Traditional stains and modern techniques for demonstrating microorganisms in histology

Abstract

Introduction Microorganisms (or microbes) are organisms which can only be individually seen by microscopy. Many do not cause disease in humans and act as normal colonizers of human hosts. Complex interactions between pathogens, which are capable of causing diseases, the host and the environment lead to clinical infections. Pathogens fall into five main groups: • Viruses • Bacteria • Fungi • Protozoa • Helminths. With the advent of new and broader spectrum antibiotics, improved environmental hygiene, and advances in microbiological techniques it was widely expected that the need for diagnosis of infectious agents in tissue would diminish in importance. This assumption underestimated the infinite capacity of infectious agents for genomic variation, enabling them to develop antimicrobial resistance and exploit new opportunities to spread infections which are created when host defenses become compromised. The following are currently the most important factors influencing the presentation of infectious diseases: • The 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. • Immunodeficiency states occurring either as part of an infection, e.g. Human Immunodeficiency Virus (HIV) which causes acquired immune deficiency syndrome (AIDS), or as an iatrogenic disease. As treatment becomes more aggressive, depression of the host’s immunity occurs, enabling organisms of low virulence to become life-threatening, and may allow latent infections, accrued throughout life, to reactivate and spread. • Emerging, re-emerging and antibiotic-resistant organisms such as the tubercle bacillus and staphylococcus are a constant and growing threat. • Adaptive mutation occurring in microorganisms allows them to jump species barriers and exploit new physical environments. Such adaptation allows infections to evade host defenses and resist agents of treatment. • Bioterrorism has become an increasing concern. The world’s public health systems and primary healthcare providers must be prepared to address varied biological agents, including pathogens which are rarely seen in developed countries. High-priority agents include organisms which pose a risk to national security because they: ◦ Can easily be disseminated or transmitted from person to person. ◦ Cause high mortality, with potential for a major public health impact. ◦ May 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 (various). These factors, acting singly or together, provide an ever-changing picture of infectious disease where clinical presentation may involve multiple pathological processes, unfamiliar organisms, and modification of the host response by a diminished immune status. Size The term ‘microorganism’ has been interpreted liberally in this chapter. Space limitation precludes a comprehensive approach to the subject, and the reader is referred to additional texts e.g. von Lichtenberg (1991) and Mandell et al. (2014) for greater depth. The organisms in Table 16.1 are discussed and techniques for their demonstration are described. Table 16.1 Size of organisms Organisms Size Viruses 20–300 nm Mycoplasmas 125–350 nm Chlamydia 200–1000 nm Rickettsia 300–1200 nm Bacteria 1–14 μm Fungi 2–200 μm Protozoa 1–50 μm Metazoans 3–10 mm Safety Most infectious agents are rendered harmless by direct exposure to formal saline. Standard fixation procedures should be sufficient to kill microorganisms, one exception being material from those with Creutzfeldt-Jakob disease (CJD) and other prion diseases. It has been shown that well-fixed tissue, paraffin-processed blocks and stained slides from CJD cases remain infectious when introduced into susceptible animals. Treatment of fixed 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 unfixed material is unavoidable. When available, unfixed tissue samples should be sent for microbiological culture, as this offers the best chance of rapid and specific identification of etiological agents, even when heavy bacterial contamination may have occurred. General principles of detection and identification The diagnosis of illness from infectious diseases generally starts with clinical presentation of the patient, and in most cases a diagnosis is made without a tissue sample being taken. Cases which rely on tissue diagnosis range from autopsy specimens, where material maybe plentiful and sampling error presents little problem, through to cytology samples where cellular material is often scarce and lesions may be easily missed. A full clinical history is always 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 may often give a clear diagnosis of infection. Those with frank pus, abscess formation, cavitation, hyperkeratosis, demyelination, pseudo-membrane formation, focal necrosis and granulomas can provide evidence of infection. These appearances are often non-specific but occasionally in hydatid cyst disease or some helminth infestations the appearances are diagnostic. The microscopic appearance of routine stained sections at low-power magnification often reveals indirect evidence of the presence of infection, e.g. neutrophil or lymphocytic infiltrates, granuloma formation, micro-abscesses, eosinophilic aggregates, Charcot-Leyden crystals and caseous necrosis. Some of these appearances may be sufficiently reliable to provide an initial, or at least provisional diagnosis, and allow treatment to be started, even if the precise nature of the suspect organism is never identified, particularly in the case of tuberculosis. At the cellular level, the presence of giant cells e.g. Warthin-Finkeldy or Langhans’ type may indicate measles or tuberculosis respectively. Other cellular changes which include intra-cytoplasmic edema of koilocytes, acantholysis, spongiform degeneration of the brain, margination of chromatin, syncytial nuclear appearance, ‘ground-glass’ changes in the nucleus or cytoplasm, or inclusion bodies, can indicate infectious etiology. At some stage in these processes, suspect organisms may be visualized. It should be understood, a well-performed hematoxylin and eosin (H&E) method will stain many organisms. Papanicolaou stain and Romanowsky stains, e.g. Giemsa, will also stain 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 specific histochemical methods is required for their detection. When organisms are few in number, fluorochromes may be used to increase the microscopic sensitivity of a technique. Finally, the following two techniques offer the possibility of specific identification of microorganisms which extend to the appropriate strain level. Immunohistochemistry (IHC) Immunohistochemistry (see Chapter 19) is now a routine and invaluable procedure in the histopathology laboratory for the detection of many microorganisms. There are many commercially available antibodies for viral, bacterial and parasitic