Initial assessment of clinical information and examination of the gross specimen may provide indications of an infective process. Some organisms, such as Actinomyces or Mycobacterium tuberculosis reveal their presence in characteristic ways. The specimen may contain areas of consolidation, caseation or necrosis, inflammatory pus or granuloma. Subsequent examination of haematoxylin and eosin (H&E) stained sections will provide a number of clues based upon tissue reaction to a suspected infection. Many infective agents cause typical tissue responses which may vary as the infection progresses from acute to chronic or disseminated phases. When considered with relevant clinical information, the histological features may provide sufficient information to confine the search to a particular type of organism or even a specific entity. The H&E stain may even reveal the presence of an organism, many organisms having basophilic staining characteristics, while many viral inclusion bodies are eosinophilic. Larger parasites may be readily visible and show differential staining of their structures and ova. Clusters of bacteria may be sufficiently stained with haematoxylin to be seen, as will some fungi. More often, however, routine stains merely provide a diagnostic puzzle which may only be resolved by the use of special staining techniques, designed to demonstrate types of organisms and occasionally specific entities.

In recent decades advances in immunopathology have led to methodologies which will specifically identify many organisms. These methods have evolved from simple immunofluorescence techniques applicable to frozen sections of unfixed tissue, through more complex immunohistochemical methods applicable to both frozen and paraffin sections of fixed tissue, to extremely sensitive hybridisation techniques using DNA and other probes. Nevertheless these procedures are often limited in terms of expedition and cost and traditional staining methods continue to play an essential and significant role in diagnostic laboratories.

The demonstration of bacteria in tissue sections
The stain technology for demonstrating bacteria in tissue sections has changed little in a century. When Paul Ehrlich turned his attention, in 1882, to the demonstration of the tubercle bacillus, the result was a method as enduring as his later development (1886) of the alum haematoxylin stain. Ehrlich's method was further developed first by Franz Ziehl¹ and then Friedrich Neelsen² and this remains the classic method for mycobacteria. At much the same time, in Denmark, Hans Christian Gram³ developed a method for broadly distinguishing bacteria into two groups, on the basis of a particular staining characteristic.

The Ziehl-Neelsen technique^1-2 for mycobacteria uses basic fuchsin to stain the organisms, while the dye used by Gram is crystal violet. Both these dyes are triaminotriphenyl methanes or rosanilins and are cationic (basic) dyes with a strong affinity for nuclear chromatin and other strongly anionic (acidic) groups. Basic fuchsin is in fact a mixture of several similar dyes differing only in the presence, and number of, methyl groups on the benzene rings, whereas crystal violet or hexamethyl pararosanilin has six methyl substitutions of the amine groups. This degree of alkyl substitution renders crystal violet about three times more alcohol soluble than basic fuchsin and also more lipophilic. These properties provide clues as to the mechanism involved in staining bacteria by the Gram technique.³

All bacteria contain DNA and possess a cell wall rich in complex lipids and glycolipids. The lipid content of the cell wall, together with the degree of saturation of the lipids present, dictate how successfully the organism will be stained. Unsaturated lipids contain acidic hydroxyl and carboxyl groups for which basic fuchsin and crystal violet have high affinity. The tubercle bacillus, as well as other mycobacteria, has a cell wall rich in unsaturated lipids, particularly of a type identified as mycolic acids, possessing hydroxyl and carboxyl groups and varying in carbon chain length from species to species.⁴ This variation in mycolic acid chain length may contribute to the different degrees of acid-fastness (see below) demonstrated by different species of mycobacteria when stained with basic fuchsin. It is of interest to note that mycobacteria, and in particular, the tubercle bacilli, are Gram positive (see below), but of the Gram-positive bacteria only mycobacteria are acid fast, as determined by the Ziehl-Neelsen technique. It is perhaps also relevant here to note that the cell walls of Gram negative bacteria contain a significantly higher lipid content than those Gram-positive bacteria.⁵

Gram's technique³ involves staining with an aqueous crystal violet solution, followed by mordanting with iodine in solution, then differentiation in alcohol or acetone. Some bacteria retain the crystal violet, staining blue/black, and are referred to as Gram positive, while others lose the crystal violet and are referred to as Gram negative. Gram negative bacteria may be visualised by the use of a counterstain, such as safranin or neutral red. It must be stressed here that when using acetone, differentiation takes only a few seconds and prolonged exposure results in all bacteria being decolourised particularly if the mordant step has been inefficient. As mentioned earlier, crystal violet is highly soluble in alcohol (and acetone) and the differentiation appears to be competition between the affinity of crystal violet for the acidic moieties in the bacterial cell wall and its solubility in alcohol. The function of the iodine is uncertain. If iodine is omitted, acetone still removes crystal violet from the bacteria, but more slowly and with less discrimination. It is possible that the iodine forms a complex with the crystal violet which is less lipophilic rendering the stain more easily removed from the lipid-rich Gram negative organisms.

Acid-fast bacilli do not reliably stain with Gram's technique although they may often be demonstrated as Gram positive. The Ziehl-Neelsen method utilises basic fuchsin which binds to the acidic groups on the unsaturated mycolic acid chains within the bacterial cell wall. Basic fuchsin is less lipophilic than crystal violet and does not readily penetrate the lipid-dense cell wall. Phenol added to the dye solution has a detergent effect on the lipid coat, allowing easier access to the dye by reducing the hydrophobic nature of the lipid. This is enhanced by prolonged staining at an elevated temperature. Once basic fuchsin has combined with acid groups in the cell wall, the dye complex is very resistant to decolourisation using mineral acids as differentiators, hence the term 'acid-fast'.

It has long been held that the leprosy bacillus, Mycobacterium leprae, is less acid fast than the more commonly encountered Mycobacterium tuberculosis. Numerous modifications of the Ziehl-Neelsen method have evolved to overcome the variable staining of the leprosy bacillus, including the use of dewaxing solvents other than xylene, minimal exposure to alcohol during staining, and the use of very dilute acid solutions for differentiation. The xylene substitutes are supposedly less deleterious on the 'fragile' cell wall of the leprosy bacillus and thus protect its limited acid-fastness. It is interesting to consider why a short exposure to xylene during dewaxing has such an adverse effect when hours in xylene or an equivalent solvent during paraffin processing does not. Presumably the same reasoning resulted in Carnoy's fixative being shunned, due to the chloroform content. The leprosy bacillus, once adequately stained, will withstand acid differentiation with the same tenacity as the tubercle bacillus. The leprosy bacillus may resist stain uptake to a greater degree than other mycobacteria, rather than being less able to retain it.

In addition to the Gram and Ziehl-Neelsen methods, most bacteria may be stained by the Giemsa technique,⁶ on the basis of their general level of basophilia. If present in sufficient numbers, bacteria may stain with haematoxylin and be visible in an H&E. Bacteria may also be demonstrated by the Feulgen technique for DNA and the methyl green-pyronin technique. That most bacteria are PAS-positive attests to the glycolipid content of the bacterial cell wall. Another characteristic of bacteria, which is especially useful when other methods fail, is that they possess a degree of argyrophilia. Spirochaetes and some other bacteria may only be successfully demonstrated by virtue of this property. The Warthin-Starry technique⁷ evolved as a paraffin section modification of the Levaditi⁸ method for spirochaetes which was developed as a tissue block impregnation technique. The Warthin Starry technique will demonstrate most bacteria and is particularly useful in some specific instances where the morphology of the organisms usually allows a specific diagnosis. Such organisms include spirochaetes, Campylobacter spp and the causative organism of granuloma inguinale, Calymmatobacterium granulomatis.

Commonly encountered pathogenic bacteria
Cocci
Most cocci are relatively uniform in size, being about 1 mµ in diameter on average.

STAPHYLOCOCCUS
Organisms in this group are Gram positive, the most significant species being Staphylococcus aureus, a common pathogen causing conditions ranging from boils to wound and burns infections of the skin, to osteomyelitis, pneumonia, cystitis, pyelonephritis and septicaemia. Many infections originate in the hospital environment as strains become resistant to antibiotics. Cell division occurs in irregular planes and the appearance in tissue sections is characteristically as irregular clusters.

