JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1976, p. 258-265Copyright © 1976 American Society for Microbiology

Vol. 4, No. 3Printed in U.S.A.

Examination of Bacterial Flagellation by Dark-FieldMicroscopy

ROBERT M. MACNABDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520

Received for publication 3 May 1976

A method is described for visualizing unstained bacterial flagella by dark-fieldlight microscopy. Since individual filaments can be seen, a genus such as

Salmonella, which is peritrichously flagellated, can readily be distinguishedfrom a polarly flagellated genus such as Pseudomonas. Polarly flagellatedbacteria generally swim much faster than peritrichously flagellated bacteria,and turn by abrupt reversals. The differences in flagellation and motilityprovide diagnostic criteria that may be useful in clinical microbiology.

Among motile rod-shaped bacteria, the num-ber of flagella per cell, and their points of originon the cell (polar versus peritrichous), is a pri-mary taxonomic criterion (9) of clinical signifi-cance (4). The type of flagellation can readily beascertained by electron microscopy, but thistechnique is frequently not available to a clini-cal laboratory. Flagellar staining proceduresfor light microscopy (5) are available, but con-sistent results are not easily obtained.

In this paper, a method is described thatallows the observation by dark-field microscopyof individual unstained flagella on whole cells.Various species of Pseudomonas have been ex-amined as examples of polar flagellation, andSalmonella typhimurium, Bacillus subtilis,and Escherichia coli as examples of peritri-chous flagellation. The substantial differencesin velocity and turning pattern between thetwo classes are also demonstrated and dis-cussed as a taxonomic criterion.

(It is commonly stated in microbiologicaltexts that because bacterial flagella have a di-ameter [typically 15 nm] that is small com-pared with the wavelength of visible light [500nm] they cannot be seen by light microscopy.Such statements are erroneous and are basedon a misconception of the limit set by the wave-length of light. This limit is one of resolution,not of detection. Any particle immersed in amedium of different refractive index will, nomatter how small it is, scatter some light andtherefore in principle be detectable. Two parti-cles separated by less than the wavelength oflight will be seen as a single scattering source,as a result of the resolution limit.)

MATERIALS AND METHODS

Bacterial cultures. These should be grown in liq-uid medium to mid-log phase (optical density at 650

nm, -0.6) and diluted to a suitable concentration formicroscope observation. There should be enoughcells in the field of view for a statistically significantobservation to be made, but they should be suffi-ciently separated to prevent overlap of the flagella ofadjacent cells. If the cells grow in a minimal me-dium they can be observed in it directly. Complexmedia such as nutrient broth give high backgroundscattering, but may be required for growth. In thiscase, better contrast in the microscope will be ob-tained by sedimenting (e.g., in a clinical centrifugefor 10 min) and then gently resuspending the cells ina simple buffer such as 10 mM potassium phosphate,pH 7, plus 100 ,tM potassium ethylenediaminetet-raacetic acid (1) or in Vogel-Bonner citrate medium(12).

Preparation of slides. Because of the very highlight intensity used, particular attention should bepaid to cleanliness of the sample. No cleaning tech-nique can remove glass blemishes; if these seemexcessive, the only recourse is to try a differentsource of slides. For cleaning, we have found a non-foaming spray-on glass cleaner such as Bon-Ami,which is wiped off with tissue after a few seconds, tobe as effective as acid washing procedures. Thecoverslip must be of the thickness (usually 0.17 mm[size 11/2]) for which the microscope objective hasbeen corrected. It can be satisfactorily cleaned byfirm wiping with double layers of dry tissue. Samplesize should be just sufficient to allow the liquid tospread to the area of the cover slip, since excessiveliquid depth lowers image quality by providing abackground of out-of-focus scattering sources. Aloopful (3-mm loop diameter) of sample is about theright amount.

