JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1985, p. 803-8070095-1137/85/110803-05$02.00/0Copyright C) 1985, American Society for Microbiology

Advantages of a Modified Toluidine Blue 0 Stain andBronchoalveolar Lavage for the Diagnosis of Pneumocystis

carinii PneumoniaLINDA L. GOSEY,' RUBY M. HOWARD,2 FRANK G. WITEBSKY,1* FREDERICK P. OGNIBENE,' TERESA C.

WU,' VEE J. GILL,' AND JAMES D. MACLOWRY'

Microbiology Service, Clinical Pathology Department' and Critical Care Medicine Department,3 Clinical Center, NationalInstitutes of Health, and Laboratory of Pathology, National Cancer Institute,2 Bethesda, Maryland 20892

Received 1 April 1985/Accepted 5 August 1985

A modified toluidine blue 0 staining technique for Pneumocystis carinii is described. An easily preparedsulfation reagent made with sulfuric and acetic acids was used. The stain can be employed for bronchoalveolarlavages and lung tissue touch preparations. Most background material was removed by the sulfation reagent,slides were generally easy to read, and time from receipt of a specimen to reporting of results was

approximately 1 h. P. carinii cysts were more easily visualized with this stain than in slides stained withmodified methylene blue, Gram, Gram-Weigert, and standard toluidine blue 0 procedures. The importance ofcertain procedural aspects of subsegmental bronchoalveolar lavage, by which most of the specimens were

obtained, is also emphasized.

Pneuimocystis carinii pneumonia (PCP) is a frequent andpotentially life-threatening complication of the acquired im-munodeficiency syndrome (AIDS) and occurs as well inother immunocompromised patients. A variety of stainingprocedures can be used to visualize the organism in speci-mens such as bronchial washes, bronchial lavages,transbronchial biopsies, and open-lung biopsies. We haveused five staining techniques on these specimens over thelast two years, including two types of toluidine blue 0 stain,the Gram-Weigert stain (GW), a modified methylene bluestain (MMB), and the Gram stain (G). Of these, we found amodification of the toluidine blue 0 stain (MTolB) to be theeasiest to read; the reagents were also much simpler toprepare than those for the standard toluidine blue 0 stain(TolB), and the modified procedure took less time to per-form. The diagnosis of PCP can frequently be made onbronchial lavage specimens or on touch preparations ofpulmonary tissue with this MTolB.The purpose of this report is to describe this staining

technique, compare the readability of the slides with that ofthe other stains, and review certain features of the techniqueof subsegmental bronchoalveolar lavage (BAL) which arecritical to obtaining a satisfactory specimen for examination.The relative sensitivities of BAL and histologic examina-

tion of pulmonary tissue for the diagnosis of PCP have beenpreviously evaluated (7, 11, 12) and will only be brieflyassessed here.

MATERIALS AND METHODS

Patients. All patients were being evaluated at the ClinicalCenter of the National Institutes of Health for diagnosis of apulmonary infiltrate consistent with that caused by P. carinii.Most patients from whom material was obtained had AIDS;a few were immunocompromised because of other underlyingconditions.BAL. All patients had bronchoscopy performed in the

supine position with an Olympus BF-IT 10 or BF-ITR adultflexible fiberoptic bronchoscope with an external diameter of

* Corresponding author.

803

6 mm and an operating channel of 2.6 mm (Olympus Corp. ofAmerica, New Hyde Park, N.Y.). After examination of thetracheobronchial tree, patients underwent BAL of a lobejudged by a roentgenograph to be affected. To perform thelavage, the tip of the bronchoscope was wedged in asubsegmental bronchus. Aliquots of 35 ml of sterile 0.9%sodium chloride solution were injected from a sterile syringethrough the biopsy channel of the bronchoscope. The in-jected saline was then aspirated into a sterile sputum trap.The total amount of sterile saline used for each lavage rangedfrom 70 to 150 ml, with the yield ranging from 40 to 70% ofthe instilled volume. Chest roentgenograms and arterialblood gas determinations were performed postbronchoscopyand thereafter as indicated. Over 200 BALs have beenperformed thus far. Approximately one-fourth of these werestained with the initially used TolB; the rest were stained bythe MTolB. Of the BALs, 27 were stained with the GW, andall were stained with the G.Specimen preparation. BALs were placed in a 50-ml Fal-

