Preparation of Tissue for Histologic Examination

Handling lung tissue: Material for culture should be obtained at the time of biopsy or resection and sent directly to the microbiology laboratory in order to prevent contamination. When the specimen arrives in the pathology laboratory, it is expanded with formalin for fixation. Whole lungs or lobes are filled with formalin via the bronchus and allowed to fix before sectioning. A small biopsy is expanded by gentle infusion of formalin via a 25-gauge needle inserted into the specimen as shown in the photo. After fixation, the lung, lobe, or small pieces are sliced and examined for lesions. Appropriate small pieces are placed in a cassette, dehydrated, embedded in paraffin, cut on a microtome, and stained.

Special studies: For potential use in special studies, small pieces of tissue are taken before formalin fixation and may be fixed in glutaraldehyde for electron microscopy (left) or frozen (-70° C) for some immunohistochemical (middle), biochemical, or molecular studies, as well as for fat stains (right) to diagnose exogenous lipoid pneumonia (pale pink stain). The need for fat stains must be recognized before all tissue is embedded in paraffin. Tissue embedded in paraffin has had all the fat dissolved out by xylene.

Special stains: Special stains for organisms are done routinely on specimens from immunocompromised patients, but only if clinically or histologically indicated on specimens from other patients.

Artifacts: Certain artifacts appear in histologic sections. Previous biopsies or needle aspirations can produce inflammation or necrosis. Transbronchial biopsies or non-distended specimens often show collapse, which should not be interpreted as fibrosis. Proper distension can mimic emphysema. Surgically-induced hemorrhage can usually be recognized by the absence of hemosiderin-filled macrophages that indicate old blood. Surgical manipulation can cause PMN clumping in capillaries, which may be confused with true inflammation or infection .

Left: Normal postmortem lung that was fixed by distending it with formalin. Right: Other undistended lung from the same patient. The atelectasis is easily mistaken for fibrosis. Distension makes examination and diagnosis easier.

Special Stains Used in Pulmonary Pathology


Comments and Uses

Hematoxylin & eosin (H&E)

The routine tissue stain

Brown-Brenn (B&B)

Tissue gram stain


Stains acid fast bacteria (AFB) red. Uses 3-5% HCl for decolorization (strong acids produce more decolorization than weak ones)


Stains AFB red. Uses 1% sulfuric acid for decolorization


Stains AFB red. Uses acetic acid for decolorization


Stains most bacteria black (non-specific). Used for legionella, but staining must be confirmed by immunostaining or culture

Gomori methenamine silver (GMS)

Stains fungi (and mucus) black

Elastic van Gieson

Stains elastic tissue black & collagen red (van Gieson)


Stains collagenous tissue blue or green and muscle red

Periodic acid-Schiff (PAS)

Stains neutral mucin & glycogen red


Glycogen is removed by diastase digestion; neutral mucin and other PAS-positive substances remain red.

Prussian blue

Stains iron in macrophages and asbestos bodies

Congo red

Stains amyloid orange: requires apple green birefringence by polarized light for confirmation


Empirical stain for mucin (red); stains capsules of cryptococci

Alcian blue

Stains acid mucin (and some neutral ones)

Alcian blue + hyaluronidase

Pretreatment of tissue with hyaluronidase specifically removes hyaluronic acid mucin. Absence of blue stain in cytoplasmic droplets that were alcian blue positive confirms a diagnosis of acid mucin, as in malignant mesothelioma


Similar to Wright stain for blood elements; also stains pneumocystis trophozoites and toxoplasma organisms

Papanicolaou (PAP )

Used to show nuclear detail in cytologic preparations


Used to show cytoplasmic detail in cytologic preparations


Stains granules of neuroendocrine cells when they are plentiful, as in carcinoid tumors


1. Colby TV, Yousem SA. Pulmonary histology for the surgical pathologist. Am J Surg Pathol 1988; 12:223-239.

2. Sheehan DC, Hrapchak BB. Theory and Practice of Histotechnology. St Louis, CV Mosby, 1980.

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