APPUED MICROBIOLOGY, Apr. 1972, p. 814-818Copyright 0 1972 American Society for Microbiology

Vol. 23, No. 4Printed in U.SA.

Laboratory Mill for Pulverizing andHomogenizing Nail Specimens as an Aid toMicroscopy and Culture Confirmation of

OnychomycosisGEORGE M. LUEDEMANN AND ERNEST LEBRETON

Department of Microbiology, and Pharmaceutical Research and Development, Schering Corp., Bloomfield,New Jersey 07003

Received for publication 27 January 1972

A small mill has been developed for reducing nail clippings to a convenientsize for microscopy and inoculation onto isolation media. The milling process

acts to pulverize and homogenize the specimen. The use of a homogeneoussample in microscopy increases the opportunity for the discovery of fungalhyphae in a specimen. The use of a pulverized specimen increases the possi-bility of culture isolation by permitting greater numbers of potentially viablefungal cells to come into direct contact with the isolation medium.

The diagnosis of onychomycosis should bebased upon microscopic confirmation of fungalelements present in the suspected nail spec-imen and isolation and identification of thepathogenic organism (2, 4, 5, 7). Preparing anail specimen for microscopic examination canbe frustrating, particularly if one is not able totake the specimen oneself. We have receivedfor laboratory diagnosis nails of all shapes andsizes, from particles and small clippings toexcised nails complete with fingernail polish.The nail is tough and resilient, and defies sub-division with tenacity. We were pleasantlysurprised one day to receive a specimen thatcontained nail particles of a discrete and uni-form size. On inquiring how this particularspecimen had been taken, we learned that aburr was used to grind down the nail. Theseparticles, when treated with 10% KOH andgently warmed, spread out rapidly and evenlybeneath a cover slip and preserved cellularmorphology and fungal filaments. With theknowledge of the size nail particle we desired,a small mill was designed and constructed.

MATERIALS AND METHODSConstruction of mil. The construction of the nail

mill is relatively simple and can be accomplished bymost machine shops. The two holders are cut from apiece of 2-inch (5-cm) stainless steel or aluminumround stock and are turned, trued, and knurled on alathe. The two milling heads are cut from a piece of%-inch (1.6-cm) stainless-steel round stock and aretrued on a lathe and then set up on a miller with a '32-

inch (0.08-cm) end mill used to construct the cut-ting surface on the milling heads (Fig. 1). The weightof the assembled mill made with aluminum holdersaverages 230 g, and with stainless-steel holders, ap-proximately 520 g.When assembling the mill, one cutting head is

inserted into the large holder and recessed approxi-mately 'A inch (1.27 cm). It is held in place with two/4-inch (0.64-cm)-20 NC by %-inch (0.95-cm) cuppoint socket set screws (Fig. 2). The second cuttinghead is then inserted into the small holder so as toprotrude approximately X inch below the holder (Fig.3). The head (on the smaller holder) is then insertedinto the milling pit of the large holder and is spacedwith a piece of shim stock [approximately 0.004 inch(0.1 mm)] to set the distance between the cuttingheads. In the laboratory, a piece of white bond paper(approximately 0.004 inch) may be substituted forthe shim stock in adjusting the distance betweencutting surfaces.

Preparation of specimen. Nail specimens areplaced in the mill cavity. A single large nail frag-ment or several small ones may be used, but careshould be taken not to overload the mill cutting sur-faces. The mill halves are firmly pressed togetherand rotated in opposite directions, about half a turn,several times. Progress of the disintegration is vis-ually monitored by removing the small holder. Themajor particle size is determined by the spacingbetween the teeth of the milling surface.The particles are concentrated on one edge of the

milling pit by gently tapping the mill half on thetable at a slight angle to the horizontal. A largenumber of nail particles may be rapidly obtained inthis manner (Fig. 4). It is convenient to transferthese small particles by using a glass rod or dis-secting needle. A drop of 10% KOH is placed on a

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microscope slide, and the transfer needle is touchedto this drop so as to moisten the tip. The transferneedle is then touched to the pile of nail fragments,a number of which readily adhere. The transferneedle and nail particles are then returned to theKOH drop on the microscope slide, and the nail par-ticles are deposited.

Gently warm the slide containing KOH and nailparticles without a cover slip and set the slide asidefor 10 to 15 min to allow the particles to soften. Thenail particles may be further reduced in size by ma-cerating with the end of a small glass rod which hasnot been fire-polished. This step is optional, as it ispossible but not practical to reduce the nail particlesto their component cells. A drop of water or KOHmay be added to reconstitute the preparation priorto depositing the cover slip. A cover slip is touchedto this mass of cell material at an angle to reducetrapping of air bubbles in the final preparation. Thecover slip may be gently pressed down on this masswith the eraser end of a pencil to flatten and spreadout the softened nail particles. Ideally, a unicellularlayer would be best for viewing, but is rarely at-tained throughout a preparation. We prefer to make

FIG. 1. Close-up of protruding cutting head with"waffle iron" type cutting pattern and toenail clip-pings prior to milling. Scale marker represents 1inch.

four to six double preparation slides from each speci-men, as a negative microscope finding based on theexamination of one slide is not very meaningful.

Isolation. When an attempt to isolate a culture ofthe pathogen from a diseased nail is made, we havefound that the milled nail particles are ideal formaking multiple inoculations. The end of a steriletransfer needle is moistened in the isolation agar andtouched to the pile of nail particles in the mill pit.The adhering particles are then embedded andstreaked acrossed the isolation agar. It is preferableto make at least a dozen inoculations with visiblenail particles deposited at each inoculation site.Generally three inoculations are made per plate, orthree slants of each medium may be used.The choice of isolation media varies with the in-

vestigator. Mycosel (BBL) or Mycobiotic Agar(Difco) are commercially available agars useful forthis purpose, as are also pH indicator media such asDTM or ink blue agar (1-3). Our personal preferenceis to use several media for each specimen. We rou-tinely include Sabouraud dextrose agar, Littmanoxgall agar, corn meal dextrose agar, and a personalconcoction (1111G agar) composed of 0.1% yeast

FIG. 4. Milled nail fragments compared againstmillimeter scale and dissecting needle used totransfer fragments to microscope slide or agarmedia.

FIG. 2. Assembled mill. Lateral view of knurled holders with channels in which socket set screws holdmilling (cutting) heads in place. Excess length of cutting head protrudes above holder. Scale marker repre-sents 1 inch.

FIG. 3. Mill halves. View of cutting surfaces as they are locked into knurled holders. The left half bearsthe recessed cutting head and and the right half bears the protruding cutting head. Scale marker represents 1inch.

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LUEDEMANN AND LEBRETON

extract, 0.1% dextrose, 0.1% soluble starch, 0.1%CaCO3, 1.5% agar, and 100 Atg of the antibiotic genta-micin per ml available as Gentamicin Reagent Solu-tion, Schering Diagnostic Division, Port Reading,N.J. 07064). Trichophyton rubrum is the fungal spe-cies most frequently isolated from nail specimens inour geographical area, as was also reported for skin,nail, and hair specimens by Rosenthal and Furnari(3). The inclusion of the last three of the abovemedia allows a rapid presumptive identification ofT. rubrum, the colonies of which appear small,bristly, and yellowish-tinged on Littman's oxgallagar; white, thin, and spreading on corn meal dex-trose agar, with a characteristic cherry-red diffusiblepigment; and white, thin, and widely spreading onthe 1111G agar, with a characteristic maroon diffus-ible pigment rapidly developing. The latter twomedia are also good places to search for the typicalTrichophyton microconidia and mycelial configura-tions, such as spiral hyphae, raquet hyphae, pec-tinate bodies, and nodular organs. Other dermato-phytic fungal species also develop characteristicallyon these media.The nail mill after use should be sterilized by

steam or boiling. A clean dissectng needle has beenfound useful to remove adhering particles from themilling surface teeth, or the mill may be disassem-bled and cleaned with a small wire buffing brush.

Photomicrographs were taken with a standardGFL Zeiss microscope equipped with phase-contrastoptics.

RESULTSThe following observations have been made

on the use of pulverized, homogenized nailparticles. (i) Often, clearly defined hyphae orportions of hyphae are found free from nailcells. It appears that nail cells often separatealong planes that are traversed by fungal fila-ments, the filaments becoming free much asindividual pebbles in a weakly consolidatedrock conglomerate may be found to be easilydislodged from the matrix (Fig. 5-8). (ii) Mostcells of the nail when subjected to millingremain intact, rupture occurring between thecells. Small clusters of a few cells are easilyscanned for fungal filaments (Fig. 5). (iii)Small clusters of nail cells are more readilysoftened by the action of KOH than are largenail fragments, which may require severalhours or overnight KOH treatment (Fig. 9 and10). Fewer bizarre structures are introducedfrom the KOH digestion by a shorter softeningperiod. (iv) Fungal hyphae appear little dam-aged by the milling treatment of the nail andare more readily detected in microscopemounts and more easily cultured from the nailparticles.

Isolation of the pathogenic organism is notalways easy (2, 3, 7) and depends upon thecondition of the nail specimen and previous

therapy which may have impaired the viabilityof the fungus. Data presented in Table 1 (G.Baker and R. Kishimoto, personal communica-tion) were obtained in a laboratory study inwhich one of the nail mills described in thispaper was used. Although representing a smallnumber of specimens, the results are in favor-able agreement with a larger number of speci-mens run in our laboratory.A case in our laboratory requiring persist-

ence involved a patient who was clinically di-agnosed for onychomycosis and treated withgriseofulvin without results. After treatmenthad been discontinued, nail specimens weresubmitted for microscopy and culture, butthey produced only a rare microscopic positiveof degenerative, bizarre-appearing hyphae andwere culture-negative except for an occasionalsaprophyte. Periodically over a 3-year period,we received additional nail specimens whichgave similar results. Finally, the patient cameto the laboratory concerned about the appear-ance of her large toe nail which was raised andunattached to the nail bed for 75% of itslength. The raised portion of the nail wasmarked off into an outer, middle, and innerportion and painlessly clipped back to thepoint of attachment to the nail bed. The threeportions of nail clippings were individuallyhomogenized in the mill and cultured. Culturewas negative for the outer portion of the nail.From the middle portion of the nail, threeunidentified cultures of sterile mycelium, oneculture of a Penicillium species, and one cul-ture of a Fusarium species were obtained.From the inner portion of the nail, 12 cultureswere recovered which produced a dark-violetdiffusible pigment and produced typical Fu-sarium-type macroconidia. Microscopic exami-nation of nail fragments from this inner por-tion of the nail revealed a number of bizarrehyphae atypical of the even-diameter hyphaefound usually in T. rubrum infections but sim-ilar to those previously reported for Fusariumisolates from nail (4, 5). Failure of the patientto respond to griseofulvin therapy was sub-stantiated by the belated isolation and cultureof this organism. Multiple isolations of an or-ganism which commonly occurs as a sapro-phyte from the area of active invasion pro-vided a confidence factor for reporting thisorganism, which upon a single culture isolationwould have been doubtfully considered as apathogen.

DISCUSSIONThe positive microscopic identification of

fungal filaments in a nail specimen is conclu-

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FIG. 5-10. Photomicrographs of KOH preparations.FIG. 5. Nail fragment from Fig. 4. The fungal filament lies in sharp relief against the surface of this small

cluster of nail cells. Original microscope magnification x250.FIG. 6. Single nail cell and fungal filament which has been separated from its intercellular position on the

nail cells. Vacuolation, wavey outline, occasional branching, and size help to distinguish fungal hyphae fromother vegetable or synthetic fibers. Initial microscope magnification x250.

FIG. 7. Single nail cell with appressed surface fungal filament which has been sheared at either end of thecell by the milling process. Initial microscope magnification x400.

FIG. 8. Chain of fungal arthrospores freed by the milling process. Initial magnification x400.FIG. 9 and 10. Larger nail fragments containing fungal hyphae. Identification of fungal hyphae in these

multicellular nail fragments requires time for the KOH to soften the intercellular binding material. Fungalfilaments may be partially obscured by this debris and nail cells. Often, in large nail fragments intercellularmaterial may appear similar to fungal hyphae and represents a potential source of error. Initial microscopemagnification, x400.

sive evidence of fungal involvement, but unfor-tunately a negative microscopic result is notabsolutely conclusive. It may mean severalthings: the diseased condition is not due to afungus (6); the fungal hyphae were not presentin the specimen taken; or the fungal filamentsin the preparation have eluded the observationof the microscopist. The problem of micro-scopically identifying fungal filaments in nailspecimens is often complicated by the numberof cell layers which must be traversed by themicroscopist before a positive identificationcan be made. Fungal filaments passing be-tween the nail cell layers often require focusingon several planes to differentiate the fungalhyphae from intercellular artifacts. The pres-ence of these multilayers of epidermal cellsoften obscures the true outline of the fungalhyphae. A single layer of nail cells, if it could

TABLE 1. Fungi isolated from nails

Speci- Without homogenizing With homogenizingmen

1 0 02 Candida albicans Candida albicans3 0 Alternaria sp.4 0 Trichophyton rubrum5 0 06 0 07 Curvularia sp. Curvularia sp.8 0 T. mentagrophytes9 0 T. rubrum10 0 011 C. albicans C. albicans

be obtained, would be ideal for microscopicexamination. Nail specimens which have beenreduced to a small particle size in the milldescribed have made positive identification of

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818 LUEDEMANN.

fungal filaments rapid and relatively free fromfalse-positive results.

Nail specimens which have been found tocontain fungal hyphae by microscopy often failto yield positive cultures (2, 7). This conditionis frustrating and often requires obtaining ad-ditional nail specimens from a patient. Theuse of a pulverized nail specimen increases theopportunity for bringing viable fungal cellsinto direct contact with the isolation mediumand aids in obtaining multiple isolations of thepathogenic fungus.A schematic diagram suitable for a ma-

chinist to reproduce this mill will be includedwith reprint requests.

AlND LEBRETON APPL. MICROBIOL.

LITEATURE CITED1. Allen, A. M., R. A. Drewry, and R. E. Weaver. 1970.

Evaluation of two new color indicator media for diag-nosis of dermatophytosis. Arch. Dermatol. 102:68-70.

2. Merz, W. G., C. L. Berger, and M. Silva-Hutner. 1970.Media with pH indicators for the isolation of dermato-phytes. Arch. Dermatol. 102:545-547.

3. Rosenthal, S. A. and D. Furnari. 1971. Efficacy of "der-matophyte test medium." Arch. Dermatol. 104:486-489.

4. Rush-Munro, F. M., H. Black, and J. M. Dingley. 1971.Onychomycosis caused by Fusarium oxysporum. Aus-tralas. J. Dermatol. 12:18-29.

5. Zaias, N. 1966. Superficial white onychomycosis. Sabour-audia 5:99-103.

6. Zaias, N. 1969. Psoriasis of the nail. Arch. Dermatol. 99:567-579.

7. Zaias, N., I. Oertel, and D. F. Elliott. 1969. Fungi in toe-nails. J. Invest. Dermatol. 53:140.

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