organisms. Most methods today utilize (strept)avidin-biotin technologies. These are based on the high affinity that (strept)avidin (Streptomyces avidinii) and avidin (chicken egg) have for biotin. Both possess four binding sites for biotin, but due to the molecular orientation of the binding sites fewer than four molecules of biotin will actually bind. The basic sequence of reagent application consists of primary antibody, biotinylated secondary antibody, followed by either the preformed (strept)avidin-biotin enzyme complex or the avidin-biotin complex (ABC) technique or by the enzyme-labeled streptavidin. Both conclude with the substrate solution. Horseradish peroxidase and alkaline phosphatases are the most commonly used enzyme labels. Molecular methods The application of molecular techniques for the detection of microorganisms has arguably revolutionized the diagnosis of infection. These methods represent a rapidly expanding and exciting field, particularly when considering novel and emerging infections. However, testing must be undertaken rationally and appropriately in order to produce meaningful results (Procop, 2007). Conventional staining may lack sensitivity and specificity to detect and speciate microorganisms. Microbial culture is not viable from formalin-fixed, paraffin-embedded (FFPE) specimens. In comparison, molecular identification of pathogens is rapid with high sensitivity and specificity and can be applied to a variety of histological specimens (Rogers et al., 2009). Common molecular techniques used include direct hybridization and nucleic acid amplification (often referred to under the umbrella term of polymerase chain reaction – PCR) (Procop, 2007). In situ hybridization (ISH) uses reporter synthetic DNA probes which hybridize and label specific RNA sequences in target microbes present in the sample. This technique is most useful when type or genus of the microorganism has been elucidated, e.g. to identify the exact species of staphylococcus or yeast. It has been used successfully to detect and accurately differentiate a range of morphologically related organisms such as Legionella spp., filamentous bacteria and fungi in tissue samples (Hayden et al., 2001, Hayden et al., 2004, Isotalo et al., 2009). PCR relies on the detection of unique regions of microbial DNA or RNA following the extraction and amplification of genetic material from specimens, and can be used to diagnose microbial infections from autopsy tissues and surgical specimens. Whilst fresh/frozen tissues provide the best-quality nucleic acids for analysis, DNA and RNA extracted from FFPE samples can be used successfully for PCR testing. A number of specific PCRs have been developed and applied to detect a range of viruses, bacteria, fungi and parasites in histopathological specimens (Denison et al., 2011, Surat et al., 2014, Rickerts, 2016, Gebhardt et al., 2015). Since formalin cross-links proteins and nucleic acids resulting in significant degradation, it is essential to begin processing of specimens as quickly as possible, ensuring that a 10% concentration of formalin is used for fixation, and making certain that fixation times are kept to less than 48 hours (von Ahlfen et al., 2007, Chung et al., 2008, Srinivasan et al., 2002). Individual PCRs are useful when a particular infecting organism is suspected; in contrast, micro-array and multiplex PCR has the ability to identify a variety of related and unrelated microorganisms simultaneously from a single sample (Fukumoto et al., 2015). Broader still, although less sensitive, are pan-bacterial and pan-fungal 16S and 18S RNA PCR probes which will detect the presence of any bacterial or fungal RNA. Further analysis and sequencing of any relevant genetic material identified is used to characterize the species. A benefit of investigating samples using PCR analysis is the generation of quantitative data which indicate the microbial burden. This aids interpretation of results, as the presence of an organism does not necessarily mean infection. Indeed, PCR positivity may be misleading if the patient has been exposed to prior antimicrobial agents or where the microorganism persists despite clinical resolution, as is the case with many respiratory viral infections (Lehners et al., 2016). Although relatively expensive, molecular methods of diagnosis are becoming increasingly routine and available with less restrictive costs. These techniques have a unique role to play in the identification of novel infectious diseases from histological samples, particularly at autopsy, for example, pandemic influenza virus (Shieh et al., 2010), and will continue to play a key role in the detection of emerging infections and bioterrorist attacks (Hajjeh et al., 2002) . In addition, as technology advances, it is now possible to obtain detailed genetic sequencing information which provides important information relating to microbial transmission, virulence and resistance mechanisms. This is increasingly important in an age of global communities and the advent of unprecedented antimicrobial resistance. However, further studies are still required to answer a more fundamental question, which is whether molecular testing improves patient outcomes, and this is an area for future work. In summary, molecular methods offer the ability to make a rapid and accurate diagnosis of infection of a broad range of potential pathogens. It is vital that these tests are used judiciously and interpreted with care. Whilst 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 stained section. The growing number of patients whose immune status is compromised, or those who can mount only a minimal or inappropriate response to infection, further complicates the picture. This justifies speculative use of special stains, such as those for mycobacteria and fungi on tissue from such patients. It should be remembered, that for a variety of reasons, negative results for the identification of an infectious agent do not exclude its presence. In particular, administration of antibiotics to the patient before a biopsy is often the reason for failure to detect a microorganism in tissue. Detection and identification of bacteria When bacteria are present in large numbers, in an abscess or vegetation on a heart valve, they appear as blue-gray granular masses with an H&E stain. However, organisms are often poorly visible, and can be 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 historical classification (Table 16.2 ). Table 16.2 A simplified classification of important bacteria Gram-positive bacteria Gram-negative bacteria Cocci Bacilli Cocci Bacilli Coccobacilli Staphylococcus Bacillus Neisseria Escherichia Brucella Clostridium Klebsiella Bordetella Streptococcus Corynebacterium Salmonella Haemophilus (inc. Pneumococcus) Mycobacteria (weak+) Shigella Lactobacillus (commensal) Proteus Listeria Pseudomonas Vibrio