con tube (Becton Dickinson Labware, Oxnard, Calif.) andcentrifuged for 15 min at 2,000 x g in an IEC Centra 7centrifuge (International Equipment Co., Needham Heights,Mass.) that was fitted with a fixed-angle rotor. The rotor wasallowed to come to a stop without applying the brake toavoid resuspending any sediment. All but the bottom 5 ml ofthe supernatant was aspirated and transferred to anothertube and held as excess for possible further studies. APasteur pipette was used to aspirate the sediment plusapproximately 1.0 ml of the remaining 5 ml of fluid. Thismaterial was transferred to a clean 15-ml centrifuge tube,gently mixed with the Pasteur pipette, and then used toprepare the slides.To prepare the slides, a drop of the concentrated specimen

was spread over approximately 1 cm2 of a microscope slide.When the specimen was thick or mucoid, the drop was

spread over the entire slide, with care being taken not tomake the smear too thick. Several slides were preparedsimultaneously, depending on the number of stains to beemployed, with a few spare slides prepared for any repeatstains which might be needed. The slides were dried on a

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heating block at 50 to 55°C and removed as soon as they haddried. Flaming or further heat fixing of the slides was notnecessary. Staining could be started as soon as the slides haddried.Lung biopsies. Touch preparations of approximately 100

specimens of pulmonary tissue (open-lung biopsies andtransbronchial biopsies) were also examined with thesestains. After the touch preparations had dried, the slideswere processed in the same manner as the slides of BALs.Approximately one-half of the slides were stained by theinitially used TolB, and the rest were stained by the MtolB.Twenty-two specimens were stained with the GW, and allwere stained with the G.

Staining. MToIB. MTolB was a modification of the TolB ofChalvardjian and Grawe (2) and used the sulfation reagentemployed in the cresyl echt violet stain for P. carinii ofBowling et al. (1). MTolB required the following reagents. (i)For sulfation reagent, 45 ml of glacial acetic acid was pouredinto a Coplin jar which had been placed into a plastic tubfilled with cool tap water (not below 10°C). A 15-ml portionof concentrated sulfuric acid was slowly added with a glasspipette, being careful not to produce splashing. The solutionwas gently mixed with a glass rod. The Coplin jar was thensealed with petroleum jelly. The sulfation reagent was keptat room temperature and could be used for 1 week. (ii) Fortoluidine blue 0 solution, 0.3 g of toluidine blue 0 (52% dyecontent-0. 16 g of dye) (Roboz Surgical Instrument Co., Inc.,Washington, D.C.) was dissolved in 60 ml of distilled waterto which was added 2.0 ml of concentrated hydrochloricacid, followed by 140 ml of absolute ethyl alcohol. Thesolution was stored at room temperature and could be usedfor 1 year. Staining was performed with all solutions inCoplin jars. (iii) For other reagents, two Coplin jars eachwere needed for 95% ethyl alcohol, absolute ethyl alcohol,and xylene. The 95% ethyl alcohol solutions were changedwhen they were no longer clear or darker than a pale blue.The absolute alcohol and xylene solutions were changedmonthly or sooner when they became cloudy or dark blue.The MTolB staining procedure was as follows. (i) Withforceps, slides were placed in the sulfation reagent for 10min. The reagent was mixed with a glass stirring rod imme-diately after insertion of the slides and again after 5 min. (ii)The slides were removed from the sulfation reagent withforceps, placed in a glass slide holder, and washed gentlyunder cold running tap water for 5 min. The excess waterwas then drained. (iii) The slides were placed in toluidineblue 0 for 3 min. (iv) The slides were dipped in and out of95% ethyl alcohol (in two Coplin jars) for approximately 10s until clean, with most of the blue dye being removed in thefirst jar. (v) The slides were dipped in and out of absoluteethyl alcohol (in two Coplin jars) for approximately 10 s forfurther decolorizing. (vi) The slides were dipped in and outof xylene (in two Coplin jars) for approximately 10 s untilclean. (vii) The back and frosted areas of the slides werewiped dry with a paper towel. With a sterile glass Pasteurpipette, a small amount of Permount (Fisher Scientific Co.,Fair Lawn, N.J.) was placed along a long edge of a largecover slip (20 by 50 mm) which was then placed on the slidewhile the specimen area was still moist with xylene. (viii)The slides were examined with 20x and 40x objectives;examination under oil was not necessary but could be doneonce the cover-slipped slides had dried. The staining proce-dure itself took approximately 20 min. Up to 4 slides couldbe stained readily simultaneously.