STREPTOCOCCUS
These organisms are Gram positive and there are several significant species. Streptococcus pneumoniae is responsible for lobar pneumonia and inflammations of the respiratory system and appears in pairs, (hence its earlier title of Diplococcus pneumoniae). Other Streptococci occur either in pairs or short chains due to replication in a single axis.

S. pyogenes causes tonsillitis, scarlet fever, rheumatic fever and glomerulonephritis as well as milder oral and nasal infections. Other streptococcal species cause lymphoid, intestinal and cardiac infections.

NEISSERIA
This group is Gram negative and less well seen in tissue sections. The organisms occur mainly in pairs and may appear slightly flattened on one side rather than spherical. Significant in this group is N. gonorrhoea or the gonococcus, causing gonorrhoea, generally sexually transmitted, but also causing eye infections in the newborn as a consequence of maternal infection. Their characteristic appearance in tissue sections (Gram-Twort^9-10, or Giemsa) is as pairs resembling coffee beans. N. meningitis, or the meningococcus, causes meningitis, an inflammation of the membranes covering the brain. This organism also generally appears in pairs with the adjacent sides flattened.

Bacilli
Whilst all rod shaped bacteria are commonly referred to as bacilli, the term bacillus has a more specific meaning as a particular genus of bacteria, including Bacillus anthracis.

MYCOBACTERIUM
This genus comprises the so called acid-fast bacilli and includes M. tuberculosis, M. leprae and other less common species. Tubercle bacilli cause tuberculosis, still one of the most widespread diseases in the world. Any tissue site may become infected, but most commonly the lungs. The disease is characterised in tissues by the formation of nodules, or tubercles, which necrose and caseate. M. leprae, or Hansen's bacillus causes leprosy which may develop in two forms, either as lepromatous (or nodular type), forming granulomatous lesions in the skin and various internal organisms, or the tuberculoid type, where the organism infects nerves. Normally, more organisms are demonstrable in the nodular lesions. Both these organisms are Gram positive and stain by the Giemsa method, but are characterised by their acid-fast staining with the Ziehl-Neelsen technique. They are of similar size, but the leprosy bacillus is somewhat finer and typically appears as parallel clusters, whereas the tubercle bacillus occurs singly, in pairs or small bundles of parallel bacilli. Both organisms may show beading and are 2.5 to 3.5 µm in length by about 0.3 µm in diameter.

The mycobacteria comprise a large number of species, of which about a dozen may be pathogenic. Mycobacteria other than M. tuberculosis and M. leprae are often referred to as 'atypical'. Those of interest include M. ulcerans and M. marinum, both of which cause granulomatous skin lesions. M. marinum infections are often the result of wound contamination from sea water, swimming pools, fish tanks, and the like. Morphologically, they are somewhat longer than the tubercle bacilli and may appear bent or kinked in tissue sections where they are often present only in small numbers making them difficult to find by light microscopy. The fluorescent technique of Kuper and May¹¹ enables the organism to be located more easily.

Gram-positive bacilli
BACILLUS ANTHRACIS
This is the causative organism of anthrax, now rarely encountered, infecting humans and animals. The skin lesion, which is less severe than the respiratory or enteric forms of the disease, may be seen in farm workers and others who work with animals or their untreated skins. The primary lesion is called a pustule. The anthrax bacillus is readily seen in tissue sections as it is large, square-ended and up to 1.3 µm in diameter and between 3 and 10 µm in length. The organisms occur singly or in chains of two or more bacilli.

CLOSTRIDIUM
Several members of this genus including C. perfringens (C. welchii) cause gas gangrene which is characterised by oedema, necrosis and gangrene of tissues accompanied by gas production. C. tetani causes tetanus (lockjaw), which is an acute, often fatal toxic poisoning resulting from introduction of the organism into deep wounds. C. botulinum causes botulism, an extremely severe poisoning caused by eating contaminated food in which the organism has been allowed to grow and release its toxin. This condition may result in muscular paralysis and death from asphyxiation. C. perfringens is 1 µm in diameter by 4 to 6 µm in length and occurs singly or in pairs. C. tetani is slender by comparison, being 0.4 to 0.5 µm in diameter by 2 to 5 µm in length. C. botulinum is large and pleomorphic, being 0.9 to 1.2 µm in diameter by 4 to 6 µm in length.

CORYNEBACTERIUM DIPHTHERIAE
This organism produces diphtheria, an acute inflammation of the throat, which may cause respiratory obstruction and death by asphyxiation. The organism produces a toxin which may damage vital organs. The organism is pleomorphic, varying considerably in size and shape. It may be slightly curved or club-shaped and occurs as parallel or angular clusters due to the longitudinally snapping mode of cell division. Another corynebacterium like organism, Listeria monocytogenes, causes an uncommon form of meningitis and septicaemia. It may also produce perinatal infection in the mother which results in a generalised condition known as granulomatous infantiseptica in the newborn child. Listeria is a short bacillus 1.0 to 1.2 µm in length, usually occurring in pairs.

Gram-negative bacilli
ESCHERICHIA COLI
This is a small bacillus, 0.5 µm in diameter by 2 to 4 µm in length, normally occuring in the colon as a commensal organism, but also causes pathogenic infections elsewhere in the body, including septicaemia and septic shock in the urinary and biliary tracts and in wounds. Some strains are responsible for enteric infection, such as childhood diarrhoea and dysentry.

E. coli is the most common organism found in hospital acquired infections.

SALMONELLA TYPHI
Salmonella strains are similar to E. coli in morphology, and cause enterocolitis, bacteremia, localised infections and typhoid. Complications include intestinal perforation and endocarditis. Shigella dysenteriae causes a severe bacterial dysentery, resulting in dehydration from diarrhoea and may cause septicaemia. Morphologically, S. dysenteriae is similar to E. coli.

KLEBSIELLA PNEUMONIAE
Also known as Friedländer's bacillus, K pneumoniae causes pneumonia and urinary tract infections. Prolonged staining in acetified gentian violet followed by careful differentiation in dilute acetic acid may show a pale blue capsule of the organism surrounding the more intense blue halo of the bacillus itself.

YERSINIA PESTIS
This is the causative organism of plague (Black Death). The severe form may develop either as bubonic or pneumonic plague. It is a short oval bacillus with rounded ends (coccobacillus), 0.7 µm in diameter by 1.5 µm in length, occurring singly or in pairs. With Giemsa or methylene blue it exhibits bipolar staining, which is an important identifying characteristic.

BORDETELLA PERTUSSIS
A small, oval coccobacillus, B pertussis causes whooping-cough, one of the most common childhood fevers affecting the respiratory tract.

HAEMOPHILUS INFLUNZAE
H. influenzae is a very small bacillus or coccobacillus, 0.3 µm in diameter by 1.5 µm in length, occurring singly or in pairs but pleomorphic forms are commonly seen. A common cause of respiratory tract infections, it is the most frequent cause of meningitis in early childhood.

LEGIONELLA PNEUMOPHILA
This is the causative organism of Legionnaires' Disease, a severe bacterial broncho-pneumonia. It is a short, blunt, pleomorphic bacillus 1 µm in diameter by 2 to 4 µm in length. It is Gram negative, but often difficult to see as the organisms tend to be distributed in extracellular debris as well as within macrophages and neutrophils. It can be stained by the Giemsa technique, but is best demonstrated by the Warthin-Starry or Dieterle¹² methods. Other similar organisms such as Haemophilus are more readily stained by the Gram technique.

CALYMMATOBACTERIUM GRANULOMATUS
This organism causes granuloma inguinale, an ulcerating granulomatous skin and subcutaneous lesion. It may be found associated with other venereal diseases. It is a Gram negative coccobacillus 0.5 to 1.5 µm in diameter and 1.5 to 2 µm in length. Staining reveals prominent polar granularity, accounting for their description as 'safety pins'. The organisms are found in large mononuclear cells in considerable numbers, as well as extracellularly. They are best demonstrated by the Warthin-Starry or Dieterle methods.

Curved, wavy or corkscrew rods
VIBRIO CHOLERAE
V. cholerae is the agent responsible for cholera, an acute intestinal infection which causes vomiting and diarrhoea leading to dehydration, and sometimes death. The organism is Gram negative and curved or comma shaped, about 0.5 µm in diameter by 1.5 to 3 µm in length.

