I Clin Pathol 1996;49:95-99 95 Leaders J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from

Mycological techniques

K G Davey, C K Campbell, D W Wamock

Introduction possible from the free edge of the nail and Laboratory tests are essential to establish the should include its full thickness. Specimens diagnosis of a fungal infection. The methods should be taken from several nails if more than used are based on three broad approaches: (1) one is affected. Debris beneath infected nails the detection ofthe pathogen in tissue on direct often contains the fungus. microscopic examination; (2) its isolation and It is sometimes helpful to use a Wood's lamp identification in culture; and (3) the detection to select infected scalp hairs for investigation. of an immunological response to the pathogen. Ifnone ofthe hairs gives the green fluorescence This Broadsheet describes the collection of which is characteristic of some microsporum clinical specimens from patients with suspected infections, a search should be made for lustre- fungal infection and outlines appropriate test less hairs or stumps and for hairs broken off procedures for the detection, isolation and at follicle mouths. Hairs should be extracted identification of fungal pathogens. It does not from the scalp using flat-ended forceps. Cut cover the serological diagnosis of fungal in- hairs without roots are unsuitable for my- fection. cological examination. Skin scrapings should also be collected from sites where fungal in- fection of hairs is suspected. Collection of specimens Another method which is useful for the col- It is important to provide the laboratory with lection of adequate material from patients with adequate specimens for investigation together suspected scalp infection not detectable under with sufficient clinical information about un- a Wood's lamp is to brush the scalp with a derlying illness, recent travel or previous res- plastic massage pad (available from most chem- idence abroad, animal contacts, and the ists), which is then pressed into the surface of patient's occupation if considered relevant. an agar plate. The pad should be sterilised in http://jcp.bmj.com/ With the exception of skin, nails and hair, all 1 % chlorhexidine for one hour, rinsed in sterile samples should be collected in sterile con- water and dried before being reused. tainers.

MUCOUS MEMBRANES SKIN, NAILS AND HAIR Swabs moistened in sterile water or saline nails and hair should be Skin, collected into should be used to collect exudate or discharge. on September 25, 2021 by guest. Protected copyright. folded squares of stiff black paper (about The swabs should be sent to the laboratory in 10 x 10 cm). These can now be purchased from a standard transport medium. a number of commercial suppliers (Dermaco, PO Box 470, Toddington, Bedfordshire LU5 6BK; MycoTrans, PO Box 1172, Biggar, Lan- EAR arkshire ML12 6NN). It is essential to clean Scrapings of material from the ear canal are to superficial lesions with 70% alcohol prior to be preferred, although swabs can also be used. This Broadsheet has been sampling if ointments, creams or powders have prepared by the authors at the been applied. Ifpityriasis versicolor is a possible invitation of the Association of EYE Clinical Pathologists who diagnosis, the affected sites should be examined Material from a corneal ulcer with a suspected reserve the copyright. Further under ultraviolet light (Wood's lamp). The copies of this Broadsheet may fungal cause should be scraped with a sterile be obtained from the Publishing characteristic golden yellow fluorescence often platinum spatula, sampling both the base of Manager, 3rournal of Clinical associated with this infection can help in the Pathology, BMA House, the ulcer and the edges. This procedure should Tavistock Square, selection of skin scrapings. London WClH 9JR. be performed by an eye specialist. Because the Material should be collected from cutaneous amount of material that can be obtained will PHLS Mycology lesions by scraping outwards from the margin be small, it is best transferred to a glass slide Reference Laboratory, of the lesion with a blunt scalpel. If there is for microscopic examination and to an agar Public Health minimal clear tape as Laboratory, scaling, adhesive such plate for culture at the bedside. The culture Kingsdown, sellotape can be used to remove material for plate should be marked to indicate the point Bristol BS2 8EL examination. The sellotape strip is pressed of inoculation. K G Davey against the lesion, peeled off and placed, ad- Swabs are not suitable for sam- C K Campbell pling comeal lesions. D W Wamock hesive side down, on a clean glass microscope slide for transportation to the laboratory. Correspondence to: Dr D W Wamock. Nail scrapings or clippings should be taken BLOOD Accepted for publication from discoloured or dystrophic parts of the Blood culture is an important part of the in- 26 September 1995 nail. Specimens should be cut as far back as vestigation of suspected deep fungal infection. 96 Davey, Campbell, Warnock