Pasteurella Use of control sections 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. For example, a pneumocystis-containing control should be used for demonstrating Pneumocystis jiroveci (previously called carinii). A Gram control should contain both Gram-positive and Gram-negative organisms. Post-mortem tissues have previously been a good source of control material, although medico-legal issues have now limited this in some countries. Alternatively, a suspension of Gram-positive and Gram-negative organisms can be injected into the thigh muscle of a rat shortly before it is sacrificed 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). The Gram stain In spite of more than a century since Gram described his technique in 1884, its chemical rationale remains obscure. Staining is due to a mixture of factors, the most important being cell wall thickness, chemical composition and the functional integrity of the 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 identification of the organism causing a lung abscess, wound infection, septicemic abscess or meningitis. Gram method for bacteria in smears (Gram, 1884) Method 1. Fix dry film by passing it three times through a flame or placing on a heat block. 2. Stain for 15 seconds in 1% crystal violet or methyl violet, and then pour off excess. 3. Flood for 30 seconds with Lugol’s iodine, pour off excess. 4. Flood with acetone for no 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 seconds with dilute carbol fuchsin, or freshly filtered neutral red for 1–2 minutes. 7. Wash with water and carefully blot section until it is dry. Results Gram-positive organisms blue/black Gram-negative organisms red Modified Brown-Brenn method for Gram-positive and Gram-negative bacteria in paraffin sections (Churukian & schenk, 1982) Sections Formalin-fixed, 4–5 μm, paraffin wax embedded sections. Solutions Crystal violet solution (commercially available) Crystal violet, 10% alcoholic 2 ml Distilled water 18 ml Ammonium oxalate, 1% 80 ml Mix and store; always filter before use. Modified Gram’s iodine commercially available, or 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-acetone solution Ethyl alcohol, absolute 50 ml Acetone 50 ml 0.5% basic fuchsin solution (stock) commercially available, or Basic fuchsin or pararosaniline 0.5 g Distilled water 100 ml Dissolve with aid of heat and a magnetic stirrer. Basic fuchsin solution (working) Basic fuchsin solution (stock) 10 ml Distilled water 40 ml Picric acid-acetone Picric acid 0.1 g Acetone 100 ml Note 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 suppliers. Acetone-xylene solution Acetone 50 ml Xylene 50 ml Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain with filtered crystal violet solution for 1 minute. 3. Rinse well in distilled water. 4. Place in iodine solution for 1 minute. 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 minute. 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. Results Gram-positive organisms, fibrin, some fungi, Paneth cell granules, kerato-hyalin, and keratin blue Gram-negative organisms red Nuclei red Other tissue elements yellow Note 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 difficult and uneven. Gram-Twort stain (Twort, 1924, Ollett, 1947) Sections Formalin fixed, paraffin wax embedded. Solutions Crystal violet solution (see previous method) Gram’s iodine (see previous method) Twort’s stain 1% neutral red in ethanol 9 ml 0.2% fast green in ethanol 1 ml Distilled water 30 ml Mix immediately before use. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in crystal violet solution for 3 minutes. 3. Rinse in gently running tap water. 4. Treat with Gram’s iodine for 3 minutes. 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, preheated to 56°C). This may take 15–20 minutes; the section should be light brown or straw colored. 7. Rinse briefly in distilled water. 8. Stain in Twort’s for 5 minutes. 9. Wash in distilled water. 10. Rinse in acetic alcohol until no more red runs out of the section; this only takes a few seconds. 11. Rinse in fresh absolute alcohol, clear, and mount. Results Gram-positive organisms blue/black Gram-negative organisms pink/red Nuclei red Red blood cells and most cytoplasmic structures green Elastic fibers black Techniques for mycobacteria These organisms are difficult to demonstrate by the Gram technique as they possess a capsule containing a long-chain fatty acid (mycolic acid) which makes them hydrophobic. The fatty capsule influences 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 which 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 identified as contaminants by their appearance as clumps, or floaters, above the microscopic focal plane of the section. Ziehl-Neelsen (ZN) stain for Mycobacterium bacilli (Kinyoun, 1915) Sections Formalin or fixative other than Carnoy’s, paraffin wax embedded. Solutions Carbol fuchsin commercially available, or Basic fuchsin 0.5 g Absolute alcohol 5 ml 5% aqueous phenol 100 ml Mix well and filter before use. Acid alcohol Hydrochloric acid 10 ml 70% alcohol 1000 ml Methylene blue solution (stock) commercially available, or Methylene blue 1.4 g 95% alcohol 100 ml Methylene blue solution (working) Methylene blue (stock) 10 ml Tap water 90 ml Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in carbol fuchsin solution for 30 minutes. 3. Wash well in tap water. 4. Differentiate in acid alcohol until solutions are pale pink. (This usually only takes 2–5 dips.) 5. Wash in tap water for 8 minutes, then dip in distilled water. 6. Counterstain in working methylene blue solution until sections are pale blue. 7. Rinse in tap water, then dip in distilled water. 8. Dehydrate, clear, and mount. Results Mycobacteria, hair shafts, Russell bodies, Splendore-Hoeppli immunoglobulins around actinomyces and some fungal organisms red Background pale blue Notes a. The blue counterstain may be patchy if extensive caseation is present. Care should be taken to avoid over-counterstaining as scant organisms can easily be obscured. b. Decalcification using strong acids can destroy acid-fastness; formic acid is recommended. c. Victoria blue can be substituted for carbol fuchsin and picric acid for the counterstain if color blindness causes a recognition problem. Fluorescent method for Mycobacterium bacilli (Kuper & May, 1960) Sections Formalin fixed, paraffin wax embedded. Solution Auramine O 1.5 g Rhodamine B 0.75 g Glycerol 75 ml Phenol crystals (liquefied at 50°C) 10 ml Distilled water 50 ml Method 1. Deparaffinize (1-part groundnut oil and 2 parts xylene for M. leprae). 2. Pour on preheated (60°C), filtered staining solution for 10 minutes. 3. Wash in tap water. 4. Differentiate in 0.5% hydrochloric acid in alcohol for M. tuberculosis, or 0.5% aqueous hydrochloric acid for M. leprae. 5. Wash in tap water, 2 minutes. 6. Eliminate background fluorescence in 0.5% potassium permanganate for 2 minutes. 