi xed, paraffi n. Refrigerate for up to 3 months. Equal parts of solutions A and B. Make fresh each time and fi lter before use. This detail is essential for critical identifi cation, and is best seen on under-impregnated sections. To avoid excess glycogen impregnation in liver sections, section may be digested prior to incubation. A water bath may be used effectively to insure an even incubation temperature. b. Borax insures an alkaline pH. c. Sodium bisulfi te removes excess chromic acid. d. Some workers prefer a light H&E counterstain. This is especially useful when a consulting case is sent with only one slide, providing morphological detail for the pathologist. e. Solutions A and B need to be made and stored in chemically clean glassware (20% nitric acid), as does the working solution. This includes graduates and Coplin jars. Do not use metal forceps. f. Allow all refrigerated solutions to reach room temperature before using. Fixation 10% NBF. 3-5-μm paraffi n sections. found in liver, appendix, lung, and neck. The individual organisms are Gram-positive, hematoxyphilic, non-acidfast, branching fi laments 1 micron in diameter. They become coated in 'clubs' of Splendore-Hoeppli protein when the organism is invasive. These clubs are eosinophilic, acid-fast, 1-15 μm wide, and up to 100 μm long, and stain polyclonally for immunoglobulins. This arrangement of a clump of actinomyces or fungal hyphae, which measures 30-3000 μm, surrounded by eosinophilic protein, is called a 'sulfur' granule and is an important identifi cation marker for certain fungal groups. These granules may be macroscopically visible and their yellow color is an important diagnostic aid. Nocardia asteroides is another actinomycete. It is fi lamentous and may be visible in an H&E stain, but is Grocott positive and variably acid-fast using the modifi ed Ziehl-Neelsen for leprosy; however, it is diffi cult to demonstrate even with the acid-fast bacillus. Its pathology is similar to that of actinomycosis, but its organisms are generally more disseminated than those of actinomycosis. Candida albicans is a common fungus, but with immunosuppression can become systemic. It infects the mouth as thrush, the esophagus, the vagina as vaginal moniliasis, the skin and nails, and is in heart-valve vegetations. It is seen as both ovoid budding yeast-form cells of 3-4 μm, and more commonly as slender 3-5-μm, sparsely septate, non-branching hyphae and pseudohyphae. While diffi cult to see on H&E, this organism is strongly Gram positive, and is obvious with the Grocott and PAS techniques. Aspergillus fumigatus is a soil saprophyte and a commensal in the bronchial tree. It may infect old lung cavities (Fig. 17. 3) or become systemic in immunosuppressed patients. The fungus has broad, 3-6-μm, parallelsided, septate hyphae showing dichotomous (45°C) branching. It may be associated with Splendore-Hoeppli protein and sometimes forms fungal balls within tissue. This fungus may be seen in an H&E stain and is demonstrated well with a PAS or Grocott. When it grows exposed to air, the conidophoric fruiting body may be seen as Aspergillus niger, a black species that can cause infection of the ear. Zygomycosis is an infrequently seen disease caused by a group of hyphated fungi belonging mainly to the genera Mucor and Rhysopus. They have thin-walled hyphae (infrequently septate) with non-parallel sides, ranging from 3 to 25 μm in diameter, branch irregularly, and often show empty bulbous hyphal swelling. Fungal cell walls and glycogen magenta to red Background pale green A solution of 5% aqueous sodium hypochlorite reduces over-staining by Schiff's. Actinomyces israelii is a colonial bacterium which can be found as a commensal in the mouth and tonsillar crypts. It can cause a chronic suppurative infection, actinomycosis, which is characterized by multiple abscesses drained by sinus tracts. Actinomycotic abscesses can be Grocott and PAS are the staining methods of choice (Figs 17.4 and 17.5) . Cryptococcus neoformans exists solely in yeast-form cells, variable in diameter (2-20 μm) with ovoid, elliptical, and crescentic forms frequently seen. There is an extensive mucopolysaccharide coat around the yeasts that is mostly dissolved during processing, but, when present, appears as a halo around the organism and is visible with special stains such as Mayer's or Southgate's mucicarmine procedures. Yeasts may be free form or within the cytoplasm of giant cells, staining faintly with an H&E stain. The PAS and Grocott procedures demonstrate these cells well. Infection is found in the lungs and in the brain within the parenchyma or in the leptomeninges. Usually these patients are immunosuppressed. Histoplasma capsulatum is another soil-dwelling yeast that can cause a systemic infection in humans called histoplasmosis. It is especially common along the southern border of the United States, and where there are