However, microbiologists should not expect Characteristics of some common invading blood cultures taken for bacterial isolation to hair

detect fungal pathogens other than Candida Arthrospore size J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from spp., , or Thichosporon Fungus (oIm) Arrangement spp. Isolation of fungi from blood depends on Microsporunm audouinii Small (2-5) Ectothrix a number of factors including the amount of Small (2-5) Ectothrix mentagrophytes Small (3-5) Ectothrix blood sampled and the method of processing. Large (4-8) Endothrix Commercial biphasic and Bactec blood culture Tichophyton violaceuni Large (4-8) Endothrix systems will often permit the detection offungal Tnchophyton verrucosum Large (5-10) Ectothrix infection.

SPUTUM AND BRONCHOALVEOLAR LAVAGE Fresh early morning samples of sputum are men consists ofmore than one piece ofmaterial, ideal. These should be collected in sterile con- it is advisable to use some of each for mi- tainers and processed within two hours of col- croscopic examination and culture. Hairs lection. If delay in processing is unavoidable, should first be examined under a Wood's lamp specimens should be stored at 4°C. More in- and fluorescent parts, if present, used for pro- vasive sampling methods are often required to cessing. Hairs should be cut about 5 mm above obtain specimens from neutropenic patients. the root and the upper portion discarded. Se- Broncheoalveolar lavage (BAL) provides useful lected fragments or roots are placed in a large samples for direct microscopic examination and drop of 20% potassium hydroxide (KOH) on culture. a microscope slide and a coverslip placed on top. The slide should be set to one side to soften and clear the material. Skin material will CEREBROSPINAL FLUID, URINE AND OTHER clear in 15 to 20 minutes, as will thin nail FLUIDS parings and sub-ungual debris, but thicker nail Cerebrospinal fluid samples of 3-5 ml are ideal, fragments can take 30 to 60 minutes to soften. but are usually smaller than this. Samples can Gentle warming ofpreparations will reduce the be centrifuged and the supematant fluid used time required for clearing, but care must be for serological tests. The sediment can be cul- taken to avoid boiling. tured, but is also useful for direct microscopic Once skin or nail material has digested, the examination. In non-catheterised patients, coverslip should be pressed down to squash fresh mid-stream specimens of urine are ad- out the fragments and render them transparent, equate for mycological investigation, provided blotting off the excess KOH. Hair specimens care is taken to ensure that vaginal or perineal should not be heated or squashed as infected

infection does not lead to contamination. In hairs will disintegrate and the diagnostic ar- http://jcp.bmj.com/ infants, suprapubic aspiration is the best rangement of the arthrospores will be lost. method of urine collection. Other fluids, Low power magnification ( x 10 objective) is whether aspirated or drained, should be col- adequate for the detection of fungal hyphae in lected into sterile containers which include a KOH preparations, but a higher power ( x 40 small amount of sterile heparin diluted 1 in objective) is often required to confirm their 1000 to prevent clotting. These specimens presence. Septate hyphae which may consist of