Light source. A high surface brightness is theimportant parameter, rather than overall flux.Short-arc xenon, mercury, or xenon-mercury lampsare suitable. Flagella can be seen using a 75-W arc,but a higher-wattage arc is preferred.An adjustable collimating lens system and spheri-

cal rear mirror are needed and are usually an inte-gral part of commercially available lamp housings.A tempered-glass heat filter is advisable to protectthe microscope optics and the sample. Because of the

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VISUALIZATION OF BACTERIAL FLAGELLA 259

sensitivity of bacterial motility to high-intensityblue light (reference 7, and see Results section be-low) a high-pass yellow filter is also needed forobservation of motility.

Microscope. Any good-quality microscope can beused. The light beam is brought in directly withoutdiffusing filters.The condenser should be an oil immersion dark-

field condenser of high numerical aperture (inner1.2, outer 1.4).

Choice of objective is critical for optimum imagequality. Considerable experimentation indicatedthat a medium power (40 x), dry fluorite objective, ofmoderately high numerical aperture (0.75), gave thebest combination of image brightness, contrast, andfreedom from flare. Specifically, the use of higherpower oil immersion objectives gave inferior imagesbecause of flare from bacterial cell bodies.

Standard eyepieces are used; we usually use 16x,but 10x are satisfactory.

Adjustment of optics. Because of the low scatter-ing power of the flagella, careful adjustments of allelements of the system are necessary to maximizeintensity; most of these will be necessary only dur-ing initial setup. A check list follows: (i) All opticalsurfaces must be kept scrupulously clean. (ii) Thearc must be centered on the collimating lens axis,and the secondary image of the arc from the spheri-cal rear mirror (if the housing has one) superposedon the primary image. (Personnel should acquaintthemselves with precautions against ultravioletlight and other hazards before making these adjust-ments.) (iii) The microscope condenser should becentered on the objective axis, using a precenteredbuilt-in light source if available. A suitable "sam-ple" is a microscope slide, heavily scratched withemery paper; a loopful of water is first added andthen the appropriate thickness cover slip. The oilconnection between the condenser and slide must befree ofbubbles and be copious enough to ensure thatthe entire beam is transmitted. Insufficient oil willmanifest itself as an off-center connection. (iv) Thebeam axis should be centered on the microscopeoptical axis. If a built-in 450 mirror is used, it ofcourse requires that the incident beam be horizon-tal. More flexibility in layout is obtained by the useof a universal mirror (e.g., Zeiss 46 51 07). (v) Thebeam from the housing should be adjusted until it isapproximately collimated, and then the microscopecondenser should be adjusted to give critical illumi-nation of the sample. This adjustment will be recog-nized by a minimum in the central illuminated area,with a corresponding maximum in intensity. Noattempt should be made to achieve uniform inten-sity over the whole field. (vi) Different degrees ofcollimation (slightly convergent, slightly divergent)and, if possible, different distances of the lamp fromthe microscope should be tried; in each case, thecondenser should be adjusted for critical illumina-tion of the central area. When the overall optimumbeam arrangement has been ascertained, it can beused from then on, the only adjustment needed on aroutine basis being the vertical position of the mi-croscope condenser, which must be made with eachnew slide.

Motility measurements. These were made photo-

graphically by measuring the contour lengths of thetracks generated during a 0.5-s exposure time (11),or less accurately by use of a stopwatch to estimatethe time required for a cell to cover a defined dis-tance on an eyepiece reticle. Calibration of the reti-cle is accomplished with a stage micrometer.Summary of optical equipment used in my labo-

ratory. The setup comprised the following: lightsource: Hanovia 500-W xenon arc, 959C-98; lighthousing: Oriel 6140, 48-mm fl.0 collimating lens;power supply: Oriel 6242; heat filter: Optical Indus-tries 03 FHA 023; yellow filter: Optical IndustriesGG495 or Corning 3-69; microscope: Zeiss standardRA; condenser: Zeiss oil immersion dark-field Ul-tracondenser, numerical aperture 1.2/1.4; objective:Zeiss Neofluar 40x, 0.75; eyepiece: Zeiss wide-angleKpl 16x; beam geometry: lamp to collimating lens= 50 mm; lens to condenser = 0.5 m (lens to imageplane in absence of condenser = 1.2 m), i.e., beamslightly convergent. However, note that geometryshould be optimized empirically in any given appa-ratus to give maximum image brightness.

