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 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

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).

Table 15.2 A simplified classification of important bacteria

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)

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 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.

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-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

Staining method

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.

4. Iodine solution, 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, keratohyalin, and keratin	blue
Gram-negative organisms	red
Nuclei	red
Other tissue elements	yellow