Traditional stains and modern techniques for demonstrating microorganisms in histology

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. and for greater depth. The organisms in Table 16.1 are discussed and techniques for their demonstration are described.

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 ( ). 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.

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 ( ). 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 ( ).

Common molecular techniques used include direct hybridization and nucleic acid amplification (often referred to under the umbrella term of polymerase chain reaction – PCR) ( ). 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 ( ).

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 ( ).

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 ( ). 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 ( ). 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 ( ). 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 ( ), and will continue to play a key role in the detection of emerging infections and bioterrorist attacks ( ) . 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.

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 ( ).

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.

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.