large bird populations. The organism is usually seen within the cytoplasm of macrophages that appear stuffed with small, regular, 2-5-μm yeast-form cells that have a thin halo around them in H&E and Giemsa stains. Langhans' giant cells forming non-caseating granulomas may be present. PAS and Grocott stains demonstrate this fungus well (Fig. 17.6) . Pneumocystis carinii. There is still some debate over the taxonomy of this organism, although recent analysis of its ribosomal RNA has placed it nearer to a fungal than a protozoan classifi cation (Edman et al 1988) . It came to prominence as a pathogen following immunosuppressive therapies associated with renal transplants in the 1960s, and has become a life-threatening complication of AIDS. It most frequently causes pneumonia, where the lung alveoli are progressively fi lled with amphophilic, foamy plugs of parasites and cellular debris. It is found rarely in other sites such as intestines and lymph nodes. The cysts are invisible in an H&E stain, and can barely be seen in a Papanicolaou stain, as they appear refractile when the microscope condenser is racked down. Specifi c antiserum is available to use; otherwise Grocott methenamine-silver is recommended. Only electron microscopy or an H&E stain on a resinembedded thin section will show their internal structure. The cysts are 4-6 μm in diameter and contain 5-8 dotlike intracystic bodies. The cysts rupture and collapse, liberating the trophozoites which can be seen as small hematoxyphilic dots in a good H&E and Giemsa stain; these attach to the alveolar epithelium by surface philopodia. Rickettsial organisms, such as those causing Q fever, Rocky Mountain spotted fever, or typhus, rarely need to be demonstrated in tissue sections. They can sometimes be seen with a Giemsa stain, or by using the Macchiavello technique which also demonstrates some viral inclusion bodies (Fig. 17.7) . While the cytopathic effects of viruses can often be seen in a good H&E stain, and may be peculiar to a single viral group, the individual viral particles are too small to be seen with the light microscope, thus requiring the electron microscope to reveal their structure. This allows a rapid and accurate diagnosis in viral infections; an outline of the value of electron microscopy in the diagnosis of viral lesions is given in Chapter 30. Some viruses aggregate within cells to produce viral inclusion bodies, that may be intranuclear, intracytoplasmic, or both. These inclusion bodies may be acidophilic and usually intranuclear, or can be basophilic and cytoplasmic. Most special staining methods are modifi ed trichromes, using Macchiavello's stain for rickettsia and viral inclusions, modifi ed (Culling 1974) Formalin fi xed, paraffi n. 1. Deparaffi nize and rehydrate through graded alcohols to distilled water. 2. Stain in 0.25% basic fuchsin, 30 min. 3. Differentiate in 0.5% citric acid, 3 seconds. 4. Wash in tap water, 2 min. 5. Counterstain in 1% methylene blue, 15-30 seconds. 6. Rinse in tap water. 7. Dehydrate, clear, and mount. contrasting acid and basic dyes to exploit these differences in charges on the inclusion body and the host cell. These methods include Mann's methyl blue-eosin stain for the Negri bodies of rabies, Machiavello's method, and more recently the elegant Lendrum's phloxine-tartrazine stain. Unfortunately, the need for optical differentiation in these methods increases the chance of technical error. The introduction of commercially available monoclonal antibodies to viruses, which are either class-or species specifi c, has revolutionized the tissue detection of viruses. Hepatitis B virus is a good example of the diagnostic value of this technique where the surface antigen (HBs or Australia antigen) and the core antigen (HBc) can be specifi cally detected immunohistochemically, providing clinically important information about the stage of this disease. More recently, nucleic acid hybridization probes have become available and can be used to detect genomically inserted viral nucleic acid in situ, in cells and tissues that are frozen or formalin fi xed. It should be remembered, however, that the detection of microorganisms using nucleic acid probes, unlike specifi c biotinylated antiserum, does not necessarily mean active disease. Phloxine-tartrazine technique for viral inclusions (Lendrum 1947) Formalin fi xed, paraffi n. This summary is presented because of the viruses that are likely to be encountered in surgical and post-mortem histopathology and cytopathology (Table 17 .3). Viral hepatitis. To date, fi ve hepatitis viruses have been reported, hepatitis viruses (HV) A, B, C, D, and E, that show great biological diversity, and three of which are incompletely characterized. The liver is the target organ and damage varies with the viral strain, ranging 5. Stain in orcein solution at room temperature, 4 hours, or in a Coplin jar of 37°C pre-heated orcein, 90 min. 6. Rinse in distilled alcohol and examine microscopically to determine desired staining intensity. 