LABORATORY DIAGNOSIS OF F U N G A L I N F E C T I O N S Glemt D. Roberts, Ph.D.*

Abstract Most hospitals do not perform mycologic examinations because laboratory personnel still believe in the "old wives' tale" tlmt mycolog), is too diffictdt to do and that tim fungi are too infectious to handle. This discussion presents methods suitable for use in laboratories of all sizes. It is hoped tlmt laboratories will take the initiative in offering diagnostic mycology services routinely. Medical mycology can be both a challenge and a rewarding experience to those who choose to become involved.

In the laboratory of a small hospital the laboratory diagnosis of fungal infection represents an occasional contact with tim unfamiliar, and it most often yields unrewarding results. To the technologist in the large medical center it represents a specialty field that is becoming more popular because it fl-equently yields definitive diagnostic i n f o r m a t i o n - o f t e n to the surprise of the clinician. This discussion presents information concerning the laboratory aspects of diagnostic mycology that may be used by clinical nficrobiology laboratories, either large or, snmll. In some instances methods useftd ilh the large laboratory nmy not be practical for the small laboratory; these are pointed out and ahernative metlmds included.

TREATMENT AND CULTURING OF S P E C I M E N S

The question of whether one should use culture tubes or Petri dishes for

fungal cultures often arises. For those laboratories not thoroughl)' acquainted with the handling of fungi, it is recommended that screw capped tubes be used. Tim slants should be thick and, after inoculation, the caps should be screwed on but left slightly loose. The disadwmtages of this method include relatively poor aeration of cultures and poor isolation of colonies as a result of the small surface area. Petri dishes may be used by those laboratories accustomed to Imndling fungal cultures, but they are not recommended for the inexperienced. The advantages of this method include good aeration of cultures and a large surface area, which allows better isolation of colonies. Tim disadvantages include an increased chance of laborator)' contamination (because the system is not closed) and an increased rate of dehydration of the nmditun. To prevent these problems, culture dishes should contain at least 40 ml. of ntedium, the lids should be taped down in two locations to prevent inadvertent opening, and the dishes should be incu-

*lnstrt.ctor" in .Microbiology and in l,aborator)' Medicine, Mayo Medical School. Consultant. Department of Microbiolog)"and of Laboratory Medicine. Mayo~lin~c and Mayo Foundation, Rochester, Minnesota.

161

HUMAN I'ATHOLOGY--VOI,UME 7, NUMBER 2 March 1976 bated in an atmosphere of 30 to 40 per cent relative hulnidity. The temperature of incubation should be 25 to 30 ~ C. T h e culture media used for the recovery of fimgi need not be elaborate. One can enhance tile recovery rate by using a large battery of media; however, the problem of practicality arises. It is imperative that antitnicrobial agents be added to certain media to Inevent bacterial overgrowth. Such overgrowth inhibits the recovery of the pathogenic fungi. The combilmtions of penicillin (20 units per ml.) with streptontycin (40 units l)el~ ml.) and gentamicin (5 /xg. per nil.) with chlorantphenicol (16 /xg. per ml.) have proved to be satisfactory. In many instances rapidly growing fungi ntay overgrow the slower growing strict pathogens. Cycloheximide (Actidione) in a concentration of 0.5 rag. per ml. is satisfactory for the prevention of this probleln; however, some of the pathogens, including Cr~'ptococcus m'q/'ormans anti Aspe~gillus Jitmigatus, may be inhibited, and it is necessary to inchlde a luediuln without cyclohexiinide in the battery of Inedia used. Cultures of blood and cerebrospinal fluid should be examined daily for visual e~'idence of growth. Cultures of other specimens should be exalnined three tilnes per week. Regardless of the type of specimen, all cultures should be incubated for a mininmm of 30 clays before being reported as negative. CEREBROSPINAI, FLUID.