Recently, we further simplified the staining procedure. Wenow use only one washing in 95% ethyl alcohol and one in

absolute alcohol. We also substituted Xyless (ColumbiaDiagnostics, Inc., Springfield, Va.) for the xylene, as Xylessmay be a less hazardous material and many find its odormore pleasant. The Xyless is allowed to evaporate from theslide almost completely before a cover slip is applied.

Prior to the use of the MTolB, slides had been preparedwith the TolB of Chalvardjian and Grawe (2). The principaldifference between the MTolB and the TolB was the replace-ment of the sulfuric acid-ether sulfation reagent of the TolBwith the sulfuric acid-acetic acid sulfation reagent of theMTolB. The GW was performed by the method of Rosen etal. (9), and the G was done by traditional methods (8) with a1:1 solution of 95% ethyl alcohol and acetone as thedecolorizer. The MMB was essentially the same as that usedby Macher et al. (A. M. Macher, S. M. Gersch, and V. J.Gill, Abstr. 22nd Intersci. Conf. Antimicrob. Agents Che-mother. 1982, abstr. no. 582, p. 168); approximately 35specimens were examined with this stain.

Controls. When available, touch preparations of rat lungwith PCP were used as controls. Otherwise, slides preparedwith a drop of a light suspension of Candida albicans wereused as controls.

RESULTSStains. G and MMB stained well only what is thought to be

the sporozoite form of P. carinii. These forms are containedwithin circular structures approximately 5 ,um in diameter,which are often surrounded by a thin, clear halo. Thesporozoites themselves appear as up to eight distinct bodieswith a darkly staining center surrounded by a clear zone.With G, the structures visualized stained a light red; thesame structures stained light blue with MMB. Very rarelywere many sporozoite-containing cysts seen; usually only afew were present, and often these were extremely difficult tofind. In many specimens, none were found even with diligentsearching. In a typical preparation from an AIDS patientwith PCP, with both stains there was a frothy, dirty back-ground within which negative images of structures sugges-tive of P. carinii cysts could sometimes be visualized.Occasionally, presumed P. carinii cysts appeared diffuselygranular with G and MMB. There might be many macro-phage-like mononuclear cells present, but polymorphonu-clear leukocytes were generally scarce. Photographs of P.carinii stained by G have been previously published (4, 5).GW (Fig. 1) stained what is thought to be the cyst form asdark blue to black structures approximately S ,um in size,with a circular or cup-shaped appearance. The cysts mightappear somewhat grainy, but no definite internal structurescould be visualized. The sporozoites either did not stain orcould not be distinguished morphologically from the cystforms. The background appeared generally pinkish, but cellnuclei might either stain pink or retain the crystal violet tovarious degrees and might sometimes be confused with P.carinii cysts. Both toluidine blue 0 stains also apparentlystained only the cyst forms. Results with the TolB are shownin Fig. 2 and 3. In Fig. 2, the cysts are lavender in color butare somwhat grainy in appearance; much background mate-rial is also evident. In Fig. 3, structures thought to be P.carinii cysts have a honeycombed appearance, and theoutlines of individual cysts are quite faint. The MTolB isillustrated in Fig. 4. The cyst forms appear as lavenderstructures approximately 5 ,um in diameter, and many arecup-shaped. The cyst outline is distinct, and the internalregion stains uniformly. The cysts were frequently observedin clusters, and they did not bud. Very little backgroundmaterial was seen with this stain, particularly with touch

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ADVANTAGES OF A MODIFIED TOLUIDINE BLUE 0 STAIN

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FIG. 1. GW. The small, dark structures are P. carinii cysts. Thisis an extreme example of the interpretive difficulties sometimespresented by this stain (magnification, x460).

preparations. Both toluidine blue 0 stains stain yeasts andother fungal elements. Nonbudding yeast cells might appearvery similar to P. carinii cysts, although the yeast cells mightappear more oval in outline.

