Vol. 17, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1983, p. 717-7210095-1137/83/050717-05$02.00/0Copyright C 1983, American Society for Microbiology

Dark-Field Microscopy for Detection of Malaria in UnstainedBlood FilmsGHAZI A. JAMJOOMt

Department of Pathology and Microbiology, College of Medicine, King Saud University, Rivadh, SaudiArabia

Received 9 July 1982/Accepted 21 September 1982

Pigment present in different forms of Plasmodium species infecting humansexhibits light scattering when blood films are viewed by dark-field microscopy.This facilitates the detection of parasite-infected cells owing to their brightlyilluminated appearance. The technique was described long ago for the detection ofcertain highly pigmented forms of malarial parasites such as schizonts andgametocytes but has not found an application in routine diagnosis. Here,modifications are described which allow the detection of all forms of humanmalarial parasites, including ring forms, in unstained blood films. The techniqueoffers the distinct advantages of rapid diagnosis, increased sensitivity, andadaptability to field work.

Routine laboratory diagnosis of malaria stillrelies totally on the microscopic examination ofblood films stained with the conventional stainssuch as Giemsa, Wright stain, Leishman stain,etc. These techniques require examination of theslide under oil (magnification, x1,000), which istime consuming. Conventional thin and thickfilms both have certain advantages and disad-vantages. Thin films only allow the scanning of arelatively small volume of blood and are there-fore less sensitive than thick films. They havethe advantage, however, of clearly revealing thedetailed morphology of the malarial parasites,which is needed for accurate identification ofspecies. Thick films require some experience toprepare and tend to be burdensome for labora-tories in areas where malaria is not encounteredregularly, especially where automated equip-ment is used for staining thin films. Moreover,owing to the distortion of certain forms, such asgametocytes and schizonts, accurate identifica-tion of species on the basis of the thick filmsalone may be difficult.

Several new procedures have been recentlyemployed to increase the sensitivity of labora-tory diagnosis of malaria. These include, forexample, fluorescent-antibody staining of bloodfilms (2, 7), tissue staining with fluorescentantibody or immunohistological techniques (4),and detection of malarial antigen by a modifiedradioimmunoassay (1) or enzyme immunoassay(5). The use of these techniques, however,seems to be suitable only for specialized labora-tories owing to their technical requirements. Theculture of the malaria parasite (8) also does not

t Present address: College of Medicine, King Saud Univer-sity, Abha, Saudi Arabia.

seem to be feasible at present as a simplediagnostic procedure. The detection of a sero-logical response to malaria (1, 3) may be usefulin retrospective diagnosis or survey, but not inthe diagnosis of a current infection.We have attempted to facilitate the micro-

scopic detection of different forms of malarialparasites by using the technique of dark-fieldmicroscopy. The technique was described sev-eral years ago for the detection of highly pig-mented forms of malarial parasites such as schi-zonts and gametocytes (6, 9). However, none ofthe described methods has found general recog-nition in the routine diagnosis of malaria. Thismay be due to the fact that the technique re-quires certain modifications to allow the easyrecognition of certain forms of malarial para-sites, especially ring forms, which contain small-er amounts of aggregated pigmented particles,particularly at early stages of development.These modifications, which are described here,also reduce artifacts such as dust and glassparticles, which can be bothersome and confus-ing. I here describe the use of this techniquewith different preparations of unstained bloodfilms in which it is possible to detect rapidly allthe recognized forms of malarial parasites, in-cluding ring forms. The advantages that thetechnique offers in simplifying and expeditingthe diagnosis of malaria are discussed.(A preliminary presentation of part of this

work was given at the Sixth Saudi MedicalConference, Jeddah, Saudi Arabia, 15 to 18March 1981.)

MATERIALS AND METHODS

Blood was obtained either directly from a fingerprick or from routine hematology tubes of anticoagu-

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FIG. 1. Use of inverted raised cover slip for view-ing thin film present on the lower side of the raisedcover slip. The same technique was also used forviewing wet-mount specimens. (A) Mounting medium;(B) specimen; (C) cover slips; (D) slide. If the coverslips supporting the raised specimen are not thickenough, a stack of two or three cover slips can beused.

lated blood. Thin blood films were spread on longcover slips (22 by 50 mm). After drying, each coverslip was placed upside down on a slide on which twoblank cover slips (22 by 22 mm) were placed to raisethe cover slip containing the specimen in a bridge-likemanner (Fig. 1). The specimen was mounted in buff-ered glycerol (90 ml of glycerol, 10 ml of H20, 0.16 g ofNa2CO3, 0.0715 g of NaHCO3) by adding the glycerolat one side so that it seeped under the cover slip. Thisprevented the formation of any bubbles.Thick films were prepared by making a medium-

thick film of blood on a slide. After drying, the filmwas lysed by dipping in distilled water, allowed to dry,and mounted in buffered glycerol. For more stablepreparations, films were fixed for 5 min in methanolbefore mounting in buffered glycerol. A few minuteswere allowed for glycerol to penetrate methanol-fixedthick films so as to subdue background illumination.