7. Wash in tap water and blot dry. 8. Dehydrate (not for M. leprae), clear and mount in a fluorescence-free mountant. Results Mycobacteria golden yellow (using blue light fluorescence below 530 nm) Background dark green Note The advantage of increased sensitivity of this technique is offset by the inconvenience of setting up the fluorescence microscope. Preparations fade over time, as a result of their exposure to UV light. Modified Fite method for Mycobacterium leprae and Nocardia Fixation 10% neutral buffered formalin (NBF). Sections Paraffin wax sections at 4–5 μm. Solutions 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 filter before use. Filter before each use with #1 filter paper. 5% sulfuric acid in 25% alcohol 25% ethanol 95 ml Sulfuric acid, concentrated 5 ml Methylene blue stock solution, commercially available, or Methylene blue 1.4 g 95% alcohol 100 ml Methylene blue working solution Stock methylene blue 5 ml Tap water 45 ml Xylene-peanut oil 1 part oil: 2 parts xylene Method 1. Deparaffinize in two changes of xylene-peanut oil for 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 90 seconds 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. Results (Fig. 16.1 ) Acid-fast bacilli including M. leprae bright red Nuclei and other tissue elements pale blue Fig. 16.1 The modified Fite procedure is necessary to demonstrate Mycobacterium leprae due to the organism’s fragile, fatty capsule. Quality control/note Do not overstain with methylene blue and do not allow sections to dry between carbol fuchsin and acid alcohol. Techniques for other important bacteria Cresyl violet acetate method for Helicobacter species Sections Formalin fixed, paraffin wax embedded. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Filter 0.1% cresyl violet acetate onto slide or into Coplin jar for 5 minutes. 3. Rinse in distilled water. 4. Blot, dehydrate rapidly in alcohol, clear, and mount. Results Helicobacter and nuclei blue/violet Background shades of blue/violet Notes This simple method allows good differentiation between Helicobacter sp. and other organisms. Gimenez method for Helicobacter pylori (Gimenez, 1964, McMullen et al., 1987) Sections Formalin fixed, paraffin wax embedded. Solutions 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 Stock carbol fuchsin Commercial cold acid-fast bacilli stain, or basic fuchsin 1 g Absolute alcohol 10 ml 5% aqueous phenol 10 ml Filter before use. Working carbol fuchsin Phosphate buffer 10 ml Stock carbol fuchsin 4 ml Filter before use. Malachite green Malachite green 0.8 g Distilled water 100 ml Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in working carbol fuchsin solution for 2 minutes. 3. Wash well in tap water. 4. Stain in malachite green for 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. Results Helicobacter pylori red/magenta Background blue/green Note The greatest problem with this method is overstaining or irregularity of staining with Malachite green. It is also valuable in demonstrating the Legionella bacillus in post-mortem lung smears. Toluidine blue in Sorenson’s buffer for Helicobacter Sections Formalin fixed, paraffin wax embedded. Solutions Toluidine blue in pH 6.8 phosphate buffer Sorenson’s phosphate buffer, pH 6.8 50 ml 1% aqueous toluidine blue 1 ml Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in buffered toluidine blue for 20 minutes. 3. Wash well in distilled water. 4. Dehydrate, clear, and mount. Results Helicobacter dark blue against a variably blue background Immunohistochemistry techniques for demonstrating Helicobacter species also exist. Warthin-Starry method for spirochetes (Warthin & Starry, 1920) Sections Formalin fixed, paraffin wax embedded. Solutions Acetate buffer, pH 3.6 Sodium acetate 4.1 g Acetic acid 6.25 ml Distilled water 500 ml Silver solution 1% silver nitrate in pH 3.6 acetate buffer Developer Dissolve 0.3 g of hydroquinone in 10 ml pH 3.6 acetate 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. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Celloidinize in 0.5% celloidin, drain and harden in distilled water for 1 minute. 3. Impregnate in preheated 55–60°C silver solution for 90–105 minutes. 4. Prepare and preheat developer in a water bath. 5. Treat with developer for 31/2 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. Results Spirochetes black Background golden/yellow Note It is wise to take a few slides through at various incubation times to ensure optimum impregnation. Modified Steiner method for filamentous and non-filamentous bacteria (Steiner & Steiner, 1944; modified Swisher, 1987) Sections Formalin fixed, paraffin wax embedded. Solutions 1.0% uranyl nitrate commercially available, or Uranyl nitrate 1 g Distilled water 100 ml 1% silver nitrate Silver nitrate 1 g Distilled water 100 ml Make fresh each time and filter with #1 or #2 filter paper before use. 0.04% silver nitrate Silver nitrate 0.04 g Distilled water 100 ml Refrigerate and discard after 1 month. 2.5% gum mastic commercially available, or Gum mastic 2.5 g Absolute alcohol 100 ml Allow to dissolve for 24 hours, then filter until clear yellow before use. Refrigerate any unused portion. Hydroquinone Hydroquinone 1 g Distilled water 25 ml Make fresh solution for each use. Reducing solution 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 filter with #4 filter paper; add 2.5 ml of 0.04% silver nitrate. Do not filter this solution. When the gum mastic is added, the solution will take on a milky appearance. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Sensitize sections in 1% aqueous uranyl nitrate at room temperature and place in microwave oven until solution is just at boiling point, approx. 20–30 seconds; do not boil. Alternatively, place in preheated 1% uranyl nitrate at 60°C in a water bath for 15 minutes, or in microwave oven and bring to almost boiling point – do not boil; 2% zinc sulfate in 3.7% formalin may be substituted. 3. Rinse in distilled water at room temperature until uranyl nitrate residue is eliminated. 4. Place in 1% silver nitrate at room temperature and microwave until boiling point is just reached. Do not boil. Remove from oven, loosely cover jar, and allow to stand in hot silver nitrate, 6–7 minutes; alternatively, preheat silver nitrate for 20–30 minutes in a 60°C water bath, add slides, and allow to impregnate for 90 minutes. 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 for 5 minutes. 8. Allow to air dry for 5 minutes. 