7. Rinse in cellosolve, stain in tartrazine, 2 min. 8. Rinse in cellosolve, clear, and mount. Hepatitis B-affected cells, elastic brown-black and some mucins Background yellow The success of this method largely depends on the particular batch of orcein used, and on freshly prepared solutions. This method relies on permanganate oxidizing of sulfur-containing proteins to sulfonate residues that react with orcein. Results compare well with those obtained using labeled antibodies, but the selectivity is inferior. Immunohistochemistry is now a routine and invaluable procedure in the histopathology lab for the detection of many microorganisms. There are many commercially available antibodies for viral, bacterial, and parasitic Herpes viruses are usually acquired subclinically during early life and enter a latent phase, to be reactivated during times of immunological stress. These viruses cause blistering or ulceration of the skin and mucous membranes, but can cause systemic diseases, including encephalitis, in immunosuppressed or malnourished individuals. The cytopathic effects of the herpes virus are well seen in Tzanck smears of blister fl uid, and include the margination of chromatin along nuclear membranes, Cowdry type A ('owl's eye') inclusion bodies, and syncytial or 'grape-like' nuclei within giant cells. Cytomegalovirus (CMV) is sometimes seen as a systemic opportunistic infection in AIDS patients. It is seen in the endothelial cells, forming prominent intranuclear inclusions that spill into the cytoplasm where they form granular hematoxyphilic clusters. The CMV virus causes obvious cytomegaly in the cells it infects. All herpes viruses have an identical electron microscopic appearance of spherical, 120-nm, membrane-coated particles. Papilloma viruses are a family of about 50 wart viruses that cause raised verrucous or papillomatous skin warts, or fl at condylomatous genital warts. Cytologically, evidence of hyperkeratosis may be present together with koilocytosis (irregular nuclear enlargement and cytoplasmic vacuolation forming perinuclear halos). Skin verrucas are associated with HPV 1-4 strains, genital condylomas with HPV 6, 11, 16, and 18, and cervical cancer with HPV 16 and 18. These uncoated viruses measure 55 nm, are mainly intranuclear, and can be detected using electron microscopy, or immunoperoxidase and gene probes on paraffi n sections. JC virus is a papova virus that causes progressive multifocal leucoencephalopathy, a demyelinating disease, in AIDS and other immunosuppressed patients. Intranuclear hematoxyphilic inclusions may be seen within swollen oligodendrocytes. Molluscum virus produces a contagious wart in children and young adults called molluscum contagiosum. Large eosinophilic, intracytoplasmic inclusion bodies can be seen in maturing keratinocytes, and are seen well with phloxine-tartrazine. The large 1-μm viral particles have a typical pox virus structure: brick-shaped with a superimposed fi gure-of-eight nucleic acid sequence. Rabies virus. This neurotrophic rhabdovirus forms intracytoplasmic eosinophilic inclusions best seen in the axonal hillocks of hippocampal neurons of the brain. Machiavello, phloxine-tartrazine, Mann's methyl blueeosin, or PAS stains are recommended. Human immunodefi ciency virus (HIV) consists of at least two retrovirus strains. The virus is best seen in cultured lymphocytes and is rarely seen in tissues from AIDS patients. It produces a distinctive neuropathological lesion in AIDS encephalitis consisting of microglial nodules, or stars, containing collections of giant cells, microglia, and astrocytes. Synthetic nucleic acid probes have been prepared to HIV genomes. Infl uenza virus (fl u) is a contagious respiratory illness caused by infl uenza viruses ( Fig. 17.8) . It can cause mild to severe illness, and at times can lead to death. According to the Centers for Disease Control (CDC), every year in the United States, on average: • 5-20% of the population suffers from the fl u • more than 200,000 people are hospitalized from fl u complications • about 36,000 people die from fl u. Some people, such as older people, young children, and people with certain health conditions, are at high risk for serious fl u complications. SARS (severe acute respiratory syndrome) is a viral respiratory illness caused by a coronavirus called SARSassociated coronavirus (SARS-CoV) (Fig. 17.9 ). SARS was fi rst reported in Asia in February 2003. Over the next few months, the illness spread to more than two dozen countries in North America, South America, Europe, and Asia before the SARS global outbreak of 2003 was contained. To date, more than eight transmissible neurodegenerative diseases have been described affecting the central nervous system (CNS). The diseases caused by prions include Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD), Germann-Straussler-Shienker disease, fatal familial insomnia, and kuru in humans, bovine spongiform encephalopathy (BSE, also known as 'mad cow disease'), scrapie (in goats and sheep), and chronic wasting disease (CWD) (in mule deer and elk). In addition, prions are not microbes in the usual sense because they are not alive, but the illness they cause can be transmitted from one animal to another. All usually produce a characteristic spongiform change, neuronal death, and astrocytosis in affected brains. The infectious agent is a prion, a small peptide, free of nucleic acid and part of a normal transmembrane glycoprotein which is not, strictly speaking, a virus. Antibodies have been prepared from prion protein that strongly mark accumulated abnormal protein in these diseases (Lantos 1992) . The CJD Surveillance Center in the USA is an invaluable source for monitoring and testing of human prion disease in the United States. The Center is supported by the CDC and by the American Association of Neuropathologists. Visit their website (http://www.cjdsurveillance.com) for details on how to submit specimens for testing; they perform these tests at no charge for laboratories in the USA. In addition, both CDC and the World Health Organization (WHO) also offer guidelines regarding the handling of suspected and known cases of prion disease. Visit http://www.cdc.gov and search for CJD for a fact sheet and other relevant information. WHO offers a manual in pdf form for downloading. It gives information about what to do should you fi nd yourself with a suspected or known positive case in your lab: http:// who.int/bloodproducts/TSE-manual2003.pdf. Remember that these types of cases should never knowingly be handled in a routine histology lab. Contact your local health department for additional guidelines. The identifi cation of protozoa is most often made on morphological appearance using H&E and, particularly, Giemsa stains. The availability of antisera against organisms such as entamoeba, toxoplasma, and leishmania has made diagnosis much easier in diffi cult cases ( Fig. 17 .10). Fig. 17 .9 Immunohistochemical method demonstrating the previously unrecognized SARS-associated coronavirus which is responsible for severe acute respiratory syndrome (SARS) (×20). Fixative is not critical, but B5 or Zenker's is preferred; thin (3 μm) paraffi n sections. (If Zenker's is not used, post-mordant in Zenker's in a 60°C oven for 1 hour before staining.) Giemsa stain powder 4 g Glycerol 250 ml Methanol 250 ml Dissolve powder in glycerol at 60°C with regular shaking. Add methanol, shake the mixture, and allow to stand for 7 days. Filter before use. Giemsa stock 4 ml Acetate buffered distilled water, pH 6.8 96 ml Method 1. Deparaffi nize and rehydrate through graded alcohols to water. 2. Rinse in pH 6.8 buffered distilled water. 3. Stain in working Giemsa, overnight. 4. Rinse in distilled water. 5. Rinse in 0.5% aqueous acetic acid until section is pink. 6. Wash in tap water. 7. Blot until almost dry. 8. Dehydrate rapidly through alcohols, clear, and mount. Protozoa and some other dark blue microorganisms Background pink-pale blue Nuclei blue Entamoeba histolytica, the organism causing amebic colitis or dysentery, can be found in ulcers that occur in infected colon and in amebic liver abscesses. The trophozoite (adult form) measures 15-50 μm, contains a small nucleus, and has a foamy cytoplasm containing ingested red cells and white cell debris. They may be seen in granulation tissue within ulcers, or in the luminal mucus overlying normal appearing mucosa, and are PAS positive; brief counterstaining in 1% aqueous metanil yellow emphasizes the ingested red cells. Toxoplasma gondii, a commonly encountered organism that is spread in cat litter, causes an acute lymphadenopathy which is often subclinical. Affected nodes show non-specifi c changes and no organisms. In AIDS and other immunosuppressed patients this protozoon causes systemic diseases, including meningoencephalitis where encysted bradyzoites and free tachyzoites can be seen in necrotic brain tissue. Cysts also occur in other tissues such as cardiac muscle, and measure up to 40 μm with tachyzoites 4-6 μm, which can be seen on H&E. A Giemsa stain can also be used, but the use of labeled specifi c antiserum is recommended. Leishmania tropica is transmitted by sandfl y bite and causes a chronic infl ammatory disease of the skin sometimes called cutaneous leishmaniasis. The injected parasite forms (2 μm), or amastigotes, are found in large numbers within the cytoplasm of multiple swollen histiocytes that congregate in early lesions in the dermis. A related organism, L. donovani, causes a systemic visceral infection, kala azar, in which the organisms are seen within histiocytes in spleen, lymph nodes, liver, and bone marrow. The organisms are hematoxyphilic