CAMPYLOBACTER
Campylobacter spp are Gram negative, curved rods which are of pathological significance in the intestinal tract and are well demonstrated by the Warthin-Starry technique. Florid growths characteristically occur on the surface of intestinal mucosa.

HELICOBACTER PYLORI
This organism survives in the mucous layer of the stomach by generating ammonia, which neutralises gastric acid secretions, from urea. The organism contains a powerful urease enzyme and is stained with Gram, Giemsa and Warthin Starry methods.

TREPONEMA PALLIDUM
A spirochaete or corkscrew rod, this organism causes syphilis. It is unusual to see the organism in tissue sections of tertiary syphilitic lesions, but may be found in the secondary stages of the disease in lymph nodes, ulcers and condylomas of the genitals. T pallidum does not stain by Gram's technique and is not readily stained by Giemsa. The method of choice is the Warthin-Starry. The organisms are 6 to 10 µm in length by 0.2 µm in diameter.

LEPTOSPIRA SPP.
The clinical manifestations of leptospirosis in humans and animals are variable, ranging from a mild, catarrh-like illness to icteric disease with severe kidney and liver involvement. Fever may be a prominent feature. Leptospiras are 0.1 µm in diameter by up to 20 µm in length and are demonstrable by the Warthin-Starry technique.

Chlamydias and rickettsias
Various chlamidias are responsible for such diseases as ornithosis (psittacosis), which is transmitted by birds, trachoma, a common cause of blindness, and lymphogranuloma venereum, a venereal disease involving the inguinal lymph nodes. The organisms are smaller than other bacteria, pleomorphic and grow within cells.

Rickettsias cause epidemic and endemic typhus, Rocky Mountain spotted fever and Q fever. They are, like chlamydias, intracellular parasites, pleomorphic, but typically rodlike and often found in pairs.

Many chlamydias and rickettsias, although close to the limit of resolution of the light microscope, can still be seen with this instrument. They are not demonstrated by the Gram, but may be stained by the Giemsa and Macchiavello¹³ techniques.

Mycelial bacteria
There are a number of bacteria of human pathological significance which form colonies of branching filaments and thus resemble fungi.

ACTINOMYCES SPP. (ACTINOMYCETES)
Commensal actinomycetes of the mouth and tonsils are the causative agents of actinomycosis a chronic suppurative disease principally affecting the face, neck and thorax. Actinomyces israelii is the most common organism associated with this condition. Dense colonies of filaments are usually surrounded by pus. Macroscopically, the colonies appear as yellow 'sulphur' granules which are basophilic in an H&E stain and surrounded by an eosinophilic periphery of club-like structures which may be a lipoid deposit formed as a host reaction. Filaments are Gram and Methenamine silver positive,^14-15 whereas the clubs react positively in the periodic acid Schiff (PAS) technique and may be acid fast. Giemsa stains both the filaments and clubs.

NOCARDIOSIS
This is a disease caused principally by the organism Nocardia asteroides. Nocardia develops delicate branching filaments, which are Giemsa, Gram and Methenamine silver positive and may be acid-fast with a Ziehl-Neelsen stain. The organism is not stained by haematoxylin or PAS. Nocardiosis is a suppurative disease of the lungs, and may spread to other organs, such as the brain and meninges. Unlike Actinomyces, Nocardia does not form dense colonies or granules, and tends to branch at right angles. Actinomyces, and species of Nocardia and the related Streptomyces are some of the causes of Mycetoma or Madura foot, which is also brought on by some species of true fungi. Nocardiosis is often associated with pneumonia in immunosuppressed patients and may be present in the lungs with other opportunistic infections.

Demonstration of fungi in tissue sections
Infections in humans, produced by true fungi, are referred to as mycoses and range from common ringworm infections of the skin and nails, to deep and often systemic infections which may be fatal. Pathogenic fungi are becoming relatively more significant as they are generally resistant to antibiotics used to combat bacterial infections. They are an increasing cause of opportunistic infection associated with immunosuppression. A clinical mycologist would classify fungi according to the nature of their somatic and sexual structures. in histopathology a fungal entity can only be identified on the basis of a two dimensional slice through the organism and it is therefore more appropriate to consider dividing fungi into categories according to simple morphological characteristics.

Moulds grow as long filaments or hyphae which branch and form a meshwork or 'mycelium'. They produce spores for reproduction and usually invade body tissues only locally, infecting new hosts by transmission of spores. Example, Aspergillus fumigatus.

Yeasts are unicellular, spherical or ellipsoidal which reproduce by budding. Example, Cryptococcus neoformans.

Yeast-like fungi exist partly as yeasts and partly as long filamentous cells joined end to end, forming a pseudomycelium. Example, Candida albicans.

Di-morphic fungi may exist either as yeast forms or as filaments, depending on conditions. As a parasite in the body, they usually exist as yeasts, but form mycelium in vitro. Example Blastomyces dermatiditis.

The majority of fungi with pathological significance belong to a class referred to as Fungi Imperfecti. They do not have a sexual growth phase, but reproduce by asexual spore formation or by budding. Fungi Imperfecti comprise members of all four morphological types mentioned previously.

Dermatophytes
Dermatophytic fungi cause superficial mycoses (ringworm or tinea) of the skin, hair and nails. There are three main genera of fungi responsible for ringworm, namely Microsporum, Trichophyton and Epidermophyton. They possess the ability to digest keratin and parasitise keratinous structures, but do not invade underlying tissues. The leading edge of a lesion on the skin usually occurs as a red inflammatory ring, hence the term 'ringworm'. Hyphae may occasionally be seen in an H&E stained section, but are difficult to discern due to the intense eosinophilia and layering of the keratin through which they grow. Most dermatophytes are intensely PAS positive. The presence of glycogen in the degenerating superficial skin layers may obscure hyphae, and diastase digestion is recommended before staining. Staining with methenamine silver gives good results provided the impregnation is light, but background staining of keratin can obscure the hyphae. Other superficial mycoses may arise from a variety of fungal types.

Chromoblastomycosis is a chronic and granulomatous infection of the skin and subcutis, which may extend to the lymphatic system. It is caused by a number of different species of pigmented fungi, usually introduced into the skin by penetration of splinters or thorns. In tissue sections, the fungi appear as round bodies and vary from pale to dark brown. This characteristic is lost when stained with PAS or methenamine silver.

Deep mycoses
Deep mycoses usually involve the skin, respiratory system, or become disseminated. Many deep mycoses occur as opportunistic infections in immunosuppressed cases and drug addicts, or may complicate a pre-existing infection. Certain occupations, such as agricultural workers, carry a greater risk of infection as many species of moulds live in the soil or on decaying organic matter.

CANDIDA ALBICANS
This occurs as a commensal organism in humans which becomes pathogenic in an opportunistic way. Infections range from skin or mucosal lesions (oral and vaginal thrush), to systemic infections involving the respiratory system and less frequently meningitis, endocarditis and pyelonephritis. The organism manifests partly as rounded yeast forms, which may show budding, and partly as a pseudomycelium of non branching filaments. The yeasts are Gram positive and both forms are strongly PAS positive and methenamine silver positive. Yeast forms may only be seen, but it is not usual to see filaments alone.

ASPERGILLUS
this species especially A. fumigatus is another commensal which may opportunistically infect diseased lungs or become systemic in immunosuppressed patients. Aspergillus is a filamentous fungus, with hyphae that are septate and show dichotomous branching. A. niger is pigmented and appears brown in H&E. Characteristic conidiophores (fruiting heads) may be present, producing spores. Aspergillus hyphae are moderately basophilic in H&E, poorly stained with PAS, but well demonstrated with methenamine silver.

MUCORMYCOSIS
Mucormycosis may also arise in immunosuppressed patients as an acute disease and may mimic aspergillus. Mucor, Rhizopus and Absidia are related fungi that cause this condition, but differ from Aspergillus species in that the hyphae are broad, irregular and rarely septate with irregular branching.