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 minutes, 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. Results (Fig. 16.2) Spirochetes, cat-scratch organisms, Donovan bodies, non-filamentous bacteria of Legionella pneumophila dark brown/black Background bright yellow to golden yellow Fig. 16.2 Syphilis Treponema pallidum bacilli (arrowed), seen with the modified Steiner technique. The resistance to coloration is shared by Helicobacter, spirochetes and Legionella. Notes 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 under 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. Some important bacteria Staphylococcus aureus is an important and common bacterial pathogen which may be resistant to common antibiotic treatments. It usually causes skin and soft tissue infections such as boils, wound and burn infections, and can also lead to a cavitating pneumonia in children and adults. Septicemic states may occur and are associated with the formation of multiple deep foci of infection including endocarditis. Microscopically staphylococci appear as Gram-positive clusters. Neisseria meningitidis (meningococcus) is a common cause of meningitis, and may produce a fulminating septicemia (meningococcal sepsis). Organisms can be seen in histological sections of meningococcal meningitis, but are difficult to identify because they are usually within neutrophil cytoplasm. Neisseria gonorrhoeae (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 difficult to find. Members of the Neisseria family are generally difficult to see in histological sections, although easily detectable in smears of fresh pus or cerebrospinal fluid (CSF), characteristically in pairs. They are easier to detect using the Gram-Twort method. Lactobacillus acidophilus (Döderlein’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 blue-stained masses on the surface of squamous cells stained by Papanicolaou’s method, with these cells being known as ‘clue cells’. Helicobacter pylori is frequently seen in gastric biopsies. This spiral Vibrio organism is implicated as the organism causing many cases of chronic gastritis. They are 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 identified from an H&E stain. However, Warthin-Starry, Steiner, Gimenez, toluidine blue or cresyl violet acetate methods demonstrate them more clearly. A commercial specific antiserum has recently become available for their demonstration. Clostridium difficile causes pseudomembranous colitis, an inflammation of the large bowel. This arises following the administration of broad-spectrum antibiotics; the balance of the normal anaerobic gut microflora is disturbed, allowing the organism to proliferate unchecked. C. difficile is difficult to stain but the ‘volcano lesions’ of purulent necrosis 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 which contain tiny intracellular rods arranged in a ‘Chinese letter’ formation, and staining variably with the Gram stain, are the hallmarks of this disease. Mycobacterium tuberculosis is the second leading cause of death from an infectious agent worldwide and remains a significant pathogen in developed countries. Disease results in the familiar caseating granulomatous lesion and its associated 1–2 μm, blunt-ended, acid- and alcohol-fast bacilli. In some regions such as Africa, tuberculosis is commonly associated with HIV infection, causing major morbidity and mortality. Mycobacterium avium/intracellulare are representatives of a group of intracellular opportunistic mycobacteria which are frequently present in the later stages of immunosuppression, particularly associated with HIV/AIDS. They frequently persist in spite of treatment, and are often fatal. The lesions produced are non-caseating and consist of collections of vacuolated macrophages which 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 HIV/AIDS patients. This group also includes M. kansasii. Mycobacterium leprae is an obligate intracellular, neurotrophic mycobacterium which attacks and destroys nerves, especially in the skin. The 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 florid granulomatous response (tuberculoid leprosy). M. leprae is only acid-fast and is best demonstrated with the modifed Fite method (see page 261). Legionella pneumophila was first identified in 1977 as the cause of a sporadic type of pneumonia with high mortality. The small Gram-negative coccobacillus is generally spread in aerosols from stagnant water reservoirs, usually in air-conditioning units. The bacterium may be difficult to stain except with the Dieterle and modified Steiner silver stains, and specific antiserum. Treponema pallidum is the organism causing syphilis, and is infrequently seen in biopsy specimens when 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 which often kinks in the center (Fig. 16.2). Dieterle, Warthin-Starry or modified Steiner methods may demonstrate the organism. In addition, a specific antiserum is also available. Leptospira interrogans is a spirochete organism causing leptospirosis or Weil’s disease. The disease 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 modified 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 the 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 modified 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, Afipia felis and Bartonella henselae have been implicated. It is difficult to demonstrate on paraffin sections because of the timing or maturation factor of the bacterium, but the modified Steiner and the Warthin-Starry methods are valuable techniques for demonstrating this organism. Fungal infections Fungi are widespread in nature, and humans are regularly exposed to the spores from many species. The most commonly encountered fungal diseases are the superficial mycoses which 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, Trichophyton and Epidermophyton groups and may appear as yeasts or mycelial forms within the keratin. They are seen quite well in the H&E stain, but are demonstrated better 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, representing opportunistic attacks by fungi, frequently of low virulence, but often fatal if untreated. When fungi grow in tissue they may display primitive asexual (imperfect) forms which appear as either spherical yeast or spore forms. Some may produce vegetative growth which appears as tubular hyphae which 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. Identification of fungi Some fungi may elicit a range of host reactions from exudative, necrotizing to granulomatous whereas 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 stains. Fungi are often weakly hematoxyphilic and can be suspected on H&E stains. Some fungi, e.g. sporothrix, may be surrounded by a stellate, strongly eosinophilic, refractile Splendore-Hoeppli precipitate of host immunoglobulin and degraded eosinophils. Fluorochrome-labeled specific antibodies to many fungi are available, and are in use in mycology laboratories for the identification of fungi on fresh and paraffin wax sections. These antibodies have not found widespread use on fixed tissue, where identification 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 identified sufficiently for diagnoses. However, there is no substitute for microbiological culture. Grocott methenamine (hexamine)-silver for fungi and Pneumocystis species (Gomori, 1946, Grocott, 1955, Swisher and Chandler, 1982) Sections Formalin fixed, paraffin wax embedded. Solutions 4% chromic acid commercially available, or Chromic acid 4.0 g Distilled water 100 ml 1% sodium bisulfite Sodium bisulfite 1 g Distilled water 100 ml 5% sodium thiosulfate Sodium thiosulfate 5.0 g Distilled water 100 ml 0.21% silver nitrate, stock solution a Silver nitrate 2.1 g Distilled water 1000 ml Refrigerate for up to 3 months. Methenamine-sodium borate, stock solution b Methenamine 27 g Sodium borate decahydrate (borax) 3.8 g Distilled water 1000 ml Refrigerate for up to 3 months. Methenamine-silver sodium borate working solution Equal parts of solutions a and b. Make fresh each time and filter before use. 0.2% light green stock solution Light green 0.2 g Distilled water 100 ml Glacial acetic acid 0.2 ml Light green working solution 0.2% light green stock solution 10 ml Distilled water 50 ml Prepare working solution fresh before each use. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Oxidize in 4% aqueous chromic acid (chromium trioxide) for 30 minutes. 3. Wash briefly in distilled water. 4. Dip briefly in 1% sodium bisulfite. 5. Wash well in distilled water 6. Place in preheated (56–60°C water bath) working silver solution for 15–20 minutes. Check control after 15 minutes. If section is ‘paper bag brown’ then rinse in distilled water and check under microscope. If it is not ready, dip again in distilled water and return to silver. Elastin should not be black. Check every 2 minutes from that point onwards (see Note a). 7. Rinse well in distilled water. 8. Tone in 0.1% gold chloride for 5 seconds. Rinse in distilled water. 9. Place in 5% sodium thiosulfate for 5 seconds. 10. Rinse well in running tap water. 11. Counterstain in working light green solution until a medium green (usually 5–15 seconds). 12. Dehydrate, clear and mount. Results Fungi, Pneumocystis, melanin black Hyphae and yeast-form cells sharply delineated in black of fungi Mucins and glycogen taupe to dark gray/brown Background pale green Notes a. Incubation time is variable and depends on the type and duration of fixation, and organism being demonstrated. Impregnation is controlled microscopically until fungi are dark brown. Background is colorless at this point. Over-incubation produces intense staining of elastin and fungi which may obscure fine internal detail of the hyphal septa. This detail is essential for critical identification, 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 ensures an alkaline pH. c. Sodium bisulfite 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. McManus’ PAS method for glycogen and fungal cell walls Fixation 10% NBF. Sections 3–5-μm paraffin wax sections. Solutions Schiff’s reagent, see page 183 or commercially available. 0.5% periodic acid solution Periodic acid 0.5 g Distilled water 100 ml 0.2% light green stock solution Light green 0.2 g Distilled water 100 ml Glacial acetic acid 0.2 ml This is the same stock solution used in the GMS. Light green working solution 0.2% light green stock solution 10 ml Distilled water 50 ml Make fresh before each use. Method 1. Deparaffinize and hydrate slides to distilled water. 2. Oxidize in periodic acid solution for 5 minutes. 3. Rinse in distilled water. 4. Place in Schiff’s reagent for 15 minutes. 5. Wash in running tap water for 10 minutes to allow pink color to develop. 6. Counterstain for a few seconds in working light green solution. 7. Dehydrate in 95% alcohol, absolute alcohol and clear in xylene. 8. Mount in resin-based mountant. Results Fungal cell walls and glycogen magenta to red Background pale green Note A solution of 5% aqueous sodium hypochlorite reduces overstaining by Schiff’s. A selection of the more important fungi and actinomycetes 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 found in the liver, appendix, lung and neck. The individual organisms are Gram-positive, hematoxyphilic, non-acid-fast, branching filaments 1 μm 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-like hyphae, which measures 30–3000 μm, surrounded by eosinophilic protein, is called a ‘sulfur’ granule and is also an important identification marker for other fungal groups. These granules may be macroscopically visible and their yellow color is an important diagnostic aid. Nocardia asteroides is another actinomycete. It is filamentous and may be visible in an H&E stain, but it is Grocott-positive and variably acid-fast using the modified ZN for leprosy. However, it is difficult to demonstrate even with the acid-fast bacillus techniques. Its pathology is similar to that of actinomycosis, but its organisms are generally more disseminated and it tends to cause invasive infection in the immunocompromised. Candida albicans is a common yeast, but with immunosuppression may become systemic. It infects the mouth (thrush), the esophagus, the vagina (vaginal moniliasis), the skin and nails, and may be found 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 pseudo-hyphae. Whilst difficult 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. 16.3 ) or become systemic in immunosuppressed patients. The fungus has broad, 3–6 μm, parallel-sided, septate hyphae showing dichotomous (45 degree) 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 which can cause infection of the ear. Fig. 16.3 A strong hematoxylin (Ehrlich’s) and eosin stain will show the fine detail of many infectious agents. The hyphal structure identifies this as Aspergillus fumigatus which was colonizing an old tuberculosis cavity in the lung. Zygomycosis is an infrequently seen disease caused by a group of hyphated fungi belonging mainly to the genera Mucor and Rhizopus. 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. Grocott and PAS are the staining methods of choice (Fig. 16.4, Fig. 16.5 ). Fig. 16.4 Rhizomucor (a cosmopolitan, filamentous fungus) is well demonstrated by this PAS stain with light green counterstain. Fig. 16.5 Immunohistochemistry is being increasingly applied to the demonstration of microorganisms using labeled specific antibody. This figure demonstrates Zygomycetes, a fast-growing fungus, with fast red chromogen. Cryptococcus neoformans exists solely in yeast-form cells, is variable in diameter, 2–20 μm, with ovoid, elliptical and crescentic forms frequently seen. There is an extensive mucopolysaccharide coat around the yeasts which 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. Often, these patients are immunosuppressed. Histoplasma capsulatum is another soil-dwelling yeast which 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 which appear stuffed with small, regular, 2–5 μm yeast-form cells which 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. 