Correlation of BAL MTolB results with tissue stain results.From January 1984, when the MTolB began to be usedroutinely, to July 1985, on 23 occasions when a trans-bronchial biopsy was positive in the histology laboratory forP. carinii, the corresponding BAL was positive on 21occasions (91%). Of these procedures, 20 were performed onAIDS patients, and 3 were performed on non-AIDS patients.One AIDS patient and one non-AIDS patient each had anegative BAL. Five patients in whom a correspondingtransbronchial biopsy was negative had a positive BAL; fourwere AIDS patients, and one was a non-AIDS patient. Ninepatients in whom a transbronchial biopsy was not done hada positive BAL; one of these patients was still positive whenpulmonary tissue was examined at autopsy. Five of thesenine patients were AIDS patients; four were non-AIDSpatients. There were no patients with a negative BAL ortransbronchial biopsy who subsequently had an open-lung

FIG. 3. TolB. Presumed P. carinii cysts showing honeycombedstructure. Such structures are very difficult to distinguish frombackground debris (magnification, x460).

biopsy that was positive or were found to be positive for P.carinii upon autopsy. Our histology laboratory routinelyexamines transbronchial biopsies with the following stains:hematoxylin and eosin, Grocott methenamine silver, peri-odic acid-Schiff, Giemsa, Fite, and toluidine blue 0.

DISCUSSION

In a study of the use of BAL in AIDS patients (11), PCPwas diagnosed by BAL in 25 of 27 cases (93%) in which atransbronchial biopsy was positive at the same time; in anadditional patient, the BAL was positive but thetransbronchial biopsy was negative. In another study of theuse of BAL in immunosuppressed patients (12), PCP wasdiagnosed by BAL in 18 of 22 cases (82%) and in oneadditional case in which the transbronchial biopsy wasnegative. Because of the clinical status of the patients in thatstudy, transbronchial biopsy could only be done in 12 of thecases. In both these studies from other institutions (11, 12),the staining procedures used included GW and thePapanicolaou stain, but the relative utility of the differentstains for the diagnosis of PCP was not assessed.

In a previous study from our institution, it has been shown

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FIG. 2. TolB. P. carinii cysts. The amorphous background mterial obscures the cyst outlines. Background staining is sometimeseven more prominent with TolB than this example illustrates (mag-nification, x460).

FIG. 4. MtolB. P. carinii cysts stain clearly and stand outprominently, as very little background material is present. Notetheir characteristic lavender color (magnification, x460).

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that BAL in AIDS patients with PCP has nearly the samesensitivity as transbronchial biopsy (7). In that study, P.carinii cysts were seen in the BAL specimen in 16 of 18specimens in which there was a corresponding trans-bronchial or open-lung biopsy in which the organisms wereseen histologically. The sensitivity of BAL for the diagnosisof PCP in that study was 89%. The staining procedure usedwas TolB.

Since January 1984, with the MTolB we have found theBAL positive on 21 occasions of 23 in which the correspond-ing transbronchial biopsy was positive (91%). In addition, infive patients in whom the corresponding transbronchialbiopsy was negative, the BAL was positive. Nine patients inwhom a transbronchial biopsy was not done had a positiveBAL.BAL is a relatively benign procedure and is less invasive

than transbronchial biopsy or open-lung biopsy. Few com-plications are attributable to BAL alone. In our experience,all patients develop a transient increase in pulmonary infil-trate at the site of the BAL after the procedure. Transientdecreases in the partial pressure of oxygen in arterial bloodand transient temperature elevations may also have beencaused at least in part by the BAL. The technical aspects ofthe procedure are of great importance in obtaining an ade-quate specimen from the alveoli themselves. If the tip of thebronchoscope is not wedged sufficiently tightly and if thereturn of lavage fluid is not satisfactory, such material as isobtained may simply reflect washings from the bronchusitself and not material obtained from the alveoli in which themajority of P. carinii are found. A negative BAL for P.carinii does not rule out PCP; other diagnostic procedures,such as open-lung biopsy, may be necessary. This is partic-ularly true for non-AIDS patients, in whom the organismburden even prior to therapy may be considerably lowerthan in most AIDS patients. AIDS patients who have beentreated may also have specimens in which very few orga-nisms can be seen. The overall sensitivity of BAL for thediagnosis of PCP appears to be in the range of 90%,compared with transbronchial biopsy. However, it shouldalso be noted that occasionally BAL is diagnostic for PCP,while the concomitantly performed transbronchial biopsyshows no P. carinii organisms. Such discrepancy is probablydue to sampling a larger number of alveoli by BAL than bytransbronchial biopsy.One or more forms in the life cycle of P. ccarinii can be