Concentrated preparations were made by mixing 0.5ml of blood with 1.5 ml of distilled water and shakinggently until complete lysis was obtained. The speci-men was then centrifuged at 3,000 rpm for 10 min. Thesupernatant was decanted, and the sediment was sus-pended, spread on a slide, dried, and mounted inbuffered glycerol. For stable preparations the film was

fixed for 5 min in methanol before mounting.Wet mounts were prepared by mixing a drop of

blood with 5 drops of glycerol containing 2.5% sodiumdodecyl sulfate and 1% Nonidet P-40 in distilled water.If complete lysis did not occur, the concentration ofsodium dodecyl sulfate was increased slightly. Twocover slips were placed on the sides of the specimen,and a third one was placed on top (Fig. 1). Thisallowed focusing on the middle part of the specimen,away from artifacts present on glass surfaces. If amore concentrated preparation was desired, a drop ofblood could be mixed with 10 to 15 [lI of a 10% solutionof sodium dodecyl sulfate in water before examinationas described above. All of the concentrated prepara-tions described above were suitable for the detectionof gametocytes and schizonts but were not particularlyreliable for the detection of ring forms, especially theweakly pigmented ring forms sometimes encounteredwith Plasmodiumfalciparum. For microscopy, a stan-dard transmission light microscope (Reichert, Diavarmodel) was used for all investigations. Dark-fieldattachments included a condenser (no. 67297, A-1,18/1.42) and x40 and x100 objectives fitted with irisdiaphragms. Viewing was done at magnifications ofx250 and x400, but photography was done at x1,000(under oil) unless specified otherwise. Cover slipsprepared in the field could be conveniently transportedby taping, face down, to pages of a clean notebook.

RESULTSFigure 2 shows the appearance of P. falci-

parum gametocytes and ring forms in an un-stained thin film prepared on a long cover slip,inverted, and mounted in glycerol while beingraised from the slide by the support of twosmaller cover slips (Fig. 1). This inverted raisedcover slip arrangement avoids most of the arti-facts present on the surface of the slide and themounting medium since it renders these artifacts

FIG. 2. P. falciparum in dark-field microscopy of unstained thin blood films. (A) Ring forms and microgame-tocytes; (B) macrogametocytes.

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FIG. 3. P. ralariae in dark-field microscopy of unstained thin blood films. (A) Ring forms; (B) trophozoites.Magnification, xl ,OOO.

out offocus. Note that the bright pigmentation inthe crescent-shaped gametocytes is concentrat-ed in the middle area. In some specimens, P.falciperum ring forms may exhibit less pigmen-tation but can still be clearly seen by this tech-nique. Double rings and applique forms are alsoclearly observed.

Figure 3A shows the ring forms of Plasmodi-um malariae in unstained thin-film preparations.These ring forms are larger than the ring formsof P. falciparum. All P. malariae ring formsobserved so far exhibited bright pigmentation.Figure 3B shows trophozoites of P. malariae,which are highly pigmented.

Figure 4 shows schizonts of Plasmodium vi-vax. This form is also highly pigmented, and thepigment is present in discrete rodlets scatteredover the whole of the infected erythrocyte. Theenlargement of the infected erythrocyte is alsoevident.More concentrated preparations such as thick

films, wet-mount preparations, and preparationsconcentrated by centrifugation after lysis of thecells with water were also used to increase thesensitivity of this method in detecting parasites.Highly pigmented forms could easily be recog-nized in these preparations (Fig. 5). Identifica-tion of ring forms, particularly when present inlow numbers or when they contained weakpigmentation, was more difficult. The thin-filmpreparation described above is more suitable forthat purpose. In addition, artifacts may occa-sionally be confused with ring forms, but notwith the more characteristic schizont and game-tocyte forms, in such concentrated preparations.

DISCUSSIONThe illuminated appearance of malarial para-

sites under dark-field microscopy is probably aresult of Tindall scattering of the incident light.This property is associated with pigmented par-

FIG. 4. P. vivax schizonts in dark-field microsco-py. Magnification, x1,000. Unstained thin films were

prepared as described in the text. If desired, thin filmsstained routinely with Giemsa or Leishman stain can

also be examined by dark-field microscopy for thecharacteristic schizonts of P. vivax. Both stained andunstained preparations give a similar appearance, butstaining gives them a sky-blue color.

