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 only individually seen by microscopy. Most do not normally cause disease in humans, existing in a state of commensalism, where there is little or no benefit to the person, or mutualism, where there is some benefit to both parties. Pathogens are agents that cause disease. These fall into five main groups:
With the advent of new and more powerful 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 exploit new opportunities to spread infections that 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. Some agents, such as Ebola, have been around for many years but the first human outbreaks were not recorded until 1976. Previous outbreaks would flare up and then burn themselves out, undetected and confined, before deforestation and the like altered this state.
• Immunodeficiency states occurring either as part of a natural disease, such as acquired immune deficiency 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 species barriers and exploit new physical environments, thus evading host defenses, and resisting 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 that are rarely seen in developed countries. High-priority agents include organisms that pose a risk to national security because they:
The following are listed by the Centers for Disease Control and Prevention (CDC) in the United States as high-risk biological agents:
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.
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 such as that of von Lichtenberg (1991) for greater depth. The organisms in Table 15.1 are discussed, and techniques for their demonstration are described.
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 patients with Creutzfeldt-Jakob disease (CJD). It has been shown that well-fixed tissue, paraffin-processed blocks, and stained slides from CJD 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 for 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 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 maybe plentiful and sampling error presents little problem, to cytology samples where cellular material is often scarce and lesions may easily be 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, such as abscesses and pus formation, cavitations, hyperkeratosis, demyelination, pseudo-membrane or fibrin 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 stains at low-power magnification often reveals indirect evidence of the presence of infection, such as 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 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, (such as Warthin-Finkeldy or Langhans’ type) may indicate measles or tuberculosis. Other cellular changes include intra-cytoplasmic edema of koilocytes, acantholysis, spongiform degeneration of brain, margination of chromatin, syncytial nuclear appearance, ‘ground-glass’ changes in the nucleus or cytoplasm, or inclusion bodies, and can indicate infectious etiology. 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 specific histochemical methods is required for their detection. When organisms are few in number, fluorochromes may be used to increase microscopic sensitivity of a technique. Finally, there are two techniques that offer the possibility of specific identification of microorganisms that extend to the appropriate strain level.
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 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 of 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 (see later chapters).
In situ hybridization, the polymerase chain reaction
In situ hybridization (ISH) has even greater potential for microorganism 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. Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) are good examples. The polymerase chain reaction (PCR) can also be a very useful technique to obtain diagnoses of microbial infections from autopsy tissues and surgical specimens. While fresh/frozen tissues provide the best-quality nucleic acids for analysis, DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues can be used quite successfully in both PCR and reverse-transcriptase PCR (Tatti et al. 2006; Bhatnagar et al. 2007; Guarner et al. 2007; Shieh et al. 2009). Since formalin cross-links proteins and nucleic acids, resulting in significant degradation, it is critical to design PCR assays targeting small amplicons, typically 500 base pairs or fewer in length (Srinivasan et al. 2002). To this end, 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 no longer than 48 hours (von Ahlfen et al. 2007; Chung et al. 2008). Furthermore, the use of real-time PCR technology, which often requires small amplicons for successful detection of products, is ideally suited for use with nucleic acid extracts from FFPE tissues (Denison et al. 2011). (Thanks are due to Amy M. Denison, PhD, for her assistance with this information.)
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 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 in vegetation on a heart valve, they appear as blue-gray granular masses with an H&E stain. However, organisms are often 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 historical classification (Table 15.2).
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. A pneumocystis containing control, for instance, 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 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 identification of the organism causing a lung abscess, wound infection, septicemic abscess or meningitis.
Gram method for bacteria in smears (Gram 1884)
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 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 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.
Modified Brown-Brenn method for Gram-positive and Gram-negative bacteria in paraffin sections (Churukian & Schenk 1982)
Crystal violet solution (commercially available)
|Crystal violet, 10% alcoholic||2 ml|
|Distilled water||18 ml|
|Ammonium oxalate, 1%||80 ml|
Modified Gram’s iodine commercially available, or
|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.
0.5% basic fuchsin solution (stock) commercially available, or
|Basic fuchsin or pararosaniline||0.5 g|
|Distilled water||100 ml|
Basic fuchsin solution (working)
|Basic fuchsin solution (stock)||10 ml|
|Distilled water||40 ml|
|Picric acid||0.1 g|
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.
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Stain with filtered crystal violet solution, 1 minute.
3. Rinse well in distilled water.
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.
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.)
|Gram-positive organisms, fibrin, some fungi, Paneth cell granules, keratohyalin, and keratin||blue|
|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 difficult and uneven.
Gram-Twort stain (Twort 1924; Ollett 1947)
Crystal violet solution (see previous method)
Gram’s iodine (see previous solution)
|1% neutral red in ethanol||9 ml|
|0.2% fast green in ethanol||1 ml|
|Distilled water||30 ml|
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Stain in crystal violet solution, 3 minutes.
3. Rinse in gently running tap water.
4. Treat with Gram’s iodine, 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, 5 minutes.
10. Rinse in acetic alcohol until no more red runs out of the section; this takes only a few seconds.
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) that 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 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 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)
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.
|Hydrochloric acid||10 ml|
|70% alcohol||1000 ml|
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Carbol fuchsin solution, 30 minutes.
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.
|Mycobacteria, hair shafts, Russell bodies, Splendore-Hoeppli immunoglobulins around actinomyces, and some fungal organisms||red|
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)
|Auramine O||1.5 g|
|Rhodamine B||0.75 g|
|Phenol crystals (liquefied at 50°C)||10 ml|
|Distilled water||50 ml|
1. Deparaffinize (1 part groundnut oil and 2 parts xylene for M. leprae).
2. Pour on preheated (60°C), filtered staining solution, 10 minutes.
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, 2 minutes.
7. Wash in tap water and blot dry.
8. Dehydrate (not for M. leprae), clear, and mount in a fluorescence-free mountant.
|Mycobacteria||golden yellow (using blue light fluorescence below 530 nm)|
Modified Fite method for M. leprae and Nocardia
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, working
|Stock methylene blue||5 ml|
|Tap water||45 ml|
|Xylene-peanut oil||1 part oil: 2 parts xylene|
1. Deparaffinize 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 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.
Results See Figure 15.1
Figure 15.1 The modified Fite’s procedure is necessary to demonstrate Mycobacterium leprae due to the organism’s fragile, fatty capsule.
|Acid-fast bacilli including M. leprae||bright red|
|Nuclei and other tissue elements||pale blue|