O l l c tO 3 llll.

of fi-eshly collected cerebrospinal fluid should be fihered through a 0.45 /zm. Swinnex fiher (Millipore Corporation) attached to ,i sterile syringe. The filter pad is removed and placed on the naedium so that the side containing the concentrate ks toucifing the agar surface. Each day the ~lter shoukl be lifted off and placed in another location oil the medium surthce. Media suitable for culturing eerebrospinal Ihlid specimens inchlde Sabouraud's agar, brain-heart infusion agar, inhibitory mold agar, and chocolate agar. Ill general, one should use any two of these media to enhance the recovery rate. BLOOD. In small laboratories it is sufficient to inoculate oil l)rain-heart infusion agar or Sal)ouraud's agar with 5 to 10 nil. of blood. The surface of the agar should be flooc!Ed with the blood, and the cultures

162

should be examined daily. In addition, if a conunercial blood cuhure bottle containing a liquid mediuln is used, it should be vented with a sterile cotton phlgged needle that is left in place for the entire 30 day incubation period. After 2,t to ,t8 IIours, subcuhures should be ntade onto brainheart infusion or Sabouraud's agar. For the large microbiology laboratory it is recommended that a bottle containing a brain-heart infusion agar slant and broth be used. ~ It is necessary that all blood cuhure bottles be vented so that sullicient oxygen is l)resent to support aerobic growth. URINE. All urine specimens should be centrifuged and the sediment placed onto the culture medium. It is probably not desirable to quantitate these cultures because there is much controversy concerning the value of tiffs measuremelm The following inedia are recommended for urine cultures: inhibitory ntold agar, brain-heart infusiola agar with gentalnicin, cldoramphcnicol, and 10 per cent sheep blood; and brain-heart infusion agar with gentmnicill, cldoralnpllenicol, cyclohexinlide, and 10 per cent sheep blood. It is necessary to use each of these media, and one ntight expect contamination by gram negative bacteria even in the presence of the antibiotics. SPUTUM, BRONCItlIAL I,VASHINGS, GASTRIC '~VASII INC,S, AND "I'RANSTRACIIEAL ASI'IRATIOXS. At least 0.5 nal. of the specimen should be inoculated onto three media (inhibitory mold agar; brain-heart inftlsion agar with gentamicin, chloramphenicol, and 10 per cent sheep blood; and brain-lleart infusion agar with gentamicin, chlorampllenicol, cycloheximide, and 10 per ccitt sheep blood). Smaller laboratories that are cost conscious might use penicillin and strel)tomycin as the antimicrobial drugs. Also Sabouraud's agar or brainheart infusion agar containing either combination of antibiotics may be used in place of inhibitory mokl agar. TISSUES AND OTHER BODV FLUIDS.

All body iluids should be centrifuged and 0.5 ml. of the sediment placed on cuhure inedia. Media used are the same as those described for sputum. SKIN SCRAI'IN(;S, NAILS, AND HAIR.

Material,obtained for cuhure sliould be

LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS--Rot~ERTS

placed on Mycosel agar and incubated at 25 to 30 ~ C. for 30 days. Cultures should be examined twice weekly. CORNEAL SCRAI'INGS ANt) EAR CULTURES. Media suitable tbr culturing mate-

rial from the eyes and ears include Sabouraud's agar and brain-heart infusion agar containing no antibiotics. Another medium containing antibiotics should be used, such as Mycosel agar, inhibitory mold agar, or brain-heart infusion agar containing either gentamicin and chloramplmnicol or penicillin, streptomycin, and cycloheximide. ORAL *IucosA. Material from lesions in the m o u t h o r plmrynx should be cultured on inedia as listed tOl" sputum. Cultures shoukl be kept for 30 days before being ri:ported out as negative. DIRECT EXAMINATION OF S P E C I M E N S

In many instances direct examination o f a specimen allows one to make a tentative diagnosis in a much shorter tilne than it takes for tire c u h u r e to grow out. A confil'nlatory diagnosis can be m a d e when

Blaslomyces dermatitidis or Coccidioides immitis is the etiologic agent. In most laboratories direct examination o f a specimen is practical. This Ires been most c o m m o n l y used with skin and nail so'apings to determine w h e t h e r a d e r m a t o p h ) ' t e is present. T h e use o f the direct examination has beera e x t e n d e d to include all clinical specimens except blood. T h e p r o c e d u r e is simple and consists o f mixing the specimen with a d r o p o f 10 per cent potassium h y d r o x i d e on a microscope slide. T h e slide is covered with a cover ,glass, gently flamed, and then observed microscopically. Bright field microscopy is suitable if the a m o u n t o f light passing througl~ the specimen is tiecreased; however, if a phase contrast microscope is available, it should be used because it permits m o r e detailed ol)servation. T h e following descriptions should provide sufficient intormation fi~r a presuml)tive diagnosis o f the c o m m o n pathogens on the basis o f the direct examination: Aspergilhts: Septate hyphae that usually show dichotomolts branching; in rare instances the ty'pKal eonidiophore may be seen.