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ticles in the parasites which are seen as brownrodlets with conventional staining. The pigmentis known to be due to the presence of hemozoin,a metabolic degradation product of hemoglobin(4).The technique described here relies on a rou-

tinely used method of microscopy and requiresvery few additions of equipment or material.One of the main advantages of the technique isits speed since examination is immediate anddoes not require any fixation or staining. Thismakes the technique particularly suitable forfield work. The technique also allows speedierscanning of relatively larger volumes of bloodunder lower magnifications (x250 or x400),which should make it more sensitive than rou-tine bright-field methods. Although a detailedcomparison between the two techniques is stillunder way, our experience so far indicates thatundethedark-field technique is very sensitive in

_ j _ j = detecting malaria-infected specimens. Althoughthe criteria applied for species identification arethe same as those used with conventional stain-ing methods, the ease of detection of certainpigmented forms such as schizonts and gameto-cytes may facilitate species identification withspecimens containing only a few of these forms.Of the different methods described above forviewing blood specimens, only the thin-filmpreparation clearly reveals the morphology ofthe parasite and of the infected erythrocyte. Thispreparation is most suitable for use while learn-ing the technique and for identification of theinfecting species, and therefore, it is most suit-able for routine use. P. vivax, for example, ischaracterized by its brightly pigmented schi-zonts filling the enlarged erythrocytes. P. malar-

Nj..,,,'..14i iae is characterized by its brightly pigmentedtrophozoites, which occupy erythrocytes of nor-mal size. P. falciparum is distinguished by itscrescent-shaped gametocytes, in which the pig-ment is often concentrated in the middle, andalso by its smaller and finer rings. Double infec-tions and high multiplicities of infection arecharacteristic of P. falciparum. Our experiencewith Plasmodium ovale is still minimal, butparasites in a suspected infection of this speciesalso exhibited bright forms.

Although the technique described here is sim-ple, it no doubt requires familiarity with dark-field microscopy, the proper microscopic setup,and some experience to avoid the confusioncaused by artifacts. The examination of a num-ber of known positive and negative specimens is

FIG. 5. Various forms of malarial parasites in con- therefore initially required.centrated preparations of lysed blood. (A) P. falci- ACKNOWLEDGMENTSparum gametocytes (thick film); magnification, I am grateful to Aqeela Zaafaran, Nour Al Houda Mustafa,x1,000. (B) P. malariae trophozoites (wet mount); Ali Mahmood, Mohammed Mahjoub, Mohammed Gaddoura,magnification, x400. (C) P. fialciparuni ring forms Ahmed Khairat, Abdul Jaleel, and Ahmed Yousif for helpful(highly pigmented); magnification, x 1,000. discussions. I am also grateful to the staff members of the

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Ministry of Health Branch in Gizan, Saudi Arabia, and AbuAreesh Hospital, particularly Emmy Rodriguez, for providingme with specimens.

LITERATURE CITED

1. Avraham, H., J. Golenser, D. T. Spira, and D. Sulitzeanu.1981. Plasmodium falciparum: assay of antigens and anti-bodies by means of a solid phase radioimmunoassay withradioiodinated staphylococcal protein A. Trans. R. Soc.Trop. Med. Hyg. 75:421-425.

2. Brooke, M., M. Melvin, and G. R. Healy. 1961. StainingPlasmodium berghei with fluorescein tagged antibodies.Proceedings of the Sixth International Congress on Tropi-cal Medicine and Malaria.

3. Collins, W. E., G. M. Jeffrey, and J. C. Skinner. 1964.Fluorescent antibody studies in human malaria. I. Develop-ment of antibodies to Plasmnodiium7 ,nalariae. Am. J. Trop.Med. Hyg. 13:1-5.

4. Galbraith, R. M., W. P. Faulk, G. M. P. Galbraith, andT. W. Holbrook. 1980. The human materno-foetal relation-ship in malaria. I. Identification of pigment and parasites in

the placenta. Trans. R. Soc. Trop. Med. Hyg. 74:52-60.

5. Mackey, L. J., I. A. McGregor, N. Paounova, and P. H.Lambert. 1982. Diagnosis of Plasmodiurm f(lciparin infec-tion in man: detection of parasite antigens by ELISA. Bull.W.H.O. 60:69-75.

6. Packer, H. 1945. The use of dark field illumination in

studies of malarial parasites. J. Natl. Malaria Soc. 4:331-340.

7. Tobie, J. E., G. R. Coatney, and C. B. Evans. 1961. Fluo-rescent antibody staining of human malaria parasites. Exp.Parasitol. 11:128-132.

8. Trager, W., and J. B. Jensen. 1976. Human malaria para-sites in continuous culture. Science 193:674-675.

9. Wolter, A. 1932. Uber die Schnelldiagnose der Maralia mitHilfe des Dunkelfeldes. Dermatol. Z. 63:69-72.

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