Aspergillus is a common mold found everywhere in soil, air, and decaying plant matter. In clinical settings, various species cause illnesses known as aspergillosis, particularly in individuals with weakened immune systems. Accurate identification of the specific Aspergillus species is important because different species respond differently to antifungal medications. Microscopy remains the foundational method for identifying these organisms, relying on the recognition of distinct structural features.
The Distinctive Morphology of Aspergillus
Identification relies on observing specific, observable structures. The fungus is characterized by its hyaline, septate hyphae, which are colorless, thread-like filaments divided by cross-walls. Specialized stalks called conidiophores arise from the hyphae and terminate in a swollen, dome-shaped structure known as the vesicle.
The vesicle forms the base of the conidial head, the most defining feature of the genus. Flask-shaped cells called phialides cover the vesicle and produce the asexual spores, or conidia, in long, dry chains. The arrangement of the phialides is a key differentiator: uniseriate species have phialides arising directly from the vesicle, while biseriate species have an intermediate layer of cells, called metulae, between the vesicle and the phialides.
The size and shape of the vesicle, along with the pattern of phialide coverage, help classify the species. Distinguishing features also include the texture and color of the conidia, which range from smooth to rough-walled and from blue-green to black. These morphological differences are the targets for microscopic analysis and allow for preliminary identification.
Routine Examination: Brightfield and Staining Techniques
Initial identification often begins with simple, cost-effective methods utilizing standard brightfield microscopy. The fungus is first grown on a culture medium to encourage the formation of its distinctive conidial heads. To prepare a specimen, a wet mount or a tape mount technique is commonly used to gently lift the fungal structures from the culture without disturbing their arrangement.
The wet mount involves transferring a portion of the colony to a slide with mounting fluid. The tape mount, often preferred for preserving the delicate conidial head structure, uses clear adhesive tape to directly sample the aerial mycelium. Samples are then submerged in a stain to increase contrast, making the transparent fungal elements visible under the brightfield microscope.
Lactophenol Cotton Blue (LPCB) is the most common stain used in mycology. It contains phenol to kill the organism, lactic acid to preserve structures, and cotton blue to stain the chitin in the fungal cell walls deep blue. Potassium hydroxide (KOH) preparations are also used, dissolving surrounding non-fungal material like host tissue or cellular debris, allowing the fungal hyphae to be seen more clearly. These routine techniques provide the first visual evidence needed to assign an isolate to the Aspergillus genus.
Advanced and Clinical Visualization Methods
When identification is challenging, or when the fungus is observed directly within patient tissue, specialized visualization techniques are employed. Fluorescence microscopy offers a higher-resolution approach, often using a stain like Calcofluor White. This stain binds specifically to the cellulose and chitin within the fungal cell walls, causing structures to emit a bright, apple-green or blue fluorescence under ultraviolet light.
For visualizing the fungus in situ within a biopsy or clinical sample, histopathology stains are necessary. The Grocott-Gomori methenamine silver (GMS) stain is highly sensitive, rendering the fungal cell walls black or dark brown against a pale green background. This stain is particularly effective at highlighting the branching, septate hyphae in tissue samples.
Periodic acid–Schiff (PAS) stain is another common histopathology method, coloring the fungal elements magenta against a light blue or green counterstain. These techniques confirm the presence of the organism and its invasive nature within the host. Digital image analysis is increasingly used with these methods, allowing for the precise measurement of structures and comparison with reference databases to aid in difficult identifications.
Applying Microscopy: Key Species Differentiation
Microscopy provides the visual evidence to separate the four most clinically relevant species: A. fumigatus, A. flavus, A. niger, and A. terreus. A. fumigatus, the most common cause of human aspergillosis, is distinguished by short conidiophores and flask-shaped vesicles. These vesicles are only covered on the upper third by a single layer of phialides (uniseriate), forming characteristically columnar and bluish-green conidial heads.
In contrast, A. flavus typically exhibits biseriate phialides (metulae and phialides) that cover the entire surface of a noticeably larger, radiating vesicle. Its conidia are yellow-green and often rough-walled. A. niger is easily recognized by its dark, often black, conidia and its large, globose vesicles entirely covered by biseriate phialides.
A. terreus is identifiable by its small vesicles covered with biseriate phialides on the upper two-thirds, forming dense, columnar heads. This species is also unique because it often produces specialized structures called aleurioconidia, or solitary spores, directly along the hyphae. These specific differences in vesicle coverage, seriation, and conidial texture provide the detailed visual criteria for accurate species-level identification.