RESULTSBy use of the methods described above, indi-

vidual flagella can readily be seen. Beforegoing on to describe the observations in moredetail, it is necessary to make some commentsabout the difficulty of photographic documenta-tion in this context. No photographic film canmatch the capabilities of the human eye interms of sensitivity, dynamic range, and per-ception of the form of a moving object. All threeof these capabilities are in demand here: (i)sensitivity, because the flagella are weak scat-tering sources; (ii) dynamic range, because thecell body and flagellum differ in brightness byabout three orders of magnitude; (iii) percep-tion of moving forms, because active movementand even Brownian movement interfere withthe formation of a sharp photographic image.Exposure time is therefore a compromise be-tween factors (i) and (iii). For these reasons, aclinical laboratory will probably not wish toattempt photographic documentation, nor isthere any need for it to do so. Photographs aregiven here for illustrative purposes, but it mustbe strongly emphasized that the quality of thedirect visual image is far higher.

Flagellation. The distinction between peri-trichous and polar flagellation should not bebased on the appearance of moving cells, sincethe helical bundle of peritrichous cells tends toform at the pole (Fig. 1). Once it has beenestablished that motile cells are present in theculture, they can be rendered stationary within1 s or so by removal of the protective yellowfilter. Under these conditions, peritrichous fla-gellation is recognized as a pattern of helicalfilaments radiating from the entire cell, as isseen in the photograph of S. typhimurium in

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260 MACNAB

FIG. 1. (A) S. typhimurium during translational movement. The peritrichous flagella form a polar bundle(see cartoon, Fig. 1B) which could be mistaken for polar flagellation. Classifications should therefore be basedon the appearance offlagella on stationary cells. Dark-field photomicrograph taken with high-intensity pulsedxenon arc. Bar equals 5 gm.

Fig. 2. The overall impression of a field of such gella radiating from the polar regions only, ascells leaves the observer in no doubt that fla- can be seen in the photographs ofPseudomonasgellation is peritrichous. stutzeri and Pseudomonas aeruginosa in Fig. 3.

Polar flagellation is seen as one or more fla- Although the point of origin of each flagellum

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VISUALIZATION OF BACTERIAL FLAGELLA 261

cannot, because of scattering from the cell, bedetermined exactly, it can be located muchmore accurately than the photographs suggest.In practice, one has little difficulty in determin-ing whether or not an individual cell is polarlyflagellated, and even less difficulty in making adecision based on a field of many cells. Furtherclassification of cells may be made on the basisof the number of flagella per pole. Thus, inagreement with established classification (3),P. stutzeri and P. aeruginosa are observed to bepredominantly monotrichous, although cellswith two polar flagella were quite frequentlyobserved (as in Fig. 3D).These statements about observation of polar

flagellation need to be qualified in one regard:Pseudomonas species sometimes possess veryshort flagella (ca. one complete helical turn),and under these circumstances scattering fromthe cell body makes flagellar observation verydifficult even in cultures that have high motil-ity.

Motility. The motility of Pseudomonas spe-cies differs markedly from that of peritri-chously flagellated cells in two respects, veloc-ity and turning pattern. The differences, de-scribed below, are being presented here becausethey are sufficient to constitute a useful diag-nostic criterion that is not presently utilized inclinical microbiology.For unknown reasons, the swimming veloc-

ity of vigorously motile cultures ofPseudomo-nas species is much higher than that of peritri-chously flagellated cells (8). Velocity measure-ments of P. stutzeri and P. aeruginosa madephotographically in the present study areshown in Table 1, along with data obtained in asimilar manner for S. typhimurium in a pre-vious study (6). Velocities comparable to, orlower than, these for S. typhimurium havebeen noted for other peritrichously flagellatedspecies, such as E. coli (2), Serratia marcescens(8), and B. subtilis (R. Macnab, unpublishedwork).