and can be emphasized with a Giemsa stain. Giardia duodenalis (lamblia) is a fl agellate protozoon that is ingested in cyst form from drinking water with fecal contamination; the trophozoites migrate to the duodenum where they may cause severe diarrhea and malabsorption. These organisms can easily be missed on an H&E stain, where they appear as eosinophilic, sickleshaped fl akes with indistinct nuclei resting on intestinal mucosa that may show little evidence of infl ammation. When seen in a fresh Giemsa-stained duodenal aspirate, they appear kite-shaped, 11-18 μm in size, binucleate, and have faint terminal fl agella. Trichomonas vaginalis is a similar fl agellate protozoon most frequently seen in a Papanicolaou stain. Infl ammatory cells and mildly dysplastic squamous cells often accompany this parasite as it causes cervicitis in the female, and urethritis in both sexes. Cryptosporidium is one of a group of protozoa (including Isospora and Microsporidium) that causes severe and relentless outbreaks of diarrhea among AIDS patients. Cryptosporidial gametes are seen on H&E stain as blue dots arranged along the mucosal surface. Mature cysts are shed into feces and are acid fast in a ZN stain of fecal smears. Schistosoma species cause the disease schistosomiasis or 'bilharzia'. Various manifestations of the disease differ according to the particular Schistosoma species involved, but granulomata containing schistosome ova are found in the liver, bowel, and bladder mucosa, and sometimes in the lungs. The ova have thick, refractile, eosinophilic walls and are easily detected in H&E-stained sections. The PAS, Grocott, and ZN techniques are positive for these ova. Where the plane of section allows, the presence of a terminal spine to the ovum indicates S. haematobium whereas S. mansoni and japonicum have lateral spines. Any good trichrome procedure will demonstrate worm development. Echinococcosis. Echinococcus granulosus is a tapeworm found in dogs; humans and sheep may become intermediate hosts and develop hydatid cyst disease. These cysts form in many organs, particularly liver and lung. The walls of the daughter cysts are faintly eosinophilic, characteristically laminated, and produced by the worm, not by its host. The walls are PAS positive and Congo red positive, showing green birefringence. The scolicial hooklets survive inside old, burnt-out cysts, are of diagnostic shape, and stain brilliant yellow with picric acid. A simple and effective method for inactivating virus infectivity in formalin-fi xed samples from patients with Creutzfeldt-Jakob diseases A colour atlas and textbook of the histopathology of mycotic diseases A method for demonstrating Gram-positive and Gram-negative bacteria Modifi ed Fite stain for demonstration of mycobacterium species in tissue sections Handbook of histopathological and histochemical techniques Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi Staining rickettsia in yolk sac cultures A new histochemical test for glycogen and mucin A stain for fungi in tissue sections and smears A note on Uhlenhuth's method for sputum examination for tubercle bacilli Detection of acid-fast organisms in tissue sections by fl uorescence microscopy From slow virus to prion; a review of the transmissible spongioform encephalopathies The phloxine-tartrazine method as a general histological stain for the demonstration of inclusion bodies Histological identifi cation of campylobacter using Gimenez technique in gastric antral mucosa A method for staining both Gram positive and Gram negative bacteria in sections Staining methods of Australia antigen in paraffi n section-detection of cytoplasmic inclusion bodies New simple silver stain for demonstration of bacteria, spirochetes, and fungi in sections of paraffi n embedded tissue blocks Modifi ed Steiner procedure for microwave staining of spirochetes and nonfi lamentous bacteria Grocott methenamine silver method for detecting fungi: practical considerations Development of staining controls for Campylobacter pylori Isolation and characterization of intestinal spirochetes An improved neutral red, light green double stain for staining animal parasites, microorganisms and tissues Pathology of infectious diseases A more rapid and improved method of demonstrating spirochetes in tissues Gonorrhea, and Veneral Diseases Immunochemical staining methods handbook Manual of histologic staining methods of the Armed Forces Institute of Pathology Ein Casuistischer Beitrag zur Lehre von Tuberkulose 1882) Zur Farbung des Tuberkelbacillus Alan Stevens contributed this chapter for the fi rst three editions, and he and Bob Francis updated the fourth edition. Billie Swisher contributed the chapter for the fi fth edition. Our acknowledgments are due to them for their contributions. I would also like to thank Sherif Zaki, Jeannette Guarner, and Mitesh Patel for their assistance with this chapter.