visualized with all the stains described above. However, inour experience the utility of G and MMB is low, as thesestains reveal clearly only sporozoite-containing cysts. OtherP. carinii cysts, presumably at a different stage in the lifecycle of the organism, are often not clearly discernable witheither stain. Therefore, we feel that neither G nor MMB isuseful as a routine diagnostic stain for PCP. GW and TolBboth stain the cysts of P. carinii, but in our opinion bothstains share several disadvantages. Both stains employ re-agents which are difficult and somewhat hazardous to pre-pare, requiring the use of a fume hood. In addition, muchbackground material is present, which can make interpreta-tion of the slides difficult. The staining of cell nuclei by GWmakes screening of such slides at a low power impossible.Although the sulfation reagent is supposed to eliminatebackground material in both the TolB and the MTolB, wefound the sulfation reagent in the TolB to be significantly lesseffective in this regard. Because of the problems withreading some of the TolB-stained slides, we investigated thefeasibility of modifying the stain. The principal change wasin the sulfation reagent, and we now use the sulfation reagent

used in the histopathology laboratory for the preparation oftissue sections stained by toluidine blue 0. Use of theMTolB resulted in much cleaner backgrounds, especiallywith touch preparations, in which almost all backgroundmaterial was removed. Lavage specimens stained with theMTolB were also cleaner than those stained with the TolB;while some crystallinelike purplish background material wasusually visible, this was less of a problem than with theTolB. Even with the MTolB, a second and rarely a third slidemust sometimes be prepared if the background stains toodarkly or if only a few questionable organisms are seen.Often all that is necessary is to stain another alreadyprepared slide; more rarely it is necessary to dilute thespecimen and prepare another slide.The sulfation reagent digests away most of the background

material in both lavages and lung touch preparations butdoes not affect either fungal elements or P. carinii. Thetoluidine blue 0 stains the residual material remaining on theslide. It may not be necessary to adjust the toluidine blue 0reagent for dye content; we have not investigated this point,but the reagent used by Chalvardjian and Grawe (2) had asignificantly higher dye content than that used by us. Thealcohol and xylene washing procedures remove excesstoluidine blue 0 from the slide. It has been our impressionthat placing the cover slip on the slide while it is still moistwith xylene produces a brighter, more easily read slide, butwe have not thoroughly examined the effect of omitting thecover slip on the readability of the slide. (When using Xylessinstead of xylene, we allow the slide to become nearly drybefore applying the cover slip.)

After using the TolB for several months, we accumulatedan increasing number of cases in which the slides were foundto be essentially uninterpretable because of the presence ofstructures which resembled P. carinii cysts but were suffi-ciently atypical in appearance that an unequivocally positivediagnosis of PCP could not be made. In some other cases, P.carinii was difficult to detect because of the presence ofbackground debris. The MTolB produced excellent resultswith control slides of rat lung tissue from animals with PCP.The stain was found to work very well on clinical specimensand was then instituted as our routine laboratory stainingprocedure for detection of P. carinii cysts. We did notperform a parallel evaluation of MTolB and TolB but havenot had any further problems with ambiguous slides sinceemploying the new stain, except for having to repeat thestain occasionally when the specimen has been applied toothickly to the slide. The sensitivity of MTolB for the diag-nosis of PCP on BALs as compared with the histologicaldiagnosis on transbronchial biopsy, 91%, is essentially thesame as previously reported with TolB (89%).

P. carinii cysts typically occur both singly and in smallclusters of closely associated organisms. When only singlecysts are seen and in specimens containing yeast cells, greatcare must be exercised in making a diagnosis of P. carinii. Inspecimens containing yeast cells, we think that a positivediagnosis of P. carinii cannot be made without finding atleast one cluster of the characteristic cysts of P. carinii.We have not examined ground preparations of pulmonary

tissue with the MTolB and so cannot comment on itsusefulness on such material.