CRYPTOCOCCUS NEOFORMANS
Cryptococcus neoformans is a true yeast which reproduces by budding and does not have a hyphal form. The organism often presents as an opportunistic lung infection, but may cause meningitis and skin lesions. The organism is 5 to 20 µm in diameter, Gram positive and surrounded by a thick mucoid capsule which allows its demonstration to be highly selective. The capsule stains light blue with haematoxylin and also reacts with a variety of mucin stains, such as PAS, Southgate's mucicarmine and alcian blue. Cresyl fast violet staining under polarised light¹⁶ produces a spectacular birefringence, which is also a feature of alcian blue staining. Methenamine silver impregnates the organism, but does not stain the capsule.

A number of di-morphic fungi cause deep mycoses in man, usually only occurring in round body or yeast form.

BLASTOMYCES DERMATITIDIS (NORTH AMERICAN BLASTOMYCOSIS)
This organism causes a chronic suppurative and granulomatous infection which occurs as a primary skin lesion or a primary lung infection which may disseminate. The organism is a thick walled round body which often shows budding and some mucoid staining of the capsule. The cell contains numerous nuclei, a feature which distinguishes it from cryptococcus.

PARACOCCIDIODES BRASILIENSIS (SOUTH AMERICAM BLASOMYCOSIS)
This is a round body organism of similar size to Blastomyces dermatitidis (5 to 15 µm in diameter) with a similar thin mucoid coat. A distinguishing feature is the phenomenon of multiple budding of a single cell. The primary infection usually involves the lungs, producing epithelioid cell granulomas with foreign body giant cells.

COCCIDIODES IMMITIS
This is an endemic infective agent in parts of the United States of America. The fungus occurs in tissues as a non-budding spherical thick walled cell, approximately 20 to 70 µm in diameter containing numerous, small (2 to 5 µm) endospores. Again the primary lesion is pulmonary, but dissemination may occur. The organism is usually detectable with H&E, but a methenamine silver method renders it clearly visible.

HISTOPLASMA CAPSULATUM
This organism causes histoplasmosis, a disease endemic in the Mississippi delta, but occurring occasionally throughout the world. In tissues it occurs as a yeast form (1 to 5 µm across), usually as an intracellular parasite in reticuloendothelial macrophages. The organism is a budding yeast, oval rather than spherical, producing primary lesions in the lungs, which may disseminate via the reticuloendothelial system. In the lungs of immune-resistant hosts, an epithelioid cell granulomatous reaction which resembles non-caseous tuberculosis develops. This organism may be seen in H&E when present in macrophages, and may be confused with Toxoplasma and Leishmania. A methenamine silver stain will demonstrate histoplasma as a fungus. Very occasionally a hyphal form may be seen in old caseous lesions.

Protozoal diseases
ENTAMOEBA HISTOLYTICA
E. histolytica is a pathogenic amoeba which causes dysentery with necrosis, ulceration and occasional perforation of the large bowel. In tissues, amoebae occur either as trophozoites, showing amoeboid shapes, or as rounded cysts, containing up to four nuclei. Both trophozoites and cysts are visible in H&E and trophozoites may contain ingested red blood corpuscles. They are well stained by Giemsa and the PAS technique which reacts with the abundant glycogen, particularly in young cysts.

GIARDIA LAMBLIA
This protozoan flagellate is associated with gastrointestinal disturbances and is usually found on the mucosal surface. In sections the organisms are about 15 µm in length and vary in shape from quadrangular to triangular or sickle shapes. They are greyish-blue in H&E and are enhanced by Giemsa staining. A rounded cyst form may contain two to four nuclei.

TRICHOMONAS HOMINIS
T. hominis is another intestinal flagellate, 10 to 15 µm in length, associated with dysentery and diarrhoea, whilst T. vaginalis causes inflammation of the vagina in females and urethritis in males.

MALARIAL PLASMODIA
There are four species of malarial parasites which invade certain human tissue cells and red blood corpuscles. In tissue sections there is often an associated malarial pigment deposition which may obscure the parasites. The pigment is similar to formalin pigment and may be removed by prolonged treatment of sections in saturated alcoholic picric acid. The trophozoite is visible in H&E as a small blue-black body in the red cell, but is better demonstrated with Giemsa or Leishman stains, or with the Feulgen technique for DNA.

LEISHMANIASIS
This condition is caused by several species of Leishmania and may present as a cutaneous lesion caused by L. tropica, a muco-cutaneous lesion caused by L. brasiliensis, or as a visceral disease (Kala-Azar) caused by L. donovani. These protozoan parasites are similar in appearance and in Kala-Azar are found as numerous small haematoxyphilic bodies in endothelial cells of the spleen, liver, bone marrow and lymphatics. In cutaneous leishmaniasis the organisms occur in large numbers in histiocytes aggregating in the dermis. They are easily seen in H&E, but are enhanced by the Giemsa stain.

PNEUMOCYSTIS CARINII
Pneumocystis carinii is an extracellular protozoan which causes interstitial plasma cell pneumonia in immunosuppressed patients. The lung consolidation may lead to death as the alveolar spaces become packed with organisms, which exist as free parasites or encysted forms. The parasites are faintly visible in H&E and are enhanced by the Giemsa technique. The cysts walls, which are 3.5 to 5 µm in diameter, stain strongly with methenamine silver.

Helminths
Nematodes
A number of nematode worms may inhabit the intestinal tract. Ascaris lumbricoides may be up to 30 cm long. Others include Trichuris trichiura and Strongyloides stercoralis. Nematode worms are readily visible in H&E stains and may appear in various cross sections. It may be possible to identify a species if ova are found, as these tend to have characteristic morphology. Enterobius vermicularis is commonly encountered as an incidental finding in appendix.

Cestodes
Cestodes can exist as intestinal tapeworms such as Taenia saginata and T. solium or inhabit other tissue sites. Echinococcus granulosus is responsible for hydatid disease, with cysts arising principally in the liver and lungs. Cysts are visible with H&E or PAS and are identifiable by the presence of scolices and hooklets, the latter also being birefringent under polarised light.

Trematodes
Perhaps the most significant trematode worms are Schistosoma mansoni and S. japonicum which are responsible for schistosomiasis or bilharzia. The worms can be identified in tissue sections but it is the ova which cause granulomata in the bladder wall, liver bowel mucosa and occasionally the lungs. Ova may be calcified in which case the calcium can be removed from sections with a suitable decalcifying agent before staining. The ova are readily seen in H&E and PAS stained preparations.

Viral diseases
Individual virus particles are too small to be visualised with the light microscope. However, during the course of replication in host cells, many viruses form visible structures, referred to as inclusion bodies, which may be up to 30 µm in diameter. The inclusion body may be in the cell nucleus, cytoplasm or occasionally both. Inclusion bodies are commonly acidophilic regardless of whether they are intracytoplasmic or intranuclear. Those viruses that do not produce inclusion bodies generally cannot be visualised by traditional staining methods in tissue sections although they do produce characteristic cellular changes which can be identified in cytology preparations. Where a virus does produce inclusion bodies the best method for screening purposes is an haematoxylin and eosin stain. Inclusion bodies may stain with either dye but are commonly eosinophilic and characteristic of a particular virus. A number of methods have been developed or modified to enhance the acidophilia of viral inclusion bodies. These include the Macchiavello technique,¹³ Mann's methyl blue-eosin,¹⁷ the phosphotungstic acid-eosin technique,¹⁸ Lendrum's phloxine-tartrazine technique¹⁹ and the modified orcein technique²⁰ which is also useful for hepatitis B antigen in liver cells. Apart from the Macchiavello technique, these methods target the protein component of the inclusion body, which imparts the acidophilia. Viruses are actually composed of an inner core of either DNA or RNA, usually surrounded by a shell of structural protein. Methods for RNA and DNA, such as Feulgen and methyl green-pyronin, are of value as demonstration techniques.

Some demonstrable viral diseases
A number of viruses of different groups may cause pneumonias, often as opportunistic infections.

HERPES GROUP
Herpes varicella-zoster and Herpes simplex virus type II both produce intranuclear inclusions in cells peripheral to the necrotic primary lesions. In H&E stained sections the inclusion body is purple to red and occupies all of the nucleus. Old inclusions become more eosinophilic and are surrounded by a distinct halo. Infections with these viruses may produce mono or multinucleated cells with visible intranuclear inclusions which are moderately eosinophilic or may be demonstrated by a suitable DNA technique.