16.6 ). Fig. 16.6 Grocott’s methenamine-silver stains a wide variety of infectious agents. Here seen with light green counterstain is the method of choice for Histoplasma capsulatum, a dimorphic endemic fungus. Pneumocystis jiroveci . There is still some debate over the taxonomy of this organism, although analysis of its ribosomal RNA has placed it nearer to a fungal than a protozoan classification (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 HIV. It most frequently causes pneumonia, where the lung alveoli are progressively filled with amphophilic, foamy plugs of parasites and cellular debris. It is found rarely in other sites such as the 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. Specific immunohistochemistry is available to use, otherwise, Grocott methenamine-silver is recommended. Only electron microscopy or an H&E stain on a resin-embedded thin section will show their internal structure. The cysts are 4–6 μm in diameter and contain 5–8 dot-like 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. The demonstration of rickettsia Rickettsial organisms, e.g. 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. 16.7 ). Macchiavello’s stain for rickettsia and viral inclusions (modified Culling, 1974) Sections Formalin fixed, paraffin wax embedded. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain in 0.25% basic fuchsin for 30 minutes. 3. Differentiate in 0.5% citric acid for 3 seconds. 4. Wash in tap water for 2 minutes. 5. Counterstain in 1% methylene blue for 15–30 seconds. 6. Rinse in tap water. 7. Dehydrate, clear, and mount. Results Rickettsia and some viral inclusions red Background blue Fig. 16.7 Immunohistochemical method demonstrating Rocky Mountain spotted fever in kidney. It is caused by the bacterium Rickettsia rickettsii, which is carried by ticks. The detection and identification of viruses Whilst the cytopathic effects of viruses can often be seen in a good H&E stain, and may be characteristic of a single viral group, the individual viral particles are too small to be seen with the light microscope, and require an electron microscope to reveal their structure. This may allow a rapid and accurate diagnosis in viral infections. Some viruses aggregate within cells to produce viral inclusion bodies which 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 modified trichromes using 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, Macchiavello’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 mono-clonal immunohistology to viruses, which are either class or species specific, 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 (also known as HBs or Australia antigen) and the core antigen (HBc) can be specifically detected immunohistochemically, providing clinically important information about the stage of the disease. More recently, nucleic acid hybridization probes and PCR testing 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 fixed. It should be remembered, however, that the detection of microorganisms using nucleic acid probes, unlike specific biotinylated antiserum, does not necessarily mean active disease. Phloxine-tartrazine technique for viral inclusions (Lendrum, 1947) Sections Formalin fixed, paraffin wax embedded. Solutions Phloxine Phloxine 0.5 g Calcium chloride 0.5 g Distilled water 100 ml Tartrazine A saturated solution of tartrazine in 2-ethoxyethanol, or cellosolve. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Stain nuclei in alum hematoxylin (Carazzi’s or Harris’s) for 10 minutes. 3. Wash in running tap water for 5 minutes. 4. Stain in phloxine solution for 20 minutes. 5. Rinse in tap water and blot dry. 6. Controlling with the microscope, stain in tartrazine until only the viral inclusions remain strongly red, 5–10 minutes on average. 7. Rinse in 95% alcohol. 8. Dehydrate, clear, and mount. Results Viral inclusions bright red Red blood cells variably orange/red Nuclei blue/gray Background yellow Notes All tissue is stained red with phloxine, which is then differentiated by displacement with the counterstain tartrazine. The red color is first removed from muscle, then other connective tissues. Paneth cells, Russell bodies, and keratin can be almost as dye retentive as viral inclusions, and can occasionally be a source of confusion. Shikata’s orcein method for hepatitis B surface antigen (modified Shikata et al., 1974) Sections Formalin fixed, paraffin wax embedded. Solutions Acid permanganate 0.25% potassium permanganate 95 ml 3% aqueous sulfuric acid 5 ml Orcein Orcein (synthetic) 1 g 70% alcohol 100 ml Concentrated hydrochloric acid (gives a pH of 1–2) 1 ml Tartrazine solution Saturated tartrazine in cellosolve (2 ethoxyethanol) Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Treat with acid permanganate solution for 5 minutes. 3. Bleach until colorless with 1.5% aqueous oxalic acid for 30 seconds. 4. Wash in distilled water for 5 minutes, then in 70% alcohol. 5. Stain in orcein solution at room temperature for 4 hours, or in a Coplin jar of 37°C preheated orcein for 90 minutes. 6. Rinse in distilled alcohol and examine microscopically to determine desired staining intensity. 7. Rinse in cellosolve, stain in tartrazine for 2 minutes. 8. Rinse in cellosolve, clear and mount. Results Hepatitis B infected cells, elastic and some mucins brown/black Background yellow Notes 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 which react with orcein. Results compare well with those obtained using labeled antibodies, but the selectivity is inferior. Viral infections Whilst not exhaustive, this brief summary reflects some viruses which are encountered in surgical and post-mortem histopathology (Table 16.3 ). Table 16.3 Viral infections seen in histopathology Virus Family Genome Disease Measles Paramyxo SS RNA Measles Varicella-zoster Herpes DS DNA Chickenpox, shingles Herpes simplex Herpes DS DNA Cold sores, genital herpes Cytomegalovirus (CMV) Herpes DS DNA Cytomegalic inclusion disease Epstein-Barr virus Herpes DS DNA Glandular fever, African Burkitt’s lymphoma Human T-cell leukemia virus (HTLV-1) Retro SS RNA Adult T-cell leukemia Human immunodeficiency virus (HIV) Retro SS RNA AIDS Human papilloma viruses (HPV) Papova DS DNA Human wart viruses JC virus Papova DS DNA Progressive multifocal leukoencephalopathy (PML) Poliovirus Picorna SS DNA Poliomyelitis Molluscum virus Pox DS DNA Molluscum contagiosum Lyssavirus Rhabdo SS RNA Rabies DS = double-stranded; SS = single-stranded. Viral hepatitis is caused by a number of viral infections. The hepatitis viruses (HV) A, B, C, D, and E show great biological diversity and are clinically the most common viruses which primarily affect the liver. The liver is the target organ and damage varies with the viral strain, ranging from massive acute necrosis to chronic ‘piecemeal necrosis’ of liver cells, leading to cirrhosis. An eosinophilic ‘ground glass’ appearance is seen in the cytoplasm of some hepatocytes due to dilated smooth endoplasmic reticulum which contains tubular HB surface antigen. It is this component which can be demonstrated using Shikata’s orcein method, or by specific immunohistochemistry. 