Several stains which we have not evaluated, including theGiemsa (10), Wright (3), acridine orange (13), methenaminesilver (6), and cresyl echt violet (1), have been used for thedetection of P. carinii organisms. The Giemsa, Wright, andacridine orange stains do not stain P. carinii cysts well andpresumably would have the same disadvantages as G and

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MMB. A rapid methenamine silver stain has been described(6), but this stain requires numerous reagents, some of whichare difficult to prepare or must be prepared fresh for each setof slides to be stained. Unlike the experience of Bowling etal. (1), we have found toluidine blue 0 to be quite satisfac-tory, and we also have not found it necessary to use acounterstain. Of the stains we have employed, we havefound the MTolB the most useful in a diagnostic microbiol-ogy laboratory for the diagnosis of PCP.The turnaround time is quite rapid with MTolB; results

may be available within an hour of receipt of a BAL in thelaboratory. The reagents are easy to prepare, and the stainitself is simple to perform and usually easy to read. Themicrobiology laboratory can thus often make a diagnosis ofPCP very quickly as compared with the pathology labora-tory, as results with silver stains or special stains on tissuesections are often not available until the next day. Thissignificantly more rapid provision of results can often bevery helpful for the optimal management of critically illpatients.

ACKNOWLEDGMENTSWe thank Dorene Dalessandro and Pamula Simms for excellent

secretarial assistance.

LITERATURE CITED1. Bowling, M. C., I. M. Smith, and S. L. Wescott. 1973. A rapid

staining procedure for Pneumocystis (arinii. Am. J. Med.Technol. 39:267-268.

2. Chalvardjian, A. M., and L. A. Grawe. 1963. A new procedurefor the identification of Pneinoc-vstis (alrinii cysts in tissuesections and smears. J. Clin. Pathol. 16:383-384.

3. Domingo, J., and H. W. Waksal. 1984. Wright's stain in rapid

diagnosis of Pneutnocystis carinii. Am. J. Clin. Pathol.81:511-514.

4. Felegie, T. P., A. W. Pasculle, and A. Dekker. 1984. Recognitionof PneitinocYstis carinii by Gram stain in impression smears oflung tissue. J. Clin. Microbiol. 20:1190-1191.

5. Macher, A. M., J. Shelhamer, J. D. MacLowry, M. Parker, andH. Masur. 1983. Pnemnocvstis (arinii identified by Gram stainof lung imprints. Ann. Intern. Med. 99:484-485.

6. Mahan, C. T., and G. E. Sale. 1978. Rapid methenamine silverstain for PneutnocYstis and fungi. Arch. Pathol. Lab. Med.102:351-352.

7. Ognibene, F. P., J. Shelhamer, V. Gill, A. M. Macher, D. Loew,M. M. Parker, E. Gelmann, A. S. Fauci, J. E. Parrillo, and IH.Masur. 1984. The diagnosis of Pnelurnocvstis carinii pneumoniain patients with the acquired immunodeficiency syndrome usingsubsegmental bronchoalveolar lavage. Am. Rev. Respir. Dis.129:929-932.

8. Paik, G. 1980. Reagents. stains, and miscellaneous test proce-dures. p. 1016-1017. In E. H. Lennette, A. Balows, W. J.Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbi-ology. 3rd ed. American Society for Microbiology, Washington,D.C.

9. Rosen, P. P., N. Martini, and D. Armstrong. 1975. Pneumocystis(arinii pneumonia. Am. J. Med. 58:794-802.

10. Smith, J. W., and M. S. Bartlett. 1982. Laboratory diagnosis ofPneutnocv'stis carinii infection. Clin. Lab. Med. 2:393-406.

11. Stover, D. E., D. A. White, P. A. Romano, and R. A. Gellene.1984. Diagnosis of pulmonary disease in acquired immunedeficiency syndrome (AIDS). Am. Rev. Respir. Dis. 130:659-662.

12. Stover, D. E., M. B. Zaman, S. I. Hajdu, M. Lange, J. Gold, andD. Armstrong. 1984. Bronchoalveolar lavage in the diagnosis ofdiffuse pulmonary infiltrates in the immunosuppressed host.Ann. Intern. Med. 101:1-7.

13. Thomson, R. B., Jr., and T. F. Smith. 1982. Acridine orangestaining of Pneiunocvystis carinii. J. Clin. Microbiol. 16:191-192.

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