CYTOMEGALOVIRUS
Infection by cytomegalovirus is typically opportunistic, causing inflammation, necrosis and subsequent focal or general consolidation of the lung tissue. The viral inclusion is intranuclear, generally eosinophilic and very obvious, with a distinct halo surrounded by the nuclear membrane and resembling an 'owls eye'. Infected cells may also show small granular, intracytoplasmic basophilic bodies.

ADENOVIRUS
Adenoviral pneumonia is most common in infants under two years old and causes bronchial tree inflammation and necrosis as well as pulmonary involvement. Intranuclear inclusion bodies are found principally in bronchiolar epithelium and alveolar cells. In H&E, the infected cells appear 'smudged' with an amorphous inclusion body. Occasionally, small 'halo' inclusions are seen.

MEASLES (RUBELLA)
Measles pneumonia is most common in school children and varies from interstitial pneumonitis to lobar pneumonia, often with secondary bacterial infection. The characteristic feature is the presence of syncitial giant cells containing both intranuclear and intracytoplasmic inclusion bodies of variable size and shape. The inclusion bodies are markedly acidophilic and stain brightly with eosin and phloxine.

VIRAL HEPTITIS
Hepatitis B antigen or Australia antigen is demonstrable by light microscopy. Liver cells show a typical 'ground glass' appearance of the cytoplasm of affected cells. These hepatocytes are often larger than normal and have an eosinophilic 'frosted glass' cytoplasm. Granular staining of the cytoplasm with the modified orcein or Gomori aldehyde-fuchsin techniques is a characteristic of these cells.

RABIES
Humans are occasional victims of rabies, an acute infectious disease of the central nervous system. The virus is of RNA type and inclusion bodies are most commonly found in Purkinje cells of the cerebellum. The characteristic inclusion body, or Negri body, is oval to round, eosinophilic, clearly defined and up to 10 µm in diameter. There may be one or multiple bodies in the cytoplasm of the infected cell.

POX VIRUSES
Smallpox and other pox viruses produce both intranuclear and intracytoplasmic acidophilic inclusions in affected cells. The cytoplasmic inclusions or Guarnieri bodies are clearly seen in cells which are otherwise vacuolated by various stages of ballooning degeneration.

Staining methods
Giemsa technique⁶ (modified)
SECTION PREPARATION
Choice of fixative is not critical, formaldehyde fixatives give good results. Paraffin sections are cut at 3 to 4 µm. A control section appropriate to the type of organism being sought may prove useful.

REAGENTS REQUIRED
1 Giemsa stock solution
Giemsa's original formula (see below) can prove difficult to prepare and commercially available dye powder dissolved in equal parts of glycerol and methyl alcohol according to the manufacturer's instructions, is recommended. Commercially prepared stock solutions are also available. Giemsa's formula:
Azur II - eosin 3.0 g
Azur II 0.8 g
Glycerol 3 ml
Methyl alcohol 3 ml
The dye powder is dissolved in the glycerol at 50°C with intermittent agitation for up to one hour. After cooling, the methyl alcohol is added and the solution mixed and filtered. The stain quality improves over the first week or two after which the solution is stable for several years (4 g of commercially available Giemsa powder may be substituted for the Azur dyes in the above formula).

2 Giemsa working solution
Dilute stock solution with distilled water at an approximate ratio of 1:25.

3 Differentiator
0.5% acetic acid in 95% ethyl alcohol.

METHOD
1 Dewax and rehydrate sections using distilled water.
2 Place in working solution kept in a coplin jar, at room temperature for 12 to 18 hours (30 to 60 minutes at 60°C gives an adequate result).
3 Rinse sections in distilled water, drain well or blot.
4 Differentiate in acetified alcohol, checking progress microscopically - see technical note.
5 Arrest differentiation by washing in absolute alcohol.
6 Clear in xylene and mount in synthetic resin.

RESULTS (Fig 1)
The method will stain many types of organism purplish blue, though metazoan parasites, such as worms, may show variation in colour. Organisms known to be demonstrated by the method include, most Gram positive and Gram negative bacteria; amoebae; acid-fast bacilli; malarial parasites; Donovan bodies (granuloma inguinale); Toxoplasma; Legionella pneumophila; Pneumocystis; Actinomyces; Nocardia asteroides; Leishmania; most pathogenic fungi; metazoan parasites; Pigmented fungi such as those found in chromoblastomycosis, and Aspergillus niger, may appear green due to over staining with blue dye.

Cell nuclei stain blue, connective tissues pink, red blood cells salmon pink, starch and cellulose sky blue.

TECHNICAL NOTES
There are numerous modifications of the Giemsa method, using pH adjusted staining solutions and a variety of differentiating agents. The key to success lies with a combination of prolonged staining in a freshly prepared dilute working solution and careful differentiation. Dilute aqueous acetic acid is commonly described as a differentiator but an alcoholic solution is slower and more controllable. Differentiation may be arrested at any time, for microscopic evaluation, by washing in absolute alcohol. Differentiation proceeds with the removal of excess blue stain to reveal to pink red staining which is masked in the undifferentiated section. Differentiation is complete when the section has an overall appearance similar to an H&E, with blue nuclei and pink connective tissue. Organisms tend to stain a purplish blue in contrast to the cell nuclei which are a clear blue.

Gram's technique for bacteria³ (modified)
SECTION PREPARATION
Choice of fixative is not critical, formaldehyde fixatives give good results. Paraffin sections are cut at 3 to 4 µm. A known positive control must be used, preferably mounted on the same slide as the test sections. Use a Gram negative control (such as E. coli) when staining for Gram negative organisms.

REAGENTS REQUIRED
1 Crystal violet solution²¹
Crystal violet (CI 42555) 2 g
95% alcohol 20 ml
Ammonium oxalate 0.8 g
Diluted water 80 ml
Dissolve the dye in the alcohol, the ammonium oxalate in the water and mix the two solutions together. Filter before use. The stain is stable for 2 to 3 years.

2 Gram's iodine
Iodine crystals 1 g
Potassium iodide 2 g
Distilled water 300 ml
As iodine is more readily soluble in a strong solution of potassium iodide, dissolve the potassium iodide in 5 ml of water and dissolve the iodine in this. Dilute to 300 ml for Gram's iodine, to 100 ml for Lugol's iodine.

3 1% aqueous neutral red (CI 50040)

4 Twort's stain
0.2% neutral red in ethanol 9 ml
0.2% fast green F.C.F. in ethanol (CI 42053) 1 ml
Distilled water 30 ml
Prepare immediately before use.

METHOD
1 Dewax and rehydrate sections in distilled water.
2 Flood with filtered crystal violet for 5 minutes.
3 Wash off crystal violet with Gram's iodine (continue until metallic precipitate is washed away) then flood with iodine and leave for 5 minutes.
4 Drain slide, blot dry and differentiate in acetone (see notes).
5 Plunge slide into running tap water and wash for 1 minute.
6 Counterstain with neutral red for 1 minute or Twort's stain for 5 minutes (see notes).
7 Rinse in distilled water, dehydrate and differentiate counterstain in absolute alcohol.
8 Clear in xylene and mount in synthetic resin.

RESULTS (Fig 2)
Gram positive bacteria blue/black
Actinomyces mycelium blue/black
Actinomyces clubs red
Gram negative bacteria red
Keratin, fibrin, calcium may be blue/black
Nuclei red

TECHNICAL NOTES
1 1% aqueous crystal violet is an adequate stain solution.
2 Gram's iodine may be diluted from a stock of Lugol's iodine, or Lugol's iodine may be used instead.
3 The method is regressive, all bacteria being stained with crystal violet before differentiation. Under-differentiation causes Gram negative organisms to remain blue, whilst over-differentiation decolourises Gram positive organisms. Differentiation in acetone is very rapid, usually two to five seconds, and acetone should be poured liberally onto the slide to ensure even destaining. As soon as the section appears macroscopically colourless it should be plunged into running tap water to stop differentiation. An alternative differentiator, which is a little slower and allows more control, is a mixture comprising equal parts acetone and ethanol. A more even differentiation may be achieved if the acetone/ethanol is placed in a jar and the slide agitated in it.
4 Prolonged washing in water after differentiation should be avoided as this may decolourise positive bacteria.
5 Twort's counterstain is useful when staining for Gram negative organisms.^9-10 The organisms are coloured by the neutral red and contrast well with the green background colour. The counterstain should be differentiated for up to 15 seconds in alcohol with 2 ml acetic acid added per 100 ml.
6 Final dehydration in absolute alcohol should be as brief as possible to avoid decolourising Gram positive organisms. As the alcohol serves to differentiate the neutral red counterstain, counterstaining should be performed critically to avoid the necessity for prolonged dehydration.
7 Gram's technique will remove mercury pigment.