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 fluid, and include the margination of chromatin along nuclear membranes, Cowdry type A (‘owl’s eye’) inclusion bodies, and syncytial (‘grape-like’) nuclei within giant cells. Cytomegalovirus (CMV) causes congenital infections of newborns and can result in systemic disease in HIV and immunosuppressed patients. It is seen in the endothelial cells forming prominent intranuclear inclusions which 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 which cause raised verrucous or papillomatous skin warts, or flat 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 paraffin wax sections. JC (John Cunningham) virus is a polyomavirus (previously known as papovavirus) which causes progressive multifocal leukoencephalopathy, a demyelinating disease, in HIV 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 on routine H&E sections, and are seen well with phloxine-tartrazine. The large 1 μm viral particles have a typical pox virus structure: brick-shaped with a superimposed figure-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. Macchiavello, phloxine-tartrazine, Mann’s methyl blue-eosin or PAS stains are recommended. Note that given the pathogenicity of these agents, if suspected, the case should be passed to a relevant diagnostic center rather than a routine laboratory. Human immunodeficiency virus (HIV) consists of at least two retrovirus strains. The virus is best seen in cultured lymphocytes and is rarely seen in tissues from affected patients. It produces a distinctive neuropathological lesion in HIV encephalitis consisting of microglial nodules, or stars, containing collections of giant cells, microglia and astrocytes. Synthetic nucleic acid probes have been prepared to identify HIV genomes. Influenza (flu) is a contagious respiratory illness caused by influenza viruses (Fig. 16.8 ). 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 flu, more than 200,000 people are hospitalized from flu complications, and about 36,000 people die from flu. More recently, concern about the influenza A H5N1 strain of bird flu has emerged. Some people, e.g. older people, young children and people with certain health conditions, are at high risk of serious flu complications. Fig. 16.8 Immunohistochemical method demonstrating influenza A virus-infected cells in the bronchus. SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) can be severe respiratory illnesses caused by a coronavirus (SARS-CoV and MERS-CoV) (Fig. 16.9 ). SARS was first 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. MERS-CoV is a novel coronavirus, first identified in Saudi Arabia in 2012 and the majority of cases emerging from the Arabian Peninsula. The risk of infection is linked to exposure to camels and subsequent outbreaks have occurred in healthcare facilities. The incidence and spread of MERS remains an ongoing matter of global public health importance. Fig. 16.9 Immunohistochemical method demonstrating the previously unrecognized SARS-associated coronavirus which is responsible for severe acute respiratory syndrome (SARS). Prion disease 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 and in animals, 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). 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 which 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 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 as 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 find yourself with a suspected or known positive case in your laboratory: http://who.int/bloodproducts/TSE-manual2003.pdf. Remember that these types of cases should never knowingly be handled in a routine histology laboratory. Contact your local health department for additional guidelines. The demonstration of protozoa and other organisms The identification 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 difficult cases (Fig. 16.10a,b ). Giemsa stain for parasites Sections Fixative is not critical, but B5 or Zenker’s is preferred; thin, 3 μm paraffin wax sections (if Zenker’s is not used, post-mordant in Zenker’s in a 60°C oven for 1 hour before staining). Solutions Giemsa stock solution, commercially available or 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. Working Giemsa solution for parasites Giemsa stock 4 ml Acetate buffered distilled water, pH 6.8 96 ml Method 1. Deparaffinize 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. Results Protozoa and some other microorganisms dark blue Background pink/pale blue Nuclei blue Fig. 16.10 (a) H&E and (b) immunohistochemical methods demonstrating the single-celled parasite Toxoplasma gondii in heart. Protozoa Entamoeba histolytica , the organism causing amebic colitis or dysentery, can be found in ulcers which 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 on routine H&E staining, or in the luminal mucus overlying normal-appearing mucosa. They are PAS-positive and brief counterstaining in 1% aqueous metanil yellow emphasizes the ingested red cells. Toxoplasma gondii , a commonly encountered organism which is spread in cat litter, causes an acute lymphadenopathy which is often subclinical. Affected nodes show non-specific 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 specific antiserum is recommended (Fig. 16.10). Leishmania tropica is transmitted by sand-fly bites and causes a chronic inflammatory 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 which 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 the spleen, lymph nodes, liver and bone marrow. The organisms are hematoxyphilic and can be emphasized with a Giemsa stain. Giardia duodenalis (lamblia) is a flagellate protozoon which 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 have been seen on an H&E stain where they appear as eosinophilic, sickle-shaped flakes with indistinct nuclei, resting on intestinal mucosa which may show little evidence of inflammation. When seen in a fresh Giemsa-stained duodenal aspirate, they appear kite-shaped, 11–18 μm in size, binucleate and have faint terminal flagella. Trichomonas vaginalis is a similar flagellate protozoon most frequently seen in a Papanicolaou stain. Inflammatory 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) which causes severe and relentless outbreaks of diarrhea among HIV 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. Worms 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 occasionally 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 S. japonicum have lateral spines. Any good trichrome procedure will demonstrate worm development. Echinococcosis . Echinococcus granulosus is a tapeworm found in dogs, but 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, they have a diagnostic shape and stain brilliant yellow with picric acid.