Rhizopus-Mucor: Large ribbon-like hyl)hae with no septa.

Dermatophytes: Septate hyphae that may show solne branching; rectangular arthrospores sometimes are present; found only in skin, nail scrapings, and hair. Candida: In most instances budding yeasts are seen; in an active infection Iwphae and )'east are often present simvhaneously. Co'ptococcus: Encapsulated )east cells that may bare one or more buds attached by a narrow constriction; the cells are spherical and often vary in size; not all of the yeasts have a large capsule. Blastomyces: I.arge budding )east that appears to have a thick, double contoured wall; buds are usually single and are characteristically attached by a broad base. Coccidioides: Very large, thick walled, round sl)herules, nlally of which contain slnaller round endospores; the sl~herules have a wide size variation. IDENTIFICATION

OF Y E A S T S

During the past few years there has been a significant increase ill the n u m b e r o f yeast infections seen at the Mayo Clinic, and this is generally representative o f the e x p e r i e n c e at o t h e r institutions. Most o f these infections are attributed to CDl~tococcus neoformans a n d Candida albicans; however, o t h e r species o f yeasts have been involved. This increase results in part f r o m the increased use o f steroid, i m m u n o supt)ressant, and antimicrobial drugs. I'atients r e c e M n g these agents become predisposed to mycotic infections, and ant" )'east becomes a potential pathogen in this situation. Because yeasts are considered to be normal, tlora in the o r o p l m r y n x and gastrointestinal tract, o n e might expect their isolation front clinical specimens such as sputum, throat swabs, bronchial washiilgs, gastric washings, and stools. In addition, they often may be isolated fi'om skin and nail scrapings, urine, and vaginal specimens. In contrast, the presence o f any micro-organism in normally sterile body tluids (for example, blood, cerebrospinai fluid, and synovial iluid) should be considered an almormaI finding. Ira o u r laboratory 70 to 80 per cent o f all specimens submitted (excluding normally sterile b o d y lluids) contain one o r m o r e species, o f yeast. "Fire most corn-

163

HUMAN PATHOLOGY--VOLUME 7, NUMBER 2 March 1976 mon is Camlida albicans, followed by Torulopsis g&brata, Candida tropicalis, Candida parapsilosis, Candida hrusei, and Saccharomyces species?

164

Because most )'casts are normal flora and others in some instances are pathogens, the question o f w h e t h e r all laboratories should identify yeasts ahvays arises. All laboratories, regardless o f size, should identify yeasts if one o f the following criteria exists: (1) yeasts are isolated f r o m blood, cerebrospinal fluid, or o t h e r normally sterile body fluids; (2) yeasts are isolated from several consecutive specimens f r o m the same patient; or (3))'casts are isolated f r o m a patient in whom tim clinician suspects a mycotic infection. This means that all laboratories should have the capability o f p e r f o r m i n g the necessary tests for )'east identification. Small laboratories that p e r f o r m few mycologic examinations should screen all )'east isolates fox" urease production. A heavy inoculum should be inoct,lated o n t o a urea agar slant in a tube and incubated at 37 ~ C. for at least 72 hours. If t, rease p r o d u c t i o n is observed, the )'east should be identified as to species by using carbohydrate and nitrate assimilation tests (to be described). This step is included to e n s u r e that all laboratories will detect Cuptococcus neoformans, if present. I f tile urease test is negative and if n o n e o f the f o r e g o i n g criteria is met, it is acceptable to send out a r e p o r t stating ")'east present." In instances in which a small laboratory must identify a )'east, it is sulficient to screen only fox" Cuptococcus neoformans and Candida albicans: Candida albicans is identified by a positive g e r m tube test (to be discnssed). I f the c u h u r e is urease negative land g e r m tube negative, the r e p o r t may be sent out as ")'east present; not C. albicans." Because c a r b o h y d r a t e assimilation and f e r m e n t a t i o n tests are simple to p e r f o r m , it is r e c o m m e n d e d that a full identification be nmde, but that should be the decision o f each laboratory. Large clinical laboratories in which mycologic examinations are p e r f o r m e d routinely should at least screen each cultuxe fox" cO'ptococcus neoformans and Candida albicans by the urease and g e r m tube tests. C a r b o h y d r a t e and niu,'ate assimilation 'tests are necessary for tentative identification o f the cryptococci, and the final