Ziehl-Neelsen technique for acid fast bacilli^1-2 (modified)
SECTION PREPARATION
Choice of fixative is not critical, but some authors caution against the use of Carnoy's fixative. Paraffin sections are cut at 3 to 4 µm. Always use a known positive control for the organism being sought, preferably mounted on the same slide as the test sections.

REAGENTS REQUIRED
1 Carbol fuchsin
basic fuchsin (CI 42500) 1 g
absolute ethanol 10 ml
phenol crystals 5 g
distilled water 100 ml
The basic fuchsin is dissolved in distilled water and the phenol in absolute ethanol. The two solutions are mixed together and filtered before use. The stain has a long shelf life. CAUTION: phenol is highly toxic and may be absorbed through the skin. Use gloves, eye protection and prepare the solution under a fume extraction hood.

2 0.5% hydrochloric acid in 70% alcohol

3 0.1% aqueous methylene blue (CI 52015)

METHOD
1 Dewax sections and rehydrate in distilled water.
2 On a stain rack, flood sections with filtered carbol-fuchsin, heat from below with a spirit flame until the solution begins to steam, leave for 10 minutes. Alternatively place the dye in a coplin jar, heat to 65°C then stain for 10 minutes. (If a coplin jar is used it is placed into cold water after 10 minutes. The slides remain in the staining solution until the dye has cooled).
3 Drain sections, blot dry and differentiate directly in acid alcohol (see notes).
4 Wash sections in tap water for 2 minutes.
5 Counterstain in methylene blue for 15 to 30 seconds.
6 Rinse in tap water.
7 Dehydrate and differentiate counterstain in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS (Figs 3 and 4a)
Acid-fast bacilli deep red
Nuclei blue
Background tissues very pale pink

TECHNICAL NOTES
1 To avoid using a naked flame sections may be placed in a coplin jar of preheated carbol-fuchsin (60 to 65°C) and left until the solution has cooled.
2 Avoid washing in tap water before differentiation as this tends to produce a heavier residual background colour.
3 Use two changes of acid-alcohol to differentiate, transferring from one to the other when the excess stain has been removed. This produces a lighter background stain. Any gross deposits of precipitated stain may be removed more readily by alternate blotting and washing in acid alcohol. Differentiation should proceed until the section is a pale pink and need take no longer than two minutes. Differentiation may be shortened further when staining Mycobacterium leprae or atypical mycobacteria.
4 The methylene blue counterstain should be light and confined to the cell nuclei. Excess methylene blue may be removed by alternate rinsing in water and alcohol.
5 An alternative procedure uses Victoria blue R instead of basic fuchsin, and Bismarck brown as a counterstain (0.5 g Bismarck brown in 80 ml absolute alcohol to which is added 10 ml 1% aqueous hydrochloric acid).

Ellis and Zabrowarny method for acid fast bacilli²¹
This method demonstrates acid fast bacilli as well as the Ziehl-Neelsen technique without the need to use phenol, which is a hazardous substance. Using the stain in a coplin jar removes the need to 'flame' stain on a slide. This method is also particularly suited to the demonstration of lipofuschins giving more intense staining than a long Z.N.

SECTION PREPARATION
Choice of fixative is not critical but Carnoy's fixative is not widely advocated. Paraffin sections are cut at 3 to 4 µm. Always use a known positive control for the organism being sought, preferably mounted on the same slide as the test section.

REAGENTS REQUIRED
1 Staining solution
Solution A
Basic fuchsin (CI42500) 1 g
Absolute ethyl alcohol 10 ml

Solution B
L.O.C. High Suds (Amway) 0.6 ml (or similar liquid organic cleaner)
Distilled water 100 ml
The two solutions can be kept as stock solution and mixed immediately before use.

2 3% hydrochloric acid in 95% ethyl alcohol
Absolute ethyl alcohol 95ml
Distilled water 2 ml
Concentrated hydrochloric acid 3 ml
Make up the alcohol solution then add the concentrated acid.

3 0.25% methylene blue in 1% acetic acid
Methylene blue (CI 52015) 0.25 g
Distilled water 99 ml
Acetic acid 1 ml

METHOD
1 Place the staining solution in a coplin jar and pre-heat to 60°C for 10 mins
2 Deparaffinise sections, bring to water and stain in the pre-heated solution for 15 mins
3 Place the COPLIN JAR containing the slides into running cold tap water for 2 mins before removing the slides from the coplin jar.
4 Remove the slides from the coplin jar and wash in running water for 1 min
5 Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs from the slide
6 Wash briefly in water to remove the acid alcohol
7 Counterstain with 0.25% methylene blue in 1% acetic acid for 15 to 30 secs
8 Wash in water, dehydrate, clear and mount in DPX

RESULT (Fig 4b)
Acid fast bacilli Red
Nuclei Blue
Other tissue constituents Blue

The Kuper and May technique for Mycobacteria¹¹
SECTION PREPARATION
Choice of fixative is not critical, formaldehyde and Zenker's formula give good results. Paraffin sections are cut at 3 to 4 µm. A suitable control for the organism being sought is essential.

REAGENTS REQUIRED
1 Auramine rhodamine solution
Auramine O (CI 41000) 1.5 g
Rhodamine B (CI 54170) 0.75 g
Glycerol 75 ml
Phenol (liquified at 50°C) 10 ml
Distilled water 50 ml
Mix solution thoroughly and filter before use. The solution is stable for at least three months.

2 0.5% hydrochloric acid in 70% alcohol

3 0.5% aqueous potassium permanganate

METHOD
1 Dewax sections and rehydrate in distilled water.
2 Stain in auramine-rhodamine solution in a coplin jar for 10 minutes at 60° C.
3 Wash in running tap water for 2 minutes.
4 Differentiate in acid-alcohol for 2 minutes.
5 Wash in running tap water for 2 minutes.
6 Flood with potassium permanganate for 2 minutes.
7 Wash in running tap water for 2 minutes.
8 Blot slides dry, dehydrate rapidly in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS (Fig 4c)
Sections are examined using ultraviolet light microscopy. Mycobacteria reddish-gold fluorescence
Fluorescent artefacts usually pale yellow
Background dark

TECHNICAL NOTES
1 It is important that the auramine-rhodamine solution is preheated to 60°C and the temperature maintained for the staining period.
2 The fluorescence will fade whether sections are mounted in DPX or Fluormount and should be examined promptly. Even when mounted in Fluormount the specific fluorescence will fade within 3 to 4 days, and non-specific background fluorescence will increase.
3 As with the Ziehl-Neelsen technique, this method will demonstrate Mycobacterium leprae as well as tubercle bacilli and a number of atypical mycobacteria. Should there be concern about the relative acid and alcohol fastness of Mycobacterium leprae, the method may be modified by dewaxing in alternate solvents, such as equal parts liquid petrolatum and rectified turpentine²² using dilute aqueous hydrochloric acid to differentiate and avoiding dehydration in alcohol.

The Warthin-Starry technique⁷ (modified)
SECTION PREPARATION
The recommended fixative is 10% buffered formalin or 10% buffered formol-saline. Paraffin sections are cut at 3 to 4 µm. An appropriate control must be used (see notes).

