Sample Collection for Fungal Infections: Which Specimen, How to Collect and Transport

A patient with a scaly, itchy ring on the arm has a skin scraping taken and sent for fungal culture. Three weeks later the culture is negative, yet the lesion is clearly a dermatophyte infection. The problem was not the laboratory. The swab that was sent skimmed the surface, when the living fungus sits at the advancing edge of the lesion and has to be scraped, not swabbed.

In fungal work, the specimen decides the result more often than the test does. This article is about collecting the right specimen, from the right place, and getting it to the laboratory in the right condition.

The specimen for diagnosing a fungal infection depends on the site of infection. Skin scrapings, nail clippings, plucked hairs, blood, cerebrospinal fluid, respiratory secretions, urine, and tissue are the common specimens. Choosing and collecting the right one, and transporting it correctly, is what makes recovery of the fungus possible.

For how these specimens are then examined and tested, see the companion article on the laboratory diagnosis of fungal infections.

Figure: Clinical Specimens for the diagnosis of fungal infections

Which specimen for which site, at a glance

Selection and collection of appropriate clinical specimens and their timely transport to the laboratory for processing is a prerequisite to ensure the recovery/identification of fungal pathogens.

Site / suspicion	Ideal specimen	How to collect it	Container and transport	Do NOT
Skin (ringworm, dermatophyte)	Skin scraping from the lesion edge	Swab with 70% alcohol first, then scrape the raised, growing margin with a slide edge or scalpel	Dry sterile container or dark paper packet; room temperature	Do not swab; do not refrigerate
Nail	Subungual debris and deep nail material	Sample from beneath the nail plate; if not possible, scrape away the surface and collect deeper shavings	Dry sterile container; room temperature	Do not send a surface clipping alone; do not refrigerate
Hair	Plucked infected hairs	Pluck (do not cut) hairs from scaling or bald patches, or those fluorescing under a Wood lamp	Dry sterile container; room temperature	Do not cut hairs off; roots are needed
Blood (disseminated infection)	Blood in a fungal or lysis-centrifugation bottle	Standard blood culture technique into fungal-specific or lysis-centrifugation bottles	Room temperature to the lab	Do not refrigerate
CSF (cryptococcal or other)	As much CSF as possible	Lumbar puncture, aseptic	Room temperature or a 30°C incubator	Do not refrigerate
Respiratory (pulmonary mycosis)	Early-morning sputum, or BAL or washings	Rinse mouth, then cough deep sputum; induced sputum if dry	Sterile screw-cap container; prompt, room temperature	Do not send 24-hour pooled sputum
Urine (renal or disseminated)	First early-morning urine	Aseptic clean collection into a sterile screw-cap container	Refrigerate at 4°C if delayed beyond 2 hours	Do not send 24-hour urine
Tissue biopsy	Fresh tissue from the affected site	Surgical or needle biopsy	Sterile container with a little sterile saline; room temperature	Do not dry out, freeze, or place in formalin (for culture)
Exudate (pustule, abscess)	Aspirate	Disinfect, aspirate with needle and syringe	Capped syringe as the transport container	Do not swab if aspirate is possible
Prostatic (some deep mycoses)	Prostatic secretions	Empty bladder, prostatic massage; also collect 5 to 10 mL urine	Sterile container; prompt	Do not omit the paired urine sample

Cerebrospinal Fluid

As much cerebrospinal fluid (CSF) as possible should be used for the culture of fungi. If processing is to be delayed, samples should be left at room temperature or placed in a 30°C incubator because CSF is an adequate fluid culture medium in which fungal elements can survive until subcultured in this environmental condition. Generally, CSF is filtered through 0.45-μm filter paper and filter paper is placed onto the surface of the appropriate culture medium with the inoculum side down. Culture media should contain no antibacterial or antifungal agents.

Respiratory Tract Secretions

Sputum, induced sputum, bronchial washings, bronchoalveolar lavage (BAL), and tracheal aspirations are commonly sent respiratory tract specimens for the diagnosis of fungal infections.

Sputum should be collected early-morning after rinsing but before breakfast.

Instruct patients to rinse their mouths vigorously with water immediately before coughing ½ to 1 oz of sputum into a sterile, screw-capped container. If adequate sputum is not obtained, induced sputum with a heated aerosol saline suspension can be used. Bronchial washings, biopsy, or bronchoalveolar lavage fluid should be transported promptly to the laboratory in sterile sealed containers.