Semiquantitative data were obtained with aneyepiece reticle and stopwatch. Measurementsmade in this way on 10 cells each ofS. typhimu-rium and P. stutzeri yielded mean velocitieswith standard deviations of the mean of 25.4(1.5) and 51.5 (3.4) ,um per s, respectively, infair agreement with the more accurate photo-graphic data given in Table 1.

Peritrichous organisms such as E. coli or S.typhimurium terminate a period of transla-tional movement by chaotic tumbling (2, 6) as-sociated with dispersal of the coordinated bun-dle of flagella (7). Pseudomonas species, on theother hand, usually undergo abrupt double re-

versals (10), as can be seen in Fig. 4. Thisabrupt reversal does not appear with peritri-chously flagellated cells although some may,after tumbling, fortuitously travel back alongtheir path.

DISCUSSIONThe procedures described allow identification

of the type of flagellation possessed by motilebacterial cultures, without resort to the use ofstains or electron microscopy. The example cho-sen in this paper is the differentiation betweenS. typhimurium, which is peritrichously flagel-lated, and Pseudomonas species, which are po-larly flagellated, but the method is applicablewhenever knowledge of morphology of flagella-tion (both the origin of the flagella on the cellbody, and the number, length, and shape offlagella) would be of assistance in identifying aculture.The ability to see individual, unsheathed,

unstained flagella (7) is a significant advancein the power of dark-field light microscopy, yetit uses relatively standard microscopic equip-ment. For example, I have tested existing fluo-rescence microscopic equipment (Leitz Dialuxmicroscope, oil immersion dark-field con-denser, 200-W mercury arc lamp) in a clinicallaboratory with satisfactory results.The operating skills can be picked up quickly

by anyone familiar with the use of the lightmicroscope. No unusual visual acuity is neededfor the recognition of peritrichous flagellation;many individuals without any prior experienceor skills have done so when visiting my labora-tory. Polar monotrichous flagellation is moredemanding for two reasons. (i) The probabilityof one flagellum being suitably oriented islower than the probability of a representativeselection of flagella being suitably oriented inthe peritrichous case. (ii) The flagella tend to bemuch shorter, and therefore are more easilyobscured by flare from the cell body. In spite ofthese difficulties, an observer with average vis-ual acuity can, with a little practice, recognizepolar flagellation in most cases. In cases wherethe observer fails to note flagella in motile cul-tures, this failure is in itself an indication ofpolar flagellation (provided control cultureshave established that the optical equipment isadequate) since peritrichous bacteria withshort flagella are poorly motile (13), presum-ably because the flagella cannot extend past thecell and form the bundle.As can be seen from Table 1, the velocity of

pseudomonads is approximately twice that ofperitrichously flagellated species. This differ-ence is sufficiently marked to be obvious on

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VISUALIZATION OF BACTERIAL FLAGELLA 263

c F

FIG. 3. Polar flagellation in pseudomonads. (A) Stationary cell ofP. stutzeri. (B) Same negative [overex-posed during printing] to show cell outline. (C) Cartoon combining information from (A) and (B). (D-F) Asfor (A-C) but with P. aeruginosa (note presence of two flagella at pole, although species is predominantlymonotrichous). Bar equals 5 ,um.

FIG. 2. Peritrichous flagellation, exemplified by S. typhimurium. Dark-field photomicrograph of station-ary unstained cells taken with continuous xenon arc. (A) Exposure chosen to show flagella. (B) Shorterexposure to show outlines of cell bodies. (C) Cartoon combining information found in (A) and (B). Arrowindicates much brighter image resulting from two flagella lying together, in phase. Bar equals 5 gm.

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264 MACNAB

FIG. 4. Motility tracks ofP. stutzeri illustrating characteristic double-reversal pattern ofpolarly flagellatedcells. Arrows indicate reversals. Exposure time, 2 s. Bar represents 20 ,um. (Similar motility tracks forPseudomonas citronellolis are given in Fig. 1 of reference 10.)

inspection of a slide. The simplest method is aqualitative side-by-side comparison with con-trol cultures, but as was shown in the Resultssection, semiquantitative data may readily beobtained with an eyepiece reticle and a stop-watch. Although mean velocities in the range

of 40 to 70 ,um per s are strong evidence forpolar flagellation, measurements in the rangeof 20 to 30 g.m per s are of course not strongevidence against this type of flagellation, sinceany of a number of adverse conditions couldreduce motility.