REAGENTS REQUIRED
1 Acetate buffer (pH 3.6)
Sodium acetate 1.64 g
Glacial acetic acid 2.5 ml
Distilled water 200 ml

2 Silver solution
Silver nitrate 0.5 g
Acetate buffer 50 ml

3 Developer
a) Dissolve 7.5 g of gelatine in 150 ml distilled water in a conical flask by heating to and maintaining at 60°C.
b) Dissolve 0.3 g quinol (hydroquinone) in 10 ml acetate buffer. The quinol solution may be warmed just prior to use to dissolve the quinol crystals in the buffer.
c) Dissolve 0.6 g silver nitrate in 30 ml distilled water.
The silver nitrate solution may be kept at room temperature.
Immediately before use (b) is added to (a) and rapidly mixed. The silver nitrate (c) is then added and the solution rapidly mixed.

The above reagents are prepared in the volumes given to allow sections to be developed in a coplin jar, rather than on a staining rack (see notes). Large numbers of slides may be developed using a glass slide rack in a staining trough.

METHOD
1 Dewax sections and rehydrate with distilled water.
2 Place in a coplin jar of acetate buffer for 5 minutes.
3 Transfer to a coplin jar of silver solution (2), preheated to 60°C, for 1 hour.
4 Drain silver solution and replace immediately with the freshly prepared developer. Stand coplin jar in a trough of hot water at approximately 60°C and agitate slides gently.
5 When the desired development has taken place (see notes), transfer sections immediately to hot tap water and agitate for 30 seconds.
6 Rinse sections in distilled water and place in coplin jar of acetate buffer for up to 5 minutes.
7 Rinse in distilled water.
8 Dehydrate in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS (Fig 5)
Spirochaetes and other bacteria black
Background tissues yellow/brown
The method will also demonstrate fungi, but is most useful in identifying bacteria that are not well demonstrated by other methods and are generally typified by their morphology and distribution. These include spirochaetes, Donovan bodies (in granuloma inguinale), Campylobacter (in intestinal infections) and Legionella.

TECHNICAL NOTES
1 A known positive control must be used, preferably mounted on the same slide as the test section. However, it is not essential to use the same control as the organism being sought. The same degree of development will demonstrate any of the organisms for which the method is used.
2 Impregnation in the silver solution (step 3) is best carried out in the dark.
3 The development is the critical stage of the technique. The developer deteriorates rapidly after the reagents are mixed, so it is important to prepare and use it immediately. It is better to prepare fresh 2% silver nitrate than to dilute an existing stock solution. The sections will begin to develop a golden brown colour almost immediately and the correct degree of development is generally attained within 20 to 60 seconds, depending on the temperature of the developer. The reaction should be halted before a dark brown is produced (correct colour a gloden brown) and it may prove helpful to stain more than one slide each being removed at longer intervals as the optimal colour is reached.
4 The development is arrested by plunging the sections into hot running tap water (cold water will congeal the gelatine). If over impregnation occurs, the excess may be removed by brief rinses in 1% aqueous potassium ferricyanide. Note however, that this differentiation is not discriminatory and will remove silver from the organisms at the same rate as the background.
5 The method will also demonstrate melanin granules very sharply and this needs to be considered when searching for organisms in sections of skin.

Grocott's modification of Gomori's methenamine silver technique for fungi^14-15
SECTION PREPARATION
Choice of fixative is not critical, but see technical notes. Cut paraffin sections at 3 to 4 µm. Use an appropriate control section.

REAGENTS REQUIRED
1 Methenamine silver solution
3% methanamine (hexamine) 25 ml
5% silver nitrate 1.25 ml
5% sodium tetraborate (borax) 2 ml
Distilled water 25 ml
Mix the reagents in the order given, a white precipitate forms on addition of silver nitrate to the methenamine, which dissolves promptly when shaken. The silver nitrate/methenamine solution may be kept at 4°C as a stock solution, but if the method is only performed occasionally it is better to prepare from separate reagents.

2 Chromic acid oxidiser
Dissolve 5 g chromium trioxide in 100 ml distilled water. This is a corrosive solution and should be handled with care.

4 0.2% aqueous yellow gold chloride

5 5% sodium thiosulphate (hypo)

6 Counterstain as desired (see notes)

METHOD
1 Dewax sections and rehydrate with distilled water.
2 Oxidise for 1 hour in chromic acid (on stain rack or in coplin jar).
3 Wash in running tap water for 5 minutes.
4 Rinse in distilled water, treat with sodium bisulphite for 1 minute.
5 Wash in tap water for 5 minutes, then in several changes of distilled water.
6 Impregnate in silver solution for up to 1 hour at 60°C (see notes).
7 Wash well in several changes of distilled water.
8 Tone with gold chloride for 5 minutes.
9 Wash well in several changes of distilled water.
10 Fix in sodium thiosulphate for 5 minutes.
11 Wash in tap water for 5 minutes.
12 Counterstain (see notes).
13 Dehydrate in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS (Figs 6a and 6b)
Fungal hyphae, spores and other elements black
Glycogen black
Mucin grey to rose colour
Background tissues according to counterstain
The method will stain all fungi black, as well as many bacteria, though results on Actinomyces are indifferent.

TECHNICAL NOTES
1 The chromic acid solution deteriorates on standing and should be replaced monthly.
2 The bisulphite wash is optional, but helps to remove excess chromic acid. If not used, prolong the wash in tap water.
3 The silver solution may be capricious and precipitate prematurely. It is important to use fresh reagents and wash all glassware in distilled water before use. If the silver solution blackens and forms a mirror on the coplin jar before sections are adequately stained, a non-specific deposition of silver will coat the sections. If this occurs as a persistent problem, take the following precautions.
A. Bring the solution to optimal temperature as quickly as possible by preheating the coplin jar empty to 60°C and heating the solution in hot water in a conical flask.
B. Use freshly prepared 5% silver nitrate.
C. Place the silver solution in the dark during impregnation.
The course of the impregnation should be monitored every 5 to 10 minutes and more frequently as the colour develops. Optimally the sections should become golden brown before the silver solution begins to deteriorate. This may take from 20 minutes to over one hour. The variation in impregnation time is dependent upon the type and length of fixation used.
4 The black impregnation against a light background lends itself to a variety of counterstains. Specific nuclear stains (such as neutral red or methylene blue) or a variety of cytoplasmic stains (such as light green, safranin or metanil yellow) can be used. The method may be successfully counterstained with haematoxylin and eosin or haematoxylin and van Gieson, or simply 2 to 3 minutes in van Gieson's stain.
5 If glycogen in the sections causes a problem when searching for fungal spores, it may be removed before staining with diastase treatment.

Cresyl violet-polaroscopy technique for Cryptococcus neoformans¹⁶
SPECIMEN PREPARATION
Buffered 10% formalin fixation. Cut paraffin sections at 4 to 5 µm. Use a known positive control.

REAGENTS REQUIRED
Cresyl violet stain
Cresyl fast violet (CI 51010) 1 g
Glacial acetic acid 3 ml
Sodium acetate (3H20) 0.205 g
Distilled water 1 l
The pH of the solution should be approximately 3.5.

METHOD
1 Dewax sections and rehydrate with distilled water.
2 Stain in cresyl violet for 15 - 30 minutes.
3 Differentiate in 95% alcohol until background is clear (1 - 3 minutes).
4 Dehydrate in absolute alcohol, clear and mount.

RESULTS (Figs 7a and 7b)
Cryptococcus neoformans pink and anisotropic
Blastomyces pink, but isotropic
Coccidioidomyces pink, but isotropic
Histoplasma pink, but isotropic
Toxoplasma (cysts) pink, but isotropic
Of the above organisms, only Cryptococcus exhibits a pink luminous centre surrounded by brilliantly birefringent spinous radiations of the capsule.

TECHNICAL NOTES
This simple technique is virtually specific for Cryptococcus when used in conjunction with polarised light. Unlike similar yeast-form fungi and the cysts of Toxoplasma, Cryptococcus is surrounded by a thick mucoid capsule which is readily stainable by a range of methods. When stained with cresyl fast violet the radiating mucoid molecules refract polarised light. Alcian blue staining also produces the same effect.

Modified orcein technique for Hepatitis B (Australia) antigen²⁰
SPECIMEN PREPARATION
Formalin fixation is satisfactory. Cut paraffin sections at 3 to 4 µm. Use a known positive control section.

REAGENTS REQUIRED
1 Acidified permanganate
0.5% potassium permanganate 47.5 ml
3.0% sulphuric acid 2.5 ml
Prepare immediately before use.