Fungal infections of the respiratory tract can resemble mycobacterial infections, and the same specimen can often be used for both. When tuberculosis and a fungal infection are both suspected, the respiratory specimen can be processed for both, since the collection is the same.

Choice of culture media: Culture media should contain antibacterial agent, to prevent overgrowth of contaminating bacteria and antifungal agent such as cycloheximide, to prevent overgrowth by rapidly growing molds.

Blood

Figure: Blood for culture

Blood is the preferred sample to diagnose disseminated fungal infections. Biphasic agar-broth bottles designed specifically for fungal cultures are superior to routine bottles used for the recovery of bacterial pathogens.

Clinical microbiology laboratories mostly rely on automated blood culture systems such as BACTEC (Becton Dickinson), Bact/ALERT (BioMerieux) for the recovery of yeasts. Lysis-centrifugation systems, such as the Isolator (Wampole Laboratories) are also highly recommended, particularly for the recovery of dimorphic fungi especially Histoplasma capsulatum.

Urine

The first early-morning urine sample is preferred; random samples are acceptable. Specimens should be collected aseptically in sterile, screw-cap containers and sent immediately for processing. If a delay in processing beyond 2 hours is anticipated, the urine sample should be refrigerated at 4°C to inhibit over-growth by rapidly growing bacteria. Urine samples are centrifuged and sediment is inoculated onto the culture media.

Choice of media: Media containing antibacterial agents (to suppress the growth of gram-negative contaminants) are used to ensure optimal recovery of fungal pathogens.

Twenty-four-hour urine samples are unacceptable for fungal culture.

Prostatic secretions

Some deep-seated mycoses, notably blastomycosis and, less commonly, histoplasmosis or coccidioidomycosis, may be diagnosed by collecting prostatic secretions. The bladder is first emptied, followed by prostatic massage. Secretions should be inoculated directly into appropriate fungal culture media; also, 5-10mL of urine should be collected in a separate container.

Exudates

The skin over pustular lesions should be disinfected and exudates aspirated using a sterile needle and syringe. The syringe may also serve as transport container if the needle is capped. Biopsy of the lesion may be necessary if the aspirate fails to yield fungi.

Skin, nails, hair

Skin scrapings or biopsies, hair and nail clippings are usually submitted in the suspected cases of dermatophyte infections. These specimens should be placed in a sterile container and should never be refrigerated.

Figure: Infected nail

1. Skin: First, swab the area of skin to be sampled with 70% alcohol to remove surface bacterial contaminants. Sample the peripheral, erythematous, growing margin of typical “ringworm” lesions, scraping with the side of a glass microscope slide or the edge of a scalpel blade.
2. Nail: Infected nails should be sampled from beneath the nail plate to obtain softened material from the nail bed. If this is not possible, scrape away the surface of the nail before collecting shavings from the deeper portions.
3. Hairs: Hairs should be collected from areas of scaling or alopecia, or those that fluoresce when viewed under a Wood’s (long-wave-length) ultraviolet lamp. Infected hairs are removed by plucking them with forceps.

Culture media: Mycosel agar (Sabouraud’s dextrose agar with cycloheximide and chloramphenicol) and dermatophyte test medium (DTM) can be used for the isolation of dermatophytes. To ensure the growth of slow-growing fungal pathogens, these media should be cultured for a minimum of 21 days before reporting as negative.

Tissue Biopsies

Tissue biopsies of suspected sites of infection should be transported in a sterile gauze moistened with physiologic, nonbacteriostatic, sterile saline solution in a screw-cap container. All tissue biopsies are processed by mincing or grinding before culturing. The specimen should be cultured as soon as possible, not be frozen or allowed to dehydrate prior to culture.

Culture media: At least 1 mL of a processed sample should be spread onto the surface of appropriate culture media and incubated for 30°C for 21 days.

Transport of Specimen for Fungal Diagnosis

Most specimens in microbiology can be refrigerated if they cannot be processed at once. Fungal specimens are the opposite for almost every site, and this is the single rule worth locking in.

• Skin, nail, and hair are never refrigerated. They go in a dry container at room temperature. Dermatophytes tolerate drying well and can be recovered from dry scrapings weeks later, but cold and moisture encourage contaminating bacteria and molds.
• CSF is never refrigerated. It is held at room temperature or in a 30°C incubator, because CSF itself acts as a holding medium in which fungal elements survive, and cold harms the fastidious yeasts.
• Blood and tissue are not refrigerated. Blood goes into fungal or lysis-centrifugation bottles at room temperature; tissue is kept moist in saline and processed promptly.
• Urine is the one exception. If processing is delayed beyond 2 hours, urine is refrigerated at 4°C, because it is an excellent growth medium for contaminating bacteria that would overgrow the fungus.

The rule in one line: nearly every fungal specimen stays at room temperature, and urine is the exception that gets refrigerated.

What not to send

• A swab in place of a scraping for skin, nail, or hair. A swab collects surface debris and misses the living fungus at the lesion edge or under the nail. Scrapings, clippings, and plucked hairs are required.
• Cut hairs. The infected part is the root and the shaft near the follicle, so hairs must be plucked, not cut.
• A surface nail clipping alone. The fungus is in the nail bed and the deeper nail, so subungual debris or deep shavings are needed.
• 24-hour pooled urine or sputum. Pooling lets contaminants overgrow. Send a fresh single sample instead.
• Tissue in formalin, when culture is wanted. Formalin kills the fungus. Send fresh tissue in saline for culture, and a separate portion in formalin only if histopathology is also requested.

How to Remember

Scrape, do not swab. For skin, nail, and hair, the living fungus is at the edge, under the nail, or at the hair root, not on the surface. A swab misses it. Scrape the lesion margin, sample under the nail, pluck the hair.

Fungal specimens stay warm; urine is the exception. Nearly every fungal specimen is kept at room temperature and never refrigerated. Urine alone is refrigerated if delayed. If you remember "warm, except urine," you have the transport rule.

Pluck hair, do not cut it. The infection is at the root. A cut hair leaves the diagnostic part behind.

Edge of the ring, not the center. In ringworm, the center is often clearing and dead. The advancing red margin is where the fungus is alive and growing. Scrape there.

Dry is fine for skin, nail, hair. Unlike most specimens, these do not need moisture or a transport medium. A clean dry container or a dark paper packet works, and the fungus survives for weeks.

Key exam facts in one table

Point	Fact
Skin specimen	Scraping from the raised, growing lesion margin
Skin prep	Wipe with 70% alcohol first to remove surface bacteria
Nail specimen	Subungual debris or deep nail shavings, not a surface clipping
Hair specimen	Plucked (not cut) infected hairs; Wood lamp helps locate them
Skin/nail/hair transport	Dry container, room temperature, never refrigerate
Blood	Fungal or lysis-centrifugation bottle (lysis-centrifugation best for Histoplasma capsulatum)
CSF	As much as possible; hold at room temperature or 30°C, never refrigerate
Respiratory	Early-morning sputum after rinsing; induced sputum if dry
Urine	First early-morning; refrigerate at 4°C if delayed >2 h
24-hour urine	Not acceptable for fungal culture
Tissue	Fresh, moist in saline, not frozen and not in formalin for culture
Exudate	Aspirate into a capped syringe
Transport rule	Room temperature for almost all; urine is the refrigerated exception
Wood lamp	Ultraviolet light that makes some infected hairs fluoresce

Where Students Get Confused

"Why send a scraping instead of a swab for a skin infection?" Because the fungus lives in the keratin at the advancing edge of the lesion, not loose on the surface. A swab picks up surface debris and often misses the fungus, giving a false-negative. Scraping the growing margin collects the living organism.

"Fungal specimens can be refrigerated like other samples if there is a delay, right?" No, and this is the commonest error. Almost all fungal specimens are kept at room temperature. Skin, nail, hair, CSF, blood, and tissue are never refrigerated. Only urine is refrigerated if processing is delayed.

"Nail clippings are easy, just cut the end of the nail?" No. The fungus is in the nail bed and the deeper nail, not the free edge. Sample subungual debris from beneath the nail plate, or scrape away the surface and collect deeper shavings.

"Why pluck hairs instead of cutting them?" Because the infection is at the root and the base of the shaft. Cutting leaves the diagnostic portion on the scalp. Plucking with forceps brings the root with it.

"The lesion looks worst in the center, so sample there?" The center of a ringworm lesion is often healing and holds little live fungus. The active, advancing red margin is where the fungus is growing, so that is where to scrape.

"Can I put the biopsy in the formalin pot with the histology sample?" Not if you want a culture. Formalin kills the fungus. Send fresh tissue in saline for culture, and a separate piece in formalin only if histopathology is also needed.

References and Further Readings

1. Procop GW, Church DL, Hall GS, et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.
2. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.