should be centrifuged and the sediment cul- chains of rectangular spores (termed ar- on September 25, 2021 by guest. Protected copyright. tured. Drain fluid from patients receiving con- throspores) are typical of in- tinuous peritoneal dialysis should be collected fection. In cases of pityriasis versicolor, the into sterile containers without heparin. fungus ( furfur) appears as clusters of round cells together with short, unbranched hyphae. If a hair specimen is found to be PUS, BONE MARROW AND TISSUE infected, the size of the arthrospores and their If possible, swabs should not be used to collect arrangement should be noted (table). material from draining abscesses or ulcers. If a Care must be taken to distinguish fungal swab must be used, then material should be hyphae from the artefact known as "mosaic". taken from as deep as possible within the lesion. This is thought to be caused by deposition of Pus from undrained subcutaneous abscesses or cholesterol and, unlike true fungus, it tends to sinus tracts should be collected with a sterile follow the outlines of the epidermal cell walls. needle and syringe. If grains are visible in the Methods for selective staining of fungal hyphae pus (as in mycetoma), these must be collected. are available, but are seldom required. Bone marrow specimens should be collected The recognition of fungal hyphae and/or ar- into sterile containers which include a small throspores by direct microscopic examination amount of sterile heparin diluted 1 in 1000. is sufficient for the diagnosis of a dermatophyte Tissue specimens should be placed in sterile infection, but gives no indication of the species saline. If possible, material should be obtained of dermatophyte involved. Isolation should be from both the middle and the edge of lesions. attempted whether or not fungus has been found on microscopic examination. If there is insufficient material for both microscopic Processing of specimens examination and culture of a specimen, the SKIN, NAILS AND HAIR former should be performed, unless the clini- Skin and nail specimens should be chopped cian has done it. It is important to remember into small fragments. If, as is usual, the speci- that while fungus found on microscopic ex- Mycological techniques 97

amination may fail to grow in culture, the level 3 laboratory. Tissues, fluids and pus need opposite is also true. not be processed inside a cabinet. However, if a

Isolation is best made on agar plates. Skin, mould is isolated and the information provided J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from nail and hair specimens should be cultured on with the specimen suggests a diagnosis ofblasto- Sabouraud's dextrose agar (glucose peptone , , , agar) supplemented with chloramphenicol , or penicilliosis, all fur- (50 mg/l) to reduce bacterial contamination ther work must be performed inside a cabinet. and cycloheximide (actidione, 50 mg/l) to sup- Swabs should be cultured on Sabouraud's press most non-dermatophyte fungi. Malt agar dextrose or malt agar supplemented with chlor- can be used in place of Sabouraud's dextrose amphenicol. Plates should be incubated at agar. Up to 20 skin or nail fragments, or hair 37°C for up to one week and examined at 48 roots should be pressed into the surface of a hour intervals. Swabs should be spread over culture plate. Ifskin or nail material is suspected the surface of a culture plate, then dipped in a of being infected with moulds, such as Scy- drop of 20% KOH on a clean slide to wash off talidium spp. or Scopulariopsis brevicaulis, it material for direct microscopic examination. is important to set up duplicate plates of Sputum should be digested prior to mi- Sabouraud's dextrose agar with and without croscopic examination and culture. An amount cycloheximide. If sellotape strips are received, of Sputasol (or similar reagent), equal to that of these are detached from the microscope slides the sputum, is added to the specimen container. and placed on the surface of a Sabouraud's The specimen is incubated at 37°C for 30 dextrose agar plate. Incubation at room tem- minutes, then centrifuged and a portion of the perature is adequate for the isolation of derma- sediment spread over the surface of a Sa- tophytes, but 27-30°C is preferred. bouraud's dextrose or malt agar plate (sup- Sabouraud's dextrose agar can be obtained plemented with chloramphenicol). This should from a number of commercial suppliers, but be incubated at 37°C for 48 to 72 hours. For as the morphological appearance of the fungi, direct microscopic examination, a portion of and pigmentation in particular, often differs sediment is added to a drop of 20% KOH on from one medium to another, it is advisable to a clean slide and a coverslip added. confine supplies to one manufacturer. This also In general, blood cultures should be in- applies to the nature of the peptone if the cubated for seven to 10 days. Those showing medium is prepared in house. apparent growth should be subjected to direct Most dermatophyte cultures can be iden- microscopic examination. Ifyeast cells are pres- tified after seven to 10 days of incubation at ent, an Indian ink preparation should be made 30°C (see later). If there is no growth after this to demonstrate the possible presence of en- time, the plate can be discarded as negative. If capsulated C neoformans cells (see below). Ir- growth is apparent, the plate should be re- respective of apparent growth, all blood

incubated until the colonies are large enough cultures should be sub-cultured on to Sa- http://jcp.bmj.com/ to be identified. If there is no growth from bouraud's dextrose agar plates for incubation material which was positive on microscopic at 30 and 37°C for two weeks. If the clinical examination and further material is available, details suggest infection with a Hazard Group it is advisable to set up a further culture. 3 pathogen (see below), screw-capped sloped Moulds other than dermatophytes are some- containers should be used instead of plates. times recovered from abnormal skin and nails. Fluids should be centrifuged and the deposit In most cases, these are casual, transient con- used for microscopic examination and culture. on September 25, 2021 by guest. Protected copyright. taminants and direct microscopic examination If cerebrospinal fluid samples are being ex- of scrapings is negative. However, certain amined, an Indian ink preparation should be moulds are capable of causing infection and made to demonstrate the possible presence of when this is so, it is important that their sig- encapsulated C neoformans cells (see later). nificance is recognised. Scytalidium dimidiatum Tissue samples should be placed in a sterile (Hendersonula toruloidea) and Scytalidium hy- Petri dish and chopped into small fragments alinum have been isolated from hand, foot and prior to culture. Selected fragments should be nail infections. Microscopic examination of in- placed in a large drop of 20% KOH on a fected material shows chains of hyaline ar- microscope slide and a coverslip placed on top. throspores which may be difficult to distinguish The slide should be set to one side to soften from those of a dermatophyte. These fungi are and clear the material. For culture, up to 20 sensitive to cycloheximide and this antibiotic fragments are pressed into the surface of should be omitted from the culture medium. Sabouraud's dextrose or malt agar plates S brevicaulis is the most common non-derma- (supplemented with chloramphenicol). These tophyte cause of nail infection. It is possible to should be incubated at 30 and 37°C for up to distinguish infections with this mould on direct two weeks and examined at 48 hour intervals. microscopic examination of nail specimens if If the clinical information provided with a the characteristic roughened oval spores are specimen suggests a diagnosis of blastomy- found. cosis, coccidioidomycosis, histoplasmosis, para- coccidioidomycosis, or penicilliosis, some modification of culture methods is advisable. OTHER SPECIMENS For safety, such specimens should be cultured Sputum and BAL specimens should be re- in screw-capped sloped containers rather than garded as hazardous, due to potential myco- on plates. Duplicate cultures should be set up bacterial infection, and should be processed on Sabouraud's dextrose or malt agar (sup- inside a class 1 safety cabinet in a containment plemented with chloramphenicol) at 30°C and 98 Davey, Campbell, Warnock