2 Orcein stain
Orcein (synthetic, B.D.H.) 1 g
Conc. hydrochloric acid 2 ml
70% alcohol 98 ml
Dissolve the acid in the alcohol, then add orcein.

3 2% oxalic acid

METHOD
1 Dewax sections and wash in distilled water.
2 Oxidise in acidified permanganate for 5 minutes.
3 Wash in distilled water.
4 Bleach in oxalic acid for 5 minutes.
5 Rinse in distilled water.
6 Wash in running tap water for 5 minutes.
7 Wash in 70% alcohol.
8 Stain in orcein for up to 1½ hours.
9 Wash and dehydrate in absolute alcohol
(see notes)
10 Clear in xylene and mount in DPX.

RESULTS (Fig 8)
Australia antigen, lipofuscin reddish brown
The distribution of Australia antigen can occur as:
1 fine granules diffusely distributed in the cytoplasm of sublobular clumps of hepatocytes.
2 round or oval cytoplasmic clumps in scattered single hepatocytes.

TECHNICAL NOTES
1 Steps 8 and 9 - sections are stained progressively in the orcein stain, controlling microscopically, until the required intensity is achieved. Excess background staining may be removed by controlled differentiation in the dehydrating alcohol.
2 The method will also demonstrate lipofuscin and elastic fibres. Lipofuscin may be distinguished by virtue of its distribution in cells and its coarse granular size. Should it be necessary lipofuscin can be stained in a separate section with the PAS, Long Ziehl-Neelsen or Ellis-Zabrowarny techniques and the slides examined in comparison with the Orcein method.
3 The orcein stain must be prepared fresh before use. For ease of preparation, a stock of 2% acid alcohol is maintained, together with small vials of 0.5 g of orcein so that a 50 ml amount may be prepared immediately as required.
4 It is important that only B.D.H. synthetic orcein is used as other synthetic or natural orceins are not as effective.

Macchiavello technique for viral inclusions¹⁷(modified)
SECTION PREPARATION
Acidophilia is generally enhanced by fixation in mercuric chloride or dichromate fixatives, though formaldehyde fixatives generally give adequate results. (Some modifications of the Macchiavello include a Mallory bleach, which would be unnecessary with formalin fixation). Cut paraffin sections at 3 to 4 µm. A suitable control section is extremely useful as differentiation must be controlled microscopically.

REAGENTS REQUIRED
1 0.5% basic fuchsin (CI 42500) in distilled water (see notes)
2 0.5% aqueous citric acid
3 0.1% aqueous methylene blue (CI 52015)

METHOD
1 Dewax sections and rehydrate with distilled water
2 Stain in basic fuchsin for 30 minutes.
3 Rinse in distilled water.
4 Differentiate in citric acid (see notes).
5 Wash in tap water for 2 minutes.
6 Counterstain with methylene blue for 10 to 20 seconds.
7 Rinse in tap water.
8 Dehydrate and differentiate in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS (Fig 9)
Some Rickettsia and Chlamydia magenta
Viral inclusion bodies magenta
Cell nuclei pale blue

TECHNICAL NOTES
1 This method is similar to the Ziehl-Neelsen technique and uses the strong basic dye to stain the DNA or RNA content of the viral inclusion bodies.
2 Some modifications favour dissolving basic fuchsin in pH 7.2 buffer, rather than distilled water.
3 The differentiation is critical, may take as long as 5 minutes and can only be performed optimally if inclusion bodies can be found and observed microscopically during differentiation.
4 The method will demonstrate many inclusion bodies, especially intracytoplasmic inclusions, such as those seen with molluscum contagiosum, but is not especially reliable for staining Negri bodies.

Lendrum's phloxine-tartrazine for viral inclusion bodies²⁰
SECTION PREPARATION
This method takes advantage of the strong acidophilia of viral inclusion bodies. Whilst most fixatives give satisfactory results, formol-sublimate or other mercuric chloride fixatives, which enhance acidophilia, produce the best result. Cut paraffin sections at 4 µm. Use a viral inclusion control where possible. The chorionic membranes of eggs injected with cowpox virus provide good controls.

REAGENTS REQUIRED
1 Weigert's iron haematoxylin
2 1% acid alcohol
3 Phloxine solution
Phloxine (CI 45410) 0.5 g
Calcium chloride (anhydrous) 0.5 g
Distilled water 100 ml
The staining solution has a long shelf life
4 Saturated solution of tartrazine in 2-ethoxyethanol(cellosolve). (Maximum solubility of tartrazine in 2-ethoxyethanol is less than 0.5%)

METHOD
1 Dewax sections and rehydrate with distilled water.
2 Stain nuclei in Weigert's iron haematoxylin for 10 minutes.
3 Wash in running tap water.
4 Differentiate in acid alcohol to achieve a light nuclear stain.
5 Wash in running tap water for 5 minutes (or use a tap water substitute) to 'blue' nuclear stain.
6 Stain in phloxine solution for 20-30 minutes, rinse off excess stain in tap water, then rinse in absolute alcohol.
7 Differentiate and counterstain in tartrazine solution (see notes).
8 Complete dehydration in absolute alcohol, clear in xylene and mount in synthetic resin.

RESULTS
Viral inclusion bodies red
Red blood corpuscles pale pink
Background yellow
Nuclei grey/black

TECHNICAL NOTES
1 Weigert's haematoxylin tends to produce a sharper nuclear stain than alum haematoxylin solutions and produces better contrast with intranuclear inclusions.
2 Phloxine, a similar dye to eosin, has a strong affinity for some viral inclusion bodies, such as measles, pox viruses and cytomegalovirus. The tartrazine solution progressively removes phloxine from tissue components depending upon their affinity for the dye. Differentiation, with microscopic control, may take up to 15 minutes.
3 The tartrazine solution also counterstains background tissues, yellow. Over a long differentiation period the counterstain may become too intense. A brief rinse in tap water will remove all tartrazine and a subsequent short exposure to tartrazine will then result in a delicate yellow counterstain.

Quality control
Control sections
The use of control sections not only validates the method, but also provides reference material when searching for less commonly encountered organisms. With some critical methods where accurate differentiation is essential, it may be appropriate to place a control section on the same slide as the test material to eliminate variation in staining from one slide to another. There must be adequate identification of the control section so it cannot be confused with the test material. It must also be borne in mind that techniques which require blotting at various stages in the procedure may result in transfer of organisms from control to test sections. It is also important that control and test sections are of the same thickness, otherwise staining and differentiation between these may vary.

Contaminants and artefactual false positivity
Contamination of tissue sections with organisms, particularly bacteria and fungi, and their subsequent demonstration with routine and special staining methods is a potential cause of false positivity and misdiagnosis. Contamination may occur during several stages of tissue processing, cutting and staining.

The most common source of contamination involves the section flotation bath, which is normally set between 45°C and 50°C and provides an environment for bacteria, algae and fungi to grow. Tap water, which may contain a variety of organisms, should not be used in flotation baths. Fresh distilled water should be used and changed at the start of each day. Baths should be cleaned daily and left empty overnight. It should be noted that if organisms are collected onto slides during section mounting, the contaminating organisms will appear beneath the focal plane of the section in stained preparations. This will also be the case if the slide adhesive used is the source of contamination. Adhesives using gelatine, albumen and poly-l-lysine are all potential culture media.

Bacteria and fungi will also grow in buffers, reagents, and stains kept at ambient temperature for long periods. Contamination of mounted tissue sections from washing or staining solutions will deposit organisms on top of the sections and above the focal plane of the section. Generally, deposition of organisms on to or under tissue sections will be randomly distributed and not confined to areas of pathological significance or even to the section itself. If a contaminant is suspected, stains should be repeated after any potential sources of contamination have been discarded and reagents freshly prepared.

Tissue specimens may also be contaminated with bacteria before fixation. If fixation is delayed or avoided growth may occur on exposed surfaces and this would be reflected in the tissue section. Autopsy specimens are particularly prone to post-mortem bacterial and fungal growth, especially in the respiratory and alimentary systems.

Some staining methods, such as Gram's stain, may produce artefactual deposition of fine dye crystals which may mimic bacterial rods. Usually, polarised light will reveal these deposits to be refractile, and therefore artefactual.

References
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