BACTERIALIMOTILITY'EDWIN 0. JORDAN, MARY E. CALDWELL AND DOROTHY REITER

Department of Hygiene and Bacteriology, The University of Chicago

Received for publication, May 8, 1933

Motility is universally recognized as a significant biologicalcharacter of certain bacteria, and is used as a fundamental basisof classification in many taxonomic systems. Significance is oftenattached to motility as an identifying character in the differentia-tion of species, and independent species have sometimes beenestablished on the basis of lack or possession of motility. Rela-tively little attention seems to have been paid to variability inmotility and the factors that influence it. Discordant statementsand opinions about the presence or absence of motility in differentspecies are, however, not difficult to find. Although the over-whelming weight of opinion today is that dysentery bacilli arenon-motile (Lentz and Prigge, 1931; Gardner, 1929), both Shigaand Flexner originally described these organisms as motile andseveral experienced observers have since reported that motilityeither occurs spontaneously in certain strains or may be inducedby the application of special methods.2 As regards other groupsof bacteria, such as the so-called Morgan bacilli, quite divergentstatements as to the presence or absence of motility are made.It is well known also that non-motile strains of predominantlymotile species sometimes occur; Smith and Reagh's classicalobservations (1903) on the distinction between somatic and fla-gellar agglutinins were based on the discovery of a strain of thehog-cholera bacillus which was non-motile but in all other respectsappeared to be identical with the motile type. The effect of en-vironmental factors on motility seems to have been little studied.

1 The present investigation was aided by a grant to the University of Chicagofrom the Rockefeller Foundation.

2 It has been found, for example (Colquhoun, D. B., and Kirkpatrick, J.,1932), that many organisms, including a dysentery strain, that are non-motilein ordinary fluid or solid media develop motility when grown on semisolid medium.

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166 E. O. JORDAN, M. E. CALDWELL AND D. REITER

Occasional references to the effect of temperature upon themotility of bacteria may be found scattered through the literature.Thus Migula (1897), in discussing the effect of slight environ-mental changes upon motility, writes: "Der Bacillus prodigiosusist beispielsweise gewohnlich unbeweglich, er wird aber sehr leb-haft beweglich, wenn er bei Blutwarme gezogen wird." Mat-zuschita (1901) noted that certain strains of B. coli and B. typho-sus evinