agar block smear preparation: a novel method of slide ... · we describe a novel method of fungal...

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2010, p. 3053–3061 Vol. 48, No. 90095-1137/10/$12.00 doi:10.1128/JCM.00917-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Agar Block Smear Preparation: a Novel Method of Slide Preparationfor Preservation of Native Fungal Structures for Microscopic

Examination and Long-Term Storage�

Patrick C. Y. Woo,1,2,3,4† Antonio H. Y. Ngan,4† Hon-Kit Chui,4Susanna K. P. Lau,1,2,3,4 and Kwok-Yung Yuen1,2,3,4*

State Key Laboratory of Emerging Infectious Diseases,1 Research Centre of Infection and Immunology,2 Carol Yu Centre ofInfection,3 and Department of Microbiology,4 The University of Hong Kong, Hong Kong

Received 6 May 2010/Returned for modification 21 June 2010/Accepted 9 July 2010

We describe a novel method of fungal slide preparation named “agar block smear preparation.” A total of510 agar block smears of 25 fungal strains obtained from culture collections, 90 QC fungal strains, and 82clinical fungal strains from our clinical microbiology laboratory, which included a total of 137 species of yeasts,molds, and thermal dimorphic fungi, were prepared and examined. In contrast to adhesive tape preparation,agar block smears preserved the native fungal structures, such as intact conidiophores of Aspergillus speciesand arrangements of conidia in Scopulariopsis brevicaulis. Furthermore, agar block smears allowed examinationof fungal structures embedded in the agar, such as the ascomata with ascomal hairs in Chaetomium funicola;pycnidium of Phoma glomerata; the intercalary ovoidal chlamydospores arranged in chains of Fusariumdimerum; and the lateral, spherical chlamydospores arranged in pairs of Fusarium solani. After 1 year ofstorage, morphological integrity was found to have been maintained in 459 (90%) of the 510 agar block smears.After 3 years of storage, morphological integrity was found to have been maintained in 72 (71%) of the 102smears prepared in 2006. Agar block smear preparation preserves the native fungal structures and allowslong-term storage and examination of fungal structures embedded in the agar, hence overcoming the majordrawbacks of adhesive tape preparation. The major roles of agar block smear should be diagnosis for difficultcases, accurate identification of fungal species for clinical management of patients and epidemiological studies,and long-term storage for transportation of slides and education purposes.

Accurate identification of fungi is the cornerstone of select-ing and prescribing appropriate antifungal drugs to patientswith fungal infections. In contrast to bacteria, for which anti-biotic susceptibility testing is routinely performed when a clin-ically significant bacterium is isolated, antifungal susceptibilitytesting is not commonly performed due to the relatively poorcorrelation between in vitro susceptibility results and clinicalresponse. On the other hand, the choice of antifungal drugs forpatients with fungal infections relies heavily on the identifica-tion of the corresponding isolates. Although molecular meth-ods, such as those using the internal transcribed spacer regionand 18S rRNA sequencing, have been increasingly used forfungal identification, these technologies are still expensive andrequire corresponding expertise for laboratory technicians (5–7). Therefore, most clinical microbiology laboratories still relyon phenotypic methods for identification of fungi.

Identification of molds in clinical microbiology laboratoriesis most commonly performed by the use of culture on agarplates followed by microscopic examination of lactophenol cot-ton blue-stained adhesive tape preparations of the fungal col-onies for direct visualization of characteristic microscopic mor-

phological features. However, adhesive tape preparations areassociated with three major drawbacks. First, fungal structuresmay be crushed and damaged during the preparation proce-dures, affecting the accurate identification of the fungus. Sec-ond, as a result of drying of the smear, adhesive tape prepa-rations can be used for microscopic examination only within afew hours of the time of the initial preparation. Third, fungalstructures embedded in the agar cannot be observed. To over-come these and other drawbacks, approaches such as the mi-croslide method are sometimes employed when greater mor-phological detail is necessary. However, microslide cultures arealso not suitable for long-term storage. Throughout the years,modifications of microslide cultures have been suggested, butthese methods have still been far from ideal (1, 2). Although animprovement to adhesive tape preparations, “double-layertape prep,” which extends the useful life of the tape prepara-tion to several weeks, was suggested recently, this is still notsuitable for long-term storage, for sending the slide out forconsultation, and for examination of fungal structures embed-ded in agar (3). In this article, we describe a novel method ofslide preparation, named “agar block smear preparation,”which preserves the native structures of molds and yeasts andallows long-term storage and examination of fungal structuresembedded in the agar, hence overcoming all three drawbacksof adhesive tape preparation.

MATERIALS AND METHODS

Strains. Twenty-five fungal strains obtained from culture collections, 90 qual-ity control (QC) fungal strains, and 82 fungal strains isolated from patients in the

* Corresponding author. Mailing address: State Key Laboratory ofEmerging Infectious Diseases, Department of Microbiology, The Uni-versity of Hong Kong, University Pathology Building, Queen MaryHospital, Hong Kong. Phone: (852) 22554892. Fax: (852) 28551241.E-mail: [email protected].

† P. C. Y. Woo and A. H. Y. Ngan contributed equally to themanuscript.

� Published ahead of print on 21 July 2010.

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TABLE 1. Fungal species used in the present study

Fungal species

Sourcea

NEQASUK QC

CAPUSA QC

Our clinicalmicrobiology

laboratoryCulture collection

Hyaline molds (nondermatophytes)Aspergillus candidus � �Aspergillus clavatus �Aspergillus flavipes �Aspergillus flavus � � � (ATCC 204304)Aspergillus fumigatus � �Aspergillus glaucus �Aspergillus niger � �Aspergillus restrictus �Aspergillus sydowii �Aspergillus terreus � � �Aspergillus ustus �Aspergillus versicolor � � �Emericella nidulans � � �Eurotium chevalieri � (CBS 522.65)Eurotium cristatum � (CBS 123.53)Eurotium rubrum �Neosartorya fischeri �Neosartorya pseudofischeri �Acremonium strictum � �Arthrinium phaeospermum �Beauveria bassiana �Botrytis cinerea �Cephalotheca foveolata �Chrysosporium keratinophilum �Fusarium chlamydosporum �Fusarium dimerum � �Fusarium nygamai � (FRC-M7492)Fusarium oxysporum � � (NRRL-28973)Fusarium proliferatum � � (FRC-M6992)Fusarium solani � � � (CBS 109028)Fusarium verticillioides � � (ATCC MYA 3629)Gliocladium species �Lecythophora hoffmannii �Malbranchea species �Monilia species �Nectria haematococca �Paecilomyces lilacinus � �Paecilomyces variotii � � (ATCC MYA 3630)Penicillium species �Scopulariopsis brevicaulis � �Thermomyces species �Trichoderma species �Trichothecium roseum �

Hyaline molds (dermatophytes)Epidermophyton floccosum � �Microsporum canis � � (CBS 113480)Microsporum ferrugineum �Microsporum gypseum �Microsporum nanum �Microsporum persicolor �Trichophyton erinacei �Trichophyton fischeri �Trichophyton mentagrophytes � � �Trichophyton rubrum � � � � (CBS 118892)Trichophyton schoenleinii �Trichophyton soudanense �Trichophyton tonsurans � � �Trichophyton verrucosum �Trichophyton violaceum � �

Dematiaceous moldsAlternaria species �

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TABLE 1—Continued

Fungal species

Sourcea

NEQASUK QC

CAPUSA QC



Aureobasidium pullulans �Bipolaris hawaiiensis �Chaetomium funicola �Chaetomium globosum � �Cladophialophora bantiana �Cladophialophora boppii �Cladosporium carrionii �Cladosporium cladosporioides �Cladosporium herbarum �Curvularia species � �Cyphellophora pluriseptata �Epicoccum species �Exophiala dermatitidis � �Exophiala jeanselmei �Exophiala moniliae �Exserohilum species �Fonsecaea pedrosoi �Hormonema dematioides �Hortaea werneckii �Lasiodiplodia theobromae �Nigrospora species �Ochroconis constricta �Ochroconis gallopavum �Phaeoacremonium parasiticum �Phialemonium curvatum �Phialophora fastigiata �Phialophora richardsiae � �Phialophora verrucosa �Phoma glomerata �Pithomyces species �Pseudallescheria boydii � � � (CBS 101.22)Pyrenochaeta unguis-hominis �Rhinocladiella aquaspersa �Scedosporium prolificans � �Scytalidium dimidiatum � �Scytalidium hyalinum �Ulocladium species �

Mucorales speciesAbsidia coerulea � (MUCL10045)Cunninghamella bertholletiae � �Cunninghamella species �Lichtheimia corymbifera � � � (MUCL10046)Lichtheimia blakesleeana � (CBS 100.28)Lichtheimia hyalospora � (CBS 173.67)Mucor species � �Rhizomucor pusillus �Rhizopus azygosporus �Rhizopus microsporus var. microsporus �Rhizopus microsporus var. oligosporus � (CBS 112586)Rhizopus microsporus var. chinensis � � (CBS 631.82)Rhizopus microsporus var.rhizopodiformis

� � (CBS 343.29)

Rhizopus oryzae � � � (CBS 112.07)Rhizopus stolonifer �Syncephalastrum racemosum �

YeastsBlastoschizomyces capitatus �Candida albicans � (ATCC 90028)Candida dubliniensis � �Candida glabrata � �Candida guilliermondii �

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VOL. 48, 2010 AGAR BLOCK SMEAR PREPARATION 3055


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clinical microbiology laboratory of Queen Mary Hospital in Hong Kong wereincluded in the present study (Table 1). A total of 137 species of yeasts, molds,and thermal dimorphic fungi were included.

Culture medium. A transparent, extremely low-nutrition culture medium fordetection of Acanthamoeba species in ophthalmologic specimens (Acanthamoebaagar) that contained 12% (wt/vol) NaCl, 4% (wt/vol) MgSO4 � 7H2O, 14.2% (wt/vol)Na2HPO4, 13.6% (wt/vol) KH2PO4, and 4% (wt/vol) CaCl2 � 2H2O was used foragar block smear preparation unless otherwise stated (4).

Agar block smear preparation method. Fungi were inoculated onto Acan-thamoeba agar in a class II biological safety cabinet and incubated under optimalconditions for the specific fungus species. Spore formations and other charac-teristic structures were checked every 2 to 7 days, depending on the growth rateof the fungus, by agar block examination using agar plates under a light micro-scope (�40 or �100 magnification) (Fig. 1a). When the fungus colony wasmature, photos were taken directly using the microscope camera and the plateswere used for subsequent agar block cutting. For known or suspected biosafetylevel 3 fungi such as Coccidioides immitis, the agar plate was fumigated using37% formaldehyde for 48 h before agar block cutting was performed.

In a class II biological safety cabinet, an agar block (15 by 15 mm) was cutusing a sterile dissecting knife and placed on a glass slide (Fig. 1b). After a dropof lactophenol cotton blue stain or of another stain such as calcofluor white stainwas added, a coverslip (18 by 18 mm) was put onto the agar block (Fig. 1c). Theagar block was examined under a light microscope (�400 or �1,000 magnifica-tion) (Fig. 1d). For long-term storage, the block was dried in air at roomtemperature in a class II biological safety cabinet until the thickness of the agarblock reached 0.5 mm, which usually took about 48 h (Fig. 1e). The four sides ofthe agar block under the coverslip were filled with Permount mounting medium(Fisher Scientific, NJ) (Fig. 1f). After the mounting medium was dried, the agarblock smear was examined under a light microscope (�400 or �1,000 magnifi-cation).

RESULTS

Direct examination of agar plates under a microscope be-fore cutting of the agar block. This allows observation of verydelicate fungal structures of diagnostic significance. It shouldbe noted that for very fragile and delicate conidial structures,their arrangements on conidiophores may still be destroyed

even when the agar block smear preparation method is used.For example, all the conidia of Fusarium chlamydosporum weredislodged from the polyphialides during agar block smearpreparation (Fig. 2a), whereas their flower-like arrangementon the polyphialides was well preserved when the whole agarplate was observed under a light microscope before cutting ofthe agar block was performed (Fig. 2b). Similarly, the conidialchains of Fusarium verticillioides that were attached to thephialides (Fig. 2c) and the unique conidial chains resultingfrom basipetal growth in Trichothecium roseum (Fig. 2d) werewell preserved when the whole agar plate was observed undera light microscope, but they were destroyed by the agar blocksmear preparation. Additionally, direct examination of theagar plate under a microscope was particularly useful for ob-servation of the conidial head of Aspergillus species and of thearrangement of sporangiophores in members of the Mucoralesorder, both being key features for species identification.Observed with a lateral light source, the conidial heads ofAspergillus niger were radiate (Fig. 2e) whereas those of Aspergil-lus fumigatus were columnar (Fig. 2f). As for the Mucoralesspecies, the sporangiophores of Lichtheimia corymbifera werebranched in an umbel formation (Fig. 2g) whereas the un-branched sporangiophores of members of the Rhizopus micro-sporus group were found directly above the rhizoids (Fig. 2 h).

Microscopic examination of slides prepared by the agarblock smear preparation method. A total of 510 agar blocksmears of 137 fungal species were prepared (Table 1), with 102smears prepared in 2006, 153 in 2007, and 255 in 2008. Slidesthat were prepared using the agar block smear preparationmethod were compared to those prepared using the adhesivetape method.

TABLE 1—Continued

Fungal species

Sourcea

NEQASUK QC

CAPUSA QC



Candida kefyr �Candida krusei � � (ATCC 6258)Candida lusitaniae � �Candida parapsilosis � � (ATCC 22019)Candida tropicalis �Cryptococcus albidus �Cryptococcus neoformans � � � � (CBS 132)Cryptococcus uniguttulatus �Geotrichum candidum � �Malassezia pachydermatis �Malassezia furfur � � � (CBS 1878)Prototheca wickerhamii �Rhodotorula rubra �Saccharomyces cerevisiae �Trichosporon species � � �Ustilago species �

Dimorphic fungiBlastomyces dermatitidis �Coccidioides immitis �Penicillium marneffei �Sporothrix schenckii �

a NEQAS UK QC, National External Quality Assessment Service (United Kingdom) quality control; CAP USA QC, College of American Pathologists (UnitedStates) quality control; FRC, Fusarium Research Center; MUCL, Mycotheque de l’Universite catholique de Louvain.



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Preservation of native structural features. Intact conidio-phores with foot cells of Aspergillus versicolor were well pre-served in agar block smears. As a result, the length of conid-iophores, which is one of the key features for Aspergillusspecies identification (Fig. 3a), could be measured easily. Onthe other hand, conidiophores, especially the longer ones, wereusually broken due to the tearing force exerted during prepa-ration of adhesive tape smear (Fig. 3b).

For Scopulariopsis brevicaulis, the conidiogenous cells (an-nelids) were well preserved in agar block smears (Fig. 3c).They were found either singly arranged or in brush-like clus-ters with short conidiophores. From the annelids, truncatedconidia in chains were basipetally produced. On the otherhand, the annelids were overlapped with each other and thenative state was destroyed as a result of the adhesive tapesmear preparation method (Fig. 3d).

In the agar block smear of Phialophora verrucosa, its phi-alides were distributed alongside the vegetative hyphae like adozen vases of flowers (Fig. 3e). The phialides are shaped likeflasks with funnel-like collarettes. Round-to-oval conidia accu-mulated in clusters at their apices. However, in the adhesivetape smear of P. verrucosa, artificial overlapping of phialideswas observed (Fig. 3f).

For Cladophialophora bantiana, the elliptical conidia ar-ranged in chains and their origins were well preserved in agarblock smears (Fig. 3g). On the other hand, many acropetal

conidial chains, with the youngest conidia at the tip, weredetached, and conidiophores were not observed in the adhe-sive tape smears (Fig. 3h).

Observation of fungal structures embedded under the agarsurface. For Chaetomium funicola, the ascomata with typicalascomal hairs that were partially embedded in agar were wellshown by the agar block smear technique (Fig. 4a). The ar-rangements of the straight, stiff ascomal hairs that were repeat-edly dichotomously branched were better preserved in the agarblock smears (Fig. 4a) than in tease mounts. They differedfrom the nonbranched, undulate ascomal hairs of a more com-mon Chaetomium species, C. globosum. Under high-poweredmagnification, the limoniform ascospore and the distinctly ver-rucose ascomal hairs were visible (Fig. 4b).

For Phoma glomerata, a coelomycete which producesconidia by the activity of conidiogenous cells lining the innercavity of its asexual fruiting bodies (i.e., its pycnidia), a spher-ical pycnidium embedded partially under the agar surface withtwo ostioles in the agar block smear was observed (Fig. 4c).The pycnidium is darkly pigmented around the ostioles fromwhich conidia were released. A brown chlamydospore withlongitudinal and transverse septa (muriform), an additionalkey structure for identification of P. glomerata, was also welldemonstrated (Fig. 4d). Pyrenochaeta unguis-hominis, also acoelomycete but differing from P. glomerata by its setose pyc-nidium, was also well shown in the agar block smear (Fig. 4e).

FIG. 1. Preparation of agar block smears. (a) Spore formations and other characteristic structures were checked by agar block examination of agarplates under a light microscope. (b) An agar block (15 by 15 mm) was cut using a sterile dissecting knife. (c) A coverslip (18 by 18 mm) was put ontothe agar block after lactophenol cotton blue staining was performed. (d) The agar block with the coverslip was examined under a light microscope. (e)The block was dried in air until the thickness of the agar block reached 0.5 mm. (f) The agar block under the coverslip was filled with mounting medium.



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Unlike other Pyrenochaeta species with setae tapering towardthe tip, the setae observed in P. unguis-hominis were obtuse attheir apices (Fig. 4f).

The arrangements and positioning of chlamydospores areamong the critical characteristics for Fusarium species identi-fication. For F. dimerum, the intercalary chlamydospores ar-ranged in a chain were ovoidal in shape (Fig. 4g), but for F.solani, the lateral chlamydospores were usually arranged inpairs and spherical in shape (Fig. 4 h). These chlamydosporeswere often found on the submerged hyphae.

Microscopic examination of agar block smears after long-term storage. After 1 year of storage, 459 (90%) out of 510agar block smears were in a good state. After 3 years of stor-age, 72 (71%) out of the 102 smears prepared in 2006 were stillin a good state.

Figure 5a and b show an agar block smear of C. immitis thathad been stored for more than 3 years. Under low-powermagnification, chains of arthroconidia with alternate disjunctorcells were well displayed (Fig. 5a). Raquet hyphae were alsovisible. Under high-power magnification, conidiogenesis of al-ternate arthroconidial chains was clearly demonstrated (Fig.5b). The chains were formed by the fragmentation of hyphaethrough the dissolution of disjunctor cells. Barrel-shapedarthroconidia were usually attached with an annular frill on eachend (Fig. 5b).

Figure 5c shows an agar block smear of Bipolaris hawaiiensisstored for more than 1 year. In contrast to those of otherBipolaris species, its macroconidia showed more than threedistosepta. They were pseudosepta and were differentiatedfrom the true septa by the cytoplasm contraction of distoseptateconidia and by their having become angular, as demonstrated by

lactophenol cotton blue staining. Figure 5d shows an agar blocksmear of Exophiala dermatitidis that had been stored for morethan 1 year. Torulose hyphae with rare spherical phialides weredemonstrated. Since this species is a dematiaceous fungus, darkpigment of melanin was obvious on its cell walls.

Figure 5e, f, g, and h show agar block smears that had beenstained with calcofluor white and stored for more than 6months. The fluorescence did not fade; the different colors offluorescence were due to the use of different barrier wave-lengths in the fluorescent microscope (430 nm for blue fluo-rescence and 520 nm for green fluorescence). Figure 5e showsan agar block smear of Graphium eumorphum, the asexualsynanamorph of Pseudallescheria boydii. Conidiophores wereaggregated into a compound stalk (i.e., a synnemata). A sterilebasal part and a fertile head producing cylindrical terminalconidia were revealed using green fluorescence. Figure 5fshows an agar block smear of Exophiala moniliae in blue flu-orescence. Annellated taping, which protruded and becamerather long with age, is clearly observable as a deeply pig-mented tip due to melanin deposits. Figure 5g shows an agarblock smear of Phialemonium curvatum in blue fluorescence.Short adelophialides without a basal septum, a characteristic ofP. curvatum, were clearly observed, unlike the results seen withAcremonium strictum, which showed long phialides with a basalseptum (Fig. 5h).

DISCUSSION

The marked increase in the density of intact native struc-tures of fungi observed in agar block smears greatly improvedthe accuracy of laboratory identification of pathogenic fungi.

FIG. 2. Direct examination of an agar plate under a microscope before agar block cutting. (a and b) Fusarium chlamydosporum, showing the conidiadislodged from the polyphialides during agar block smear preparation (a) and the well-preserved flower-like arrangement of the polyphialides when thewhole agar plate was observed under a light microscope before the agar block was cut (b). (c) Fusarium verticillioides, showing the conidial chains attachingto the phialides. (d) Trichothecium roseum, showing the unique conidial chains resulting from basipetal growth. (e) Aspergillus niger, showing the radiatingconidial heads. (f) Aspergillus fumigatus, showing the columnar conidial heads. (g) Lichtheimia corymbifera, showing the sporangiophores that arebranched in umbel. (h) Rhizopus microsporus, showing the unbranched sporangiophores directly above the rhizoids.



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The integrity and the native arrangements of conidiophoresand sporulating structures are of paramount importance formicroscopic species identification of pathogenic fungi. Thelength of conidiophores is important for species identificationof some fungi, such as the Aspergillus species, whereas thearrangement of conidia is crucial to identification of others,such as S. brevicaulis. During the process of adhesive tapesmear preparation, which primarily demonstrates fungal struc-tures on the surface of agar plates, many native structures offungi were crushed and torn, leading to breaking of most co-nidiophores and loss of the native arrangement of the sporu-lating structures. As for fungal structures partially or com-pletely embedded inside the agar, such as the fruiting bodies ofsome ascomycetes and coelomycetes, although they could bedug out by the use of tease mounts, the native orientation ofthe fruiting structures was often destroyed. On the other hand,when agar block smears are prepared, the whole thickness ofagar is gradually compressed to 0.5 mm. This allows the pres-ervation and examination of the native structures of mostfungi, both those formed on the surface and those embeddedin the agar. Furthermore, it also markedly increases the chanceof recognizing rare structures, such as the spherical phialidesof Exophiala dermatitidis. As for the medium, when the agarblock smear technique was first conceived in 2006, a number oftransparent culture media, including Sabouraud dextrose agar,carnation leaf medium, and Acanthamoeba medium, were em-ployed in efforts to grow the fungi for agar block smear prep-arations. Our preliminary study showed that Acanthamoebamedium was best for promoting conidiogenesis of most molds

because of its extremely low level of available nutrition. Forslow-growing dermatophytes, a longer incubation time was re-quired for spore formation. For example, at least 3 weeks ofincubation was necessary for the observation of the macro-conidia of Microsporum canis and Microsporum gypseum.

Good preservation of the native fungal structures in agarblock smears over long time periods allows slides to be sent outfor consultation and education purposes. In July 2006, theconcept of agar block smear was conceived and the first agarblock smear was made from a strain of Fusarium solani. Up tothe time of writing, more than 500 smears had been preparedfrom 137 fungal species of yeasts, hyaline molds with septatehyphae, dematiaceous molds with septate hyphae, Mucoralesspecies with rarely septate hyphae, and thermal dimorphicfungi. In our experience, more than 90% of the smears werestill in good condition after the first year. After 1 year ofstorage, the most serious problem that could affect the qualityof the smears was air leakage through the mounting medium,with the resultant air bubbles and drying effect jeopardizing thespore structures. Other minor problems include fading of staincoloring and degeneration of the mycelium.

The major roles of the agar block smear technique should bediagnosis of difficult cases, accurate identification of fungalspecies for epidemiological and clinical studies, and long-termstorage for transportation of slides and education purposes.Despite the advantages of the agar block smear technique, itcannot replace the adhesive tape smear technique. The adhe-sive tape smear technique is easy, quick to perform, and inex-pensive. With an agar plate culture, preliminary identification

FIG. 3. Preservation of native structural features of fungi by agar block smears. (a) Aspergillus versicolor, prepared by the agar block smearmethod, showing intact conidiophores with well-preserved foot cells (arrow). (b) A. versicolor, prepared by the adhesive tape method, showingbroken conidiophores. (c) Scopulariopsis brevicaulis, prepared by the agar block smear method, showing its annelids arranged either singly or inbrush-like clusters and truncated conidia basipetally produced in chains. (d) S. brevicaulis, prepared by the adhesive tape method, showing annelidsartificially overlapping each other (arrow). (e) Phialophora verrucosa, prepared by the agar block smear method, showing its flask-shaped phialideswell distributed along the vegetative hyphae with funnel-like collarettes (arrow). (f) P. verrucosa, prepared by the adhesive tape method, showingartificial overlapping of philalides and air bubbles (arrow). (g) Cladophialophora bantiana, prepared by the agar block smear method, showingelliptical conidia arranged in chains from indistinct conidiophores. (h) C. bantiana, prepared by the adhesive tape method, showing many detachedacropetal conidial chains with the youngest conidia at the tip (arrow) and the presence of tape ripples, making it impossible to take photographsat the same focal plane.



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of molds to the genus level can often be achieved within 15 minusing the adhesive tape smear technique. This preliminaryidentification of clinically significant molds is of paramountimportance in choosing antifungal agents and has a great im-

pact on patient management. On the other hand, agar blocksmear preparation requires subculturing of the fungus and thetime for preparation of the smear. Therefore, the agar blocksmear technique cannot replace the adhesive tape smear tech-

FIG. 4. Observation of fungal structures embedded under the agar surface in agar block smears. (a) Chaetomium funicola, showing theascomata with typical straight, stiff, dichotomously branched ascomal hairs partially embedded in agar. (b) Chaetomium globosum, showing thedistinctly verrucose ascomal hairs (arrow) and its limoniform ascospore (arrow). (c and d) Phoma glomerata, showing a spherical pycnidiumpartially embedded under the agar surface with two ostioles (arrows) and a brown chlamydospore with longitudinal and transverse septa. (e andf) Pyrenochaeta unguis-hominis, showing the characteristic setose pycnidium and setae with obtuse apices (arrow). (g) Fusarium dimerum, showingthe intercalary ovoidal chlamydospores arranged in chains. (h) Fusarium solani, showing the lateral spherical chlamydospores arranged in pairs.

FIG. 5. Microscopic examination of agar block smears after long-term storage. (a and b) Coccidioides immitis, showing chains of arthroconidiawith alternate disjunctor cells, racquet hyphae (arrow), and alternate arthroconidial chains with barrel-shaped arthroconidia, usually with anannular frill (arrow) on each end. (c) Bipolaris hawaiiensis, showing macroconidia with more than three distosepta (arrow). (d) Exophialadermatitidis, showing the torulose hyphae with rare spherical phialides (arrow) and the dark pigment of melanin on its cell walls. (e) Graphiumeumorphum stained with calcofluor white, showing conidiophores aggregated into a compound stalk (synnemata) and cylindrical terminal conidia.(f) Exophiala moniliae stained with calcofluor white, showing annellated taping (arrow), which protruded and became rather lengthy with age. (g)Phialemonium curvatum stained with calcofluor white, showing the short adelophialides without a basal septum (arrow). (h) Acremonium strictumstained with calcofluor white, showing the long phialides with a basal septum (arrow).



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nique in clinical microbiology laboratories for use on a day-to-day basis because of the turnaround time and economic con-siderations. On the other hand, for accurate identification atthe species level to elucidate, for example, the differentialsusceptibilities of different species to antifungal agents, theagar block smear technique would be preferred to the adhesivetape smear technique.

ACKNOWLEDGMENTS

This work was partly supported by a Research Grants Council grant;the University Development Fund, The University of Hong Kong; andthe Hong Kong Special Administration Region (HKSAR) ResearchFund for the Control of Infectious Diseases of the Health, Welfare andFood Bureau.

REFERENCES

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