identification should include tile reaction on niger seed agar. It is r e c o m m e u d e d that all )'casts be identificd because their significance in clinical specimens has not yet been d e t e r m i n e d . In o u r laboratory, we identify all )'east isolates and follow tile m e t h o d o f Dolan.'-' In this system )'east is f r s t screened by the germ tube test; if it is not CandMa albicans, the isolate is placed on cornmeal agar containing T w e e n 80. In most instances the microscopic morphologic characteristics o f the c o m m o n species are p r o d u c e d and the final identification can be made. However, c a r b o h y d r a t e assimilation and fermentation tests, as well as nitrate assilnilation, are used in instances in which tim cornmeal-Tween 80 agar fails to provide a definitive identification. This m e t h o d is accurate, is relatively easy to p e r f o r m , and provides an identification in a slmrt period. However, it requires considerable e x p e r i e n c e and is t h e r e f o r e not recomm e n d e d for laboratories that process few specimens. T h e following paragraphs present methods reconamended for )'east identification. UREASZ TEST. A large inoculum fl'otn a single )'east colony is placed onto the surface o f the slant in a tube o f Christensen's urea agar. T h e cap is slightly tightened a n d tlm tube is incubated for 72 hours at 37 ~ C. T h e presence o f a pink or red color is indicative o f urease p r o d u c t i o n and is suggestive o f Co'ptococcus species. GERM TUBE TEST. A small inoculum f r o m a single )'east colony is placed in a sterile tube containing 0.5 ml. o f sterile normal h u m a n serum and is incubated for three hours at 37 ~ C. T h e SUSl)ension is observed microscopically for tim presence o f g e r m tubes e x t e n d i n g from single )'east cells. T h e i r presence provides a definitive identification o f Camlida albicans. CARBOHYDRATE ASSIMILA'X'XON (AuxANOGRAI'XlIC PLATF). T h e m e d i u m used in the carbohydx'ate assimilation tests is p r e p a r e d by a d d i n g 88 ml. o f filter sterilized )'east nitrogen base (Difco), pH 6.2 to 6.4, to 100 ml. o f filter sterilized 0.04 per cent bromcresol' purple (Difco). This is then a d d e d to I liter o f sterile 2 p e r cent agar and p o u r e d into Petri dishe.s (15 ml. per dish}. A suspension o f the )'east equivalent

LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS--RonERTS to a McFarland standard no. 4 is used to flood tim surface o f the agar. T h e excess inocuhun is r e m o v e d with a transfer pipet, and the lid is left ajar for 10 to 15 minutes to allow tile surface to dr)'. C a r b o h y d r a t e i m p r e g n a t e d disks (Difco), 6 ram. in diameter, are placed evenly apart on the agar surface.* T h e cultures are incubated for 2,t to 48 hours. A color change from purple to yellow a r o u n d the disks indicates assinfilation o f the carbohydrates. In rare instances, growth appears a r o u n d the disk without an a c c o m p a n y i n g color change; tiffs is also i n t e r p r e t e d as assimilation. This m e t h o d is used by o u r laboratory and is r e c o m m e n d e d for laboratories o f any size. CARBOIIYDRATE ASSIMILATION (CARBOttYDRATE SLANT). A recent rel)ort b)' Adams ",and C o o p e r s l)resents an alternate m e t h o d that may be advantageous in a small laboratory. Tim medinna used for the slant lnethod consists o f 2 gm. o f Noble agar in 90 nil. o f distilled water to wlfich 0.2 ml. o f 1.6 p e r cent bromcresol p u r p l e and 1.0 ml. o f 0.1 N sodiuln h y d r o x i d e have been added. This is autoclaved and dispensed into screw c a p p e d tubes (4.5 ml. p e r tube), and 0.5 ml. o f filter sterilized c a r b o h y d r a t e solution is a d d e d to each. Each c a r b o h y d r a t e solution is 10 p e r cent (wt./vol.) in )'east nitrogen base (Difco). T h e inoculnm is p r e p a r e d by suspending a 2 ram. loopflfl o f )'east cells in 9 ml. o f sterile distilled water. Each slant is inoculated with 0.1 ml. o f the suspension, incubated at 25 ~ C., and observed at seven day intervals for at least 14 days. Assimilation is shown by a change in color o f the indicator f r o m p u r p l e to yellow. NITRATE ASSIMILATION. It is necessary to p e r f n r m nitrate assimilation tests when oi~e is dealing with a )'east suspected o f being C~yptococcus.T h e r e have been numerous problems associated with currently available methods; this p r o m p t e d us to evaluate sonte o f t h e s e : Tile lnethod presented h e r e appears to have resoh'ed past problems and is r e c o m l n e u d e d tor use by all laboratories. T h e inedium consists o f 12.5 gin. o f *If cO'ptococcusis suspected, dextrose, maltose, sucrose, lactose, iqositol, and rallinose are used. If a )east other thap CD~)tococcusis suspected, dextrose, maltose, sucrose, la'ctose, raflinose, and trehalose are used.

indole nitrate mediutn, 5.0 gill. o f soluble starch, 2.5 gm. o f s o d i u m chloride, 10.0 gin. o f gelatin, 0.5 gin. o f dibasic potassium pllosl)hate, a n d 500 ml. o f distilled water. This is dispensed into tubes (2 ml. per tube) and autoclaved. T h e tubes are heavily inoculated a n d incnbated at 30 ~ C. for 48 to 72 hours. T l l e n six drops each o f the sulfanilic acid and c~-naphthylanfine reagents described by Edwards and Ewing s are a d d e d to each tube; each is shaken anti observed for the presence o f the red color that indicates nitrate reduction. Zinc dust is a d d e d to each negative test for confirmation: CARBOIIYDRATE FERMENTATION. Most laboratories are familiar with carbohydrate f e r m e n t a t i o n methods, and occasionally they are n e e d e d when carbohydrate assimilation reactions alone do not yield a definitive identitication. In o u r laboratory, we use the m e t h o d described by Dolan and Roberts; r however, many others are acceptable, such as the m e t h o d o f Silva-Hutner and Cooper. 8 NIGER SEEO AGAR TEST. An optional test that is usefld for the definitive identification o f Co'ptococcus ne~formans is tlle niger seed agar test developed by Staib and Senska? T h e m e d i n m is composed o f 50 gin. o f Guizottia abyssinica (niger or tlfistle) seeds,* 1 gin. o f c r e a t i n i n e , 15 gin. o f agar, 10 gin. o f dextrose, 1 gm. o f dibasic potassinm phospltate, and 1 gin. o f chloramphenicol. T h e seed is a d d e d to I000 nil. o f distilled water and pulverized in a blender. This is boiled for 30 minutes and filtered t h r o u g h a p a p e r filter, and then tile v o h n n e is b r o u g h t up to 1000 Inl. with additional distilled water. Tile remaining ingredients are a d d e d , the p H is adjusted to 5.5 nsing 1 N hydrochloric acid, and the nfixture is autoclaved f o r f i 5 nfinutes at 110 ~ C. and p o u r e d into Petri dishes before cooling occurs. Plates o f naedium are heavily inoculated and incubated at 37 ~ C. for at least one week. T h e p r o d u c t i o n o f a brown piglnent on this mediuln is diagnostic o f Coptococcus neoformans. T h e brown pigm e n t nsnally a p p e a r s first in the area o f heaviest inoculum a n d later on the renminder o f the agar surface. *Available frdm'I Philadelphia Seed Company, l'hiladelphia, Pennsyh'ania..

165

IIUMAN I)ATHOLOGY-VOLUME 7, NUMBER 2 March 1976 Tiffs mediuln is recolnlnended for all large laboratories and should be used in small laboratories as well; Itowever, the availability of the niger seed may limit its use in the latter. CO.~t.~tEX'r. Table 1 presents biocltemical reactions for most of the common )'casts isolated fi'om clinical specimens. In the preceding section we have made no attempt to describe all available procedures for )'cast identification, but methods representative of those currently available were included. If laboratories choose to use others, this is certainly acceptable. Those listed here are silnply the ones tiffs author thinks are the Inost practical for both large and small clinical microbiology laboratories.

The laboratory identification of the filamentous fnngi should be restricted only to those laboratories that have biologic safety hoods. Inadvertent opening of a culture outside a hood can resuh in a serious laboratory acquired infection. SInaller laboratories that do not have adequate facilities tor handling filamentons fimgi should send suspected pathogens to a reference laboratory, such as a state health laboratory or the Center for Disease Control, torfinal identification. In general, the growth rate for strict patlmgens illcluding Histoplasma capsulaturn, Blastomyces dermatitidis, and Coccidioides immitis is slow, and one to three weeks are required before colonies becolne visible. CoccidioMes immitis inay produce visible growth within three to five days in SOlne instances. However, the others usually require the extended incubation period, and it is recolnlnended that all cuhures be kept for 30 days before being discarded. ?Fhe colonial morphology is of little value in identifying the filamentous fimgi, becanse it varies depending on the mediuln used. For exalnple, Blastomyces dermatitidis often appears to be yeastlike on primary isolation on a blood enriched mediutn, but it grows as a white to tan, fluffy colony on Sabouraud's agar or a similar inedinm. One usually can gain inore information from the growth rate than from the colo-

I D E N T I F I C A T I O N OF FILAMENTOUS FUNGI

For many )'ears the filamentons flmgi have been considered to be either pathogens or saprobes. It recently has been shown that saprobes previously thought to be nonl3athogenic have prodt, ced severe, often thtal, infections in colnprolnised patients. It is now necessary that we consider all filamentons fungi as potential pathogens and that they be handled with extreme caution in the laboratory. TABLE

1. (]nARAC'FERISTIC FEATURES AND BIOCHEMICAL REACTIONS OF YEASTS COMMONLY ISOI.ATED FROM CLINICAl. SI'ECIMENS

Organism

Germ

Nitrate dssim.

Tube

Niger Seed Agar

Capsule

Clyplococcu~ neofm marts (2. a l b i d u s var. a l b M u s C . a l b M u s var. diJ]luens (2. IIttcoln.~

+ + + +

-

+ + -

+ -

+ + + +

C. b t t t t e n t i i Candida albitam:~

+ -

+

-

-

+ -

C. tro/,icali~ C. pamp.~il,.~i.~

. .

. .

. .

. .

(2. hYItM'i

+t

C. g u i l l c r m m u l i i

.

.

.

.

C. p . w n d o t r o p k a l i s Rhodolorula rubra "lbrulop.ds glahrata

. + .

.

.

.

(;cotrichum

.

.

amdMum

"l'richo~poron c u r a r e , urn

166

Urease

+

.

.

. .

.

. .

.

. . .

.

. .

. .

. .

. . .

.

.

*FG-=" ferlnentation detectecl by gas productkm. "l'Str,~in vaniation. * C a n d i d a . q e l l a t o i d i a is inchnded with C. a l b i c a m here; tile only difference is in sucrose assimilation.

LABORATORY DIAGNOSIS OF FUNGAL INFECTION--RoI~rRTS nial morphology. Definitive identification of the filamentous fungi can be made on the basis of the characteristic nficroscopic inorphology (type and arrangement of spores) and demonstration of both the saprobic and parasitic forlns for the dimorphic fimgi. *lost of the filngi exhibit microscopic features that are characteristic of the genus, and these usually are quite different from each other. Tiffs makes the identification SOlnewhat easier for this group of fungi than for the yeasts, all of which appear similar. In many instances it is necessary to subculture the primary isolate to Sabouraud's agar or a silnilar medium to obtain characteristic sporu]ation. Our laboratory has found it helpfill to use the adhesive transparent plastic tape method of preparing cultures for microscopic exalnination. ~~ The adhesive side of the tape is touched to the surface of a colony and is then adhered to a slide on which a drop of lactophenol-cotton blue dye has been added. This mounting inediuln consists of 15 gin. of polyvinyl alcohol, 100 ml. of distilled water, 39 ml. of lactic acid, 39 ml. of phenol (inched), and 0.1 gm. of cotton blne. T h e polyvinyl alcoltol is added to the water and is heated at 80 ~ C. tmtil clearing occurs. The lactic acid is added, followed by the phenol and cotton blue. Tiffs techniqne allows one to observe the structures arranged solnewhat as they were when part of the colon)'. One should

TABLE

1.

YEASTS

first examine the hyphae to determine whether they are septate. If no septa are present, the organism can be classified as a phycomycete-Mucor, Rhizopus, or Absidia. The size of the hyphae is important also; the phycomycetes usually Imve large, wide, ribbon-like hyphae whereas those of the strict pathogens appear quite small. Most other fimgi, including ~.lspe~L611us, have hyphae of moderate size, ratermediate between those of the phycolnycetes and the strict pathogens. The most ilnportant feature needed for the identification of the filamentous fungi is the arrallgelnent of the spores. The identification usually can be nmde by direct exalnination of the tape preparation, but in some instances no characteristic arrangement can be seen and it is necessary to prepare a microslide culture. A small agar block is placed on a sterile slide, the four corners are lightly inoculated, and a cover slip is placed on top. The culture is incubated in a moist environlnent. When adequate growth occurs, the cover slip is removed and placed on a slide containing a drop of lactopbenolcotton blue. This allows one to obscrve the nndisturbed spores as arranged during growth under the cover slip. The definitive identification of the dintorphic fungi, including Ilistopla,sma

capsulatttm, Bla.stomyces dermalitidis, Sporothrix schenchii, and Coccidioides immitis, requires that both the saprobic and parasitic

CIIARACTERISTIC FEATURES A N D COMMONL'~"

ISOL.~,TED

FROM

BIOCnEMICAL

CLINICAL

(Continued)

Assimilation lh,xtro.~e ;Maltose Sucro.w

l.atto.w

OF

REACTIONS

SPECIMENS

Fermentation*

IlaJ]ino.se Trchalo.w

Ino.sih,I I)e.xlro.w Mallo.w Sucrose

+

+

+

--

-4-

+

+

.

+

+

+

+t

+

+t

+

.

+

+

+

-

+

+

+

.

.

.

.

+

+

+

--

--t

+

+

.

.

.

.

+

+

+

+

+t

+

+

.

+

+

+~

-

-

+

-

F(;

F(;

-t

-

FG

+

+

+

-

-

+

-

F(;

FG

F(;

-

F(;

+

+

+

-

-

+

-

FG

-

-

-

FGt

+

.

F(;

.

+

+

+

-

+

+

-

F(;

-

1~(;

-

FG

+ +

+

+ +

+ -

+ +

+

.

F(;

-

F(; .

FG

F(;

+

.

+

-

F(;

+

+t

+t

+t

.

.

.

.

.

+t

.

.

.

+

+t

.

.

.

.

Lacto.w (;alaclo.w

.

.

.

.

.

.

. . .

.

.

.

. .

.

.

.

.

.

.

.

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H U M A N P A T H O I . O G Y - V O L U M E 7, NUMBER 2 March 1976 f o r m s be o b s e r v e d . C o n v e r s i o n to the parasitic f o r m m a y be a c c o m p l i s l i e d by p l a c i n g a large i n o c u h n n o f the filam e n t o t t s f o r m o n t o the s u r f a c e o f a fi'esh, moist slant o f b r a i n - h e a r t infusion a g a r with o r w i t h o u t 10 pet" c e n t s h e e p blood. C u h u r e s are i n c u b a t e d at 37 ~ C., a n d t r a n s f e r s are m a d e to fresh slants as s o o n as g r o w t h a p p e a r s . T h e inoculuna f o r the t r a n s f e r s h o u l d be t a k e n f r o m the p o r t i o n o f g r o w t h timt a p p e a r s m o s t c r e a m y in consistency. Several t r a n s f e r s a r e usually n e c e s s a r y f o r c o m p l e t e conver.sion, alt h o u g l l it Ires b e e n accoxnplished in as s h o r t a time as two days. T h i s p r o c e d u r e is suitable f o r d e m o n s t r a t i n g the )'east f o r m s o f all the d i m o r p l f i c f u n g i e x c e p t Coccidioi&'s immitis (this lacks the yeast , f o r m b u t has a s p h e r u l e form). A n i m a l inocnlation, an a h e r n a t i v e m e t h o d tlmt m u s t be u s e d to c o n v e r t CoccidioMes immitis to the sphertfle f o r m , also m a y be used to c o n v e r t the o t h e r d i m o r p l f i c f u n g i to the yeast f o r m . T h i s is a c c o m p l i s h e d by i n j e c t i n g a sntall a m o u n t o f a s u s p e n s i o n o f the o r g a n i s n t intraperitoneally. Mice are m o s t o f t e n used; t h e y a r e killed a n d a u t o p s y p e r f o r m e d two weeks a f t e r injection. T l t e spleen, liver, a n d lesions in the o m e n t t n n are rem o v e d , a n d m e t h e n a m i n e silver stained histologic sections a r e m a d e . T h e c h a r acteristic tissue f o r m s o f the o r g a n i s m s s h o u l d be readily visible; their p r e s e n c e c o n f i r m s the identification. R e c o g n i t i o n o f the diagnostic microscopic f e a t u r e s is d e t e r m i n e d by the exp e r i e n c e o f the observer. It r e q u i r e s a c a r e f u l c o m p a r i s o n o f the cultures with the p h o t o g r a p h s a n d d e s c r i p t i o n s that a r e available in c u r r e n t texts. I t is n o t within t h e s c o p e o f this discussion to p r e s e n t l n d w l d u a l descriptions f o r the filantentous f u n g i ; h o w e v e r , they m a y be f o u n d in several publications, r ' t t - t 5 With a little time a n d effort o n e s h o u l d be able to i d e n t i f y m o s t o f the c o m m o n l y isolated fltngi.

REFERENCES I. Roberts, G. D., and Washington,.l.A., II: Detection of fimgi in blood cuhures. J. CIin. Microbiol., 1:309, 1975. 2. I)olan, C. T.: A practical approach to identification of yeast-like organisms. Am.J. Clin. l'athol., 55:580, 1971. 3. Adams, E. D.,Jr., and Cooper, B. H.: Evaluation of a modified Wickerham medium for identifying medically important yeasts. Am. J. Med. Technol., 40:377, 1974. 4. Rhodes,J. C., and Roberts, G. D.: Comparison of four methods for determining nitrate utilizatioq by cryptococci. J. Clin. Microbiol., 1:9, 1975. 5. Edwards, 1'. R., and Ewing, W. lI.: hlentification of Enterobacteriaceae. Ed. 3. Minneapolis, Burgess l'ublishing Company, 1972. 6. Yu, P., Birk, R. J., and Washington, J. A., II: Evahmtion of a reagent-itnpregnated strip for detectitm of nitrate reduction by bacterial isolates. Am. J. CIin. l'athol.. 52:791, 1969. 7. Dolan, C. T., and Roberts, G. D.: Identification procedures. In Washington, J. A., II (Editor): I,aboratory I'rocedures in Clinical Microbiology. Boston. Little. Brown & Company, 1974. pp. 153-164. 8. Siha-ilutner. M., and Cooper, B. H.: Medically important yeasts. In Lennette. E. 1I.. Spaulding, E. II., and Truant. J. I'. (Editors): Manual of Clinical Microbiology. Ed. 2. Waslfington, D.C., American Society for Microbiology, 197,t, pp. ,t91-507. 9. Staib, F., and Senska, M.: Der Braunfarbeffekt (BFE) bei Coptococcu~ net?[brman~auf Gtfizzotia abyssinica-Kreatinin-Agar in Abhfingigkeit yore Ausgangs-pH-Wert. Zentralbl. Bakteriol. [Orig. A], 225:! 13, 1973. 10. Rebell, G., anti Taplin, D.: l)ermatophytes: Their Recognition and Identification. Coral Gables, University of Miami Press, 1970, p. 114. 1 I. Emmons, C. W., Binford, C. tt., and Utz, J. P.: Medical Mycology. Ed. 2. l'hiladelplfia, Lea & Febiger, 1970. 12. I)olan, C. T., Funkhouser,J. W., Koneman, E. W., Miller, N. G., and Roberts, G. D.: Atlas of Clinical Mycology. Chicago, American Society of Clinical l'athologist, 1975. 13. Conant, N. F., Smith, D. T., Baker, R. D., and Callaway, J. L.: Mam,al of Clinical Mycology. Ed. 3. i'hiladelphia, W. B. Sat,ndei's Company, 1971. i,t. Beneke, E. S., and Rogers, A. L.: Medical Mycolog)" Manual. Ed. 3. Minneapolis, Burgess Publishing Company, 1971. 15. Wilson, J. W., and l'hmkett, O. A.: The FtlllgOl.lS Diseases of Man. Berkeley, University of California Press, 1965. Section of Clinical Microbiology Mayo Clinic and Ma~'o Foundation Rochester, Minnesota'55901

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