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VISUALIZATION OF BACTERIAL FLAGELLA 265

TABLE 1. Swimming velocity ofpolarly and peritrichously flagellated bacteria

Mean velocitya (I±m per s)

Species Type of flagellation No. of cells insample Total sample Upper tenpercentile

Pseudomonas stutzeri Polar 90 48.5 (1.5) 75.1P. aeruginosa Polar 92 58.3 (1.7) 88.2S. typhimuriumb Peritrichous 45 28.8 (0.8) 36.9

a Calculated from photographically recorded motility tracks taken at 24°C. Data from several photo-graphs are included in each sample. Nonmotile cells are excluded from the calculations. Results are givenwith the standard deviation of the mean given in parentheses.

b Data of Macnab and Koshland (6).

As far as turning behavior is concerned,translational movements, followed by tumblingin the case of peritrichously flagellated cellsand by abrupt reversal in the case of polarlyflagellated cells, are easily distinguished withlittle practice, and would seem to be a simplecriterion which has been underutilized, butwhich is of course restricted to cultures that arehighly motile.

ACKNOWLEDGMENTSI wish to acknowledge the assistance of May Ornston and

Daniel Linzer in photographic documentation and motilitymeasurements, and to thank Alexander von Graevenitz forhelpful discussion, provision ofPseudomonas cultures, andthe opportunity to examine cultures with the microscope inhis clinical laboratory.

This research was supported by Public Health Servicegrant AI 12202 from the National Institute of Allergy andInfectious Diseases.

LITERATURE CITED1. Adler, J. 1973. A method for measuring chemotaxis and

use of the method to determine optimum conditionsfor chemotaxis in Escherichia coli. J. Gen. Microbiol.74:77-91.

2. Berg, H. C., and D. A. Brown. 1972. Chemotaxis inEscherichia coli analyzed by three-dimensional track-ing. Nature (London) 239:500-504.

3. Buchanan, R. E., and N. E. Gibbons (ed.), Bergey's

manual of determinative bacteriology, 8th ed., p. 220.1970. The Williams & Wilkins Co., Baltimore.

4. Hynes, M. 1968. Medical microbiology, 9th ed., p. 152-184. Churchill, London.

5. Leifson, E. 1960. Atlas of bacterial flagellation, p. 3-7.Academic Press Inc., New York.

6. Macnab, R., and D. E. Koshland, Jr. 1972. The gra-dient-sensing mechanism in bacterial chemotaxis.Proc. Natl. Acad. Sci. U.S.A. 69:2509-2512.

7. Macnab, R., and D. E. Koshland, Jr. 1974. Bacterialmotility and chemotaxis: light-induced tumbling re-sponse and visualization of individual flagella. J.Mol. Biol. 84:399-406.

8. Schneider, W. R., and R. N. Doetsch. 1974. Effect ofviscosity on bacterial motility. J. Bacteriol. 117:696-701.

9. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg.1970. The microbial world, 3rd ed. Prentice-Hall,Inc., Englewood Cliffs, N.J.

10. Taylor, B. L., and D. E. Koshland, Jr. 1974. Reversalof flagellar rotation in monotrichous and peritrichousbacteria: generation of changes in direction. J. Bacte-riol. 119:640-642.

11. Vaituzis, Z., and R. N. Doetsch. 1969. Motility tracks:technique for quantitative study of bacterial move-ment. Appl. Microbiol. 17:584-588.

12. Vogel, H. J., and D. M. Bonner. 1956. Acetyl-ornithinase of Escherichia coli. Partial purificationand some properties. J. Biol. Chem. 218:97-106.

13. Stocker, B. A. D., and J. C. Campbell. 1959. The effectof non-lethal deflagellation on bacterial motility andobservations on flagellar regeneration. J. Gen. Mi-crobiol. 20:670-685.

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