blood agar or brain heart infusion agar at 37°C. uppermost. The agar block is then removed The slopes should be incubated for three to and discarded, leaving adherent mycelium on four weeks before being discarded as negative. the slide. Lactophenol cotton blue is added J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from and a clean coverslip applied. The preparation can be sealed for long term preservation. Identification methods MOULDS AND DERMATOPHYTES Moulds and dermatophytes are identified on the basis oftheir colonial and microscopic mor- Special media phological characteristics. Macroscopic fea- A medium which will assist the non-specialist tures, such as colonial form, surface colour to distinguish dermatophytes from other and the production of pigments, are helpful in moulds is Dermatophyte Test Medium. A identification: moulds with blue, green or black change in the amber colour of the phenol red colonies are not dermatophytes. In some cases, indicator incorporated in the medium to pink for instance Thichophyton verrucosum and Tri- or red denotes a change in pH and permits the chophyton soudanense, the diagnostic char- presumptive identification of the isolate as a acteristics are most easily seen by examining dermatophyte. It is important to remember the reverse side of the plate under the low that some non-dermatophyte moulds can also power objective of a microscope. produce this colour change. The urease test can often be used to dis- tinguish isolates. A small Needle mount portion of growth is removed from a culture This is the most important part of the ex- plate with a rigid needle and used to inoculate amination of a culture. If well prepared, a a Christensen's urea slope. This is incubated needle mount will often give sufficient in- at 30°C for one week. If the colour of the formation on the form and arrangement of medium changes to red, the test is positive and spores and other structures for identification the isolate is not T rubrum. However, it is of the fungus to be made. Using a sterilised important to note that the granular form of T rigid sharp needle, some of the surface growth rubrum, like most other dermatophytes, gives is removed from a culture plate and placed in positive results. a drop of lactophenol cotton blue stain on a clean microscope slide. The material is then teased apart with two sharp needles and a coverslip is applied. Gentle pressure is used to spread out the preparation before it is examined A number of simple rapid tests have been under a microscope using the x 10 and x 40 devised for the identification of some of the

objectives. If no spores are found in slide most important fungi. These include the http://jcp.bmj.com/ mounts, it is sometimes worthwhile to remove serum germ tube test for and the lid from the culture plate and examine the the urease test for C neoformans. However, the colonies for evidence of sporulation under the first step is to note the colour and appearance low power objective of a microscope. of the colonies. Pink or coral colonies are sug- gestive ofRhodotorula spp. while cream or white colonies are suggestive of Candida spp., Crypto- Sellotape mounts coccus spp., Thichosporon spp., or Geotrichum on September 25, 2021 by guest. Protected copyright. These are useful for examining sporulating cul- spp. Brown or black colonies are suggestive of tures. Using sterilised forceps, a small piece of Exophiala spp. sellotape, adhesive side down, is pressed on to the surface of the culture. The coated sellotape is then placed, adhesive side up, on to a drop of lactophenol cotton blue on a microscope Indian ink preparation slide. A further drop oflactophenol cotton blue If the information provided with the specimen is placed on the preparation and a coverslip suggests the culture is C neoformans, an Indian applied. ink preparation should be set up. C neoformans produces round to oval or lemon-shaped cells with polysaccharide capsules. These capsules Slide culture can be detected when the cells are mounted in The slide culture technique is useful for ob- a pigmented colloidal mounting fluid, such serving the intact arrangement of spores or as Indian ink, which does not penetrate the spore bearing structures. A thin square block capsular envelope. A light loopful of inoculum of agar is placed on a sterile microscope slide is taken from the culture and suspended in a supported on a bent glass rod in a Petri dish. drop of 50% aqueous Indian ink on a mi- The four sides of the agar block are then in- croscope slide. If a capsule is present, it should oculated with portions of mycelium of the be visible as a clear halo around the cells. fungus to be identified. The block is then The presence of a capsule gives a presumptive covered with a sterile coverslip, sterile distilled identification of C neoformans, but does not water is added to the base of the Petri dish, provide a definitive identification because other the lid is replaced and the plate is incubated at yeasts may be encapsulated. Moreover, not all 30°C. Once adequate sporulation has occurred C neoformans isolates have prominent capsules the cover slip is removed from the agar and and the capsule is often lost following sub- placed to one side with the adherent mycelium culture of the organism. Mycological techniques 99

Urease test mycelium, arthrospores, and chlamydospores. Most C neoformans isolates possess the enzyme Chlamydospores (which can take up to 96

urease. A light loopful of inoculum is taken hours to develop) are indicative of C albicans. J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from from the original isolation plate and spread Addition of 1% Tween 80 to the cornmeal agar over the surface of a Christensen's urea slope, stimulates their production. If arthrospores are which is then incubated at 30°C for up to four present, the isolate is a presumptive Tri- days. A colour change from amber to pink chosporon sp. If only pseudomycelium is found, permits the presumptive identification of the the isolate is a Candida sp. other than C albicans. isolate as C neoformans. However, It should be noted that Candida guilliermondii spp. can give a positive result and bacterial needs 72 to 96 hours of incubation to produce contamination can also result in a change in pseudomycelium while the colour of the medium. needs a similar amount of time to produce arthrospores.

Germ tube test The formation of germ tubes in serum is a Further tests characteristic of most clinical isolates of C al- For those yeasts which are germ tube negative, bicans. A light loopful of inoculum is taken identification may be made using one of the from a culture plate and suspended in 0-5 ml commercial identification systems, such as API of horse serum. This is incubated at 37°C for 20 C (BioMerieux) or AUXACOLOR (Sanofi two to three hours. A drop of the suspension Diagnostics Pasteur). However, it is essential is then placed on a microscope slide, a coverslip to examine the morphological characteristics of is placed on top and the preparation examined all yeast isolates to avoid errors in identification, under the microscope. In many instances of a whatever system is used. Yeasts which cannot positive test, fewer than 10% of yeast cells will be identified with commercial systems should produce germ tubes. be sent to a reference laboratory for iden- It is important to remember that about 5% tification using traditional assimilation and fer- ofC albicans isolates fail to produce germ tubes. mentation procedures. Moreover, over-inoculation of the serum can result in inhibition of germ tube formation and too short an incubation period can lead to false HAZARDOUS FUNGI negative results: two hours is a minimum not Five dimorphic fungi are classified as hazard a maximum incubation time. Pseudohypha group 3 pathogens. These are Blastomyces production can be mistaken for germ tube dermatitidis, immitis, Histoplasma production. This can be recognised by a con- capsulatum, Paracoccidioides brasiliensis, and striction at the junction between the mother Penicillium marneffei. These organisms must be

cell and the false germ tube. handled inside a class 1 cabinet in a con- http://jcp.bmj.com/ tainment level 3 laboratory and should be cul- tured in sloped containers. Although the yeast of a Cornmeal agar culture form these pathogens presents lower risk Examination of the morphological char- of infection, these organisms will revert to a acteristics of yeast isolates under the micro- mycelial form after several days of incubation scope is essential if species with identical at 300C. Plates suspected to contain these or- physiological profiles are to be distinguished. ganisms must be sealed at once and removed on September 25, 2021 by guest. Protected copyright. Growth in microaerophilic conditions on corn- to a containment level 3 laboratory. Such cul- meal or other starch-containing media, such as tures are best sent to a specialist mycology rice agar, stimulates the formation ofmycelium, laboratory for identification. pseudomycelium, arthrospores, and chlamy- dospores in those species able to produce them. Further reading Using a loop, touch five colonies on a plate Campbell CK, Johnson EM, Philpot CM, Wamock DW. Iden- and mix them on the agar surface. Use this to tification ofpathogenic fungi. London: Public Health Laborat- ory Service, 1996 (in press). make a streak inoculation on the surface of a Evans EGV, Richardson MD (eds). Medical mycology: a practical cornmeal agar plate. The centre of the streak approach. Oxford: IRL Press at Oxford University Press, 1989. Kwon-Chung KJ, Bennett JE. Medical mycology. 4th edn. Phil- is then covered with a sterile coverslip and the adelphia: Lea and Febiger, 1992. plate incubated for at least 48 hours at 30°C. Richardson MD, Wamock DW. Fungal infection: diagnosis and management. Oxford: Blackwell Scientific Publications, 1993. The lid is then removed and the plate examined Rippon JW. Medical mycology: the pathogenic fungi and the pa- under a microscope for true mycelium, pseudo- thogenic actinomycetes. 3rd edn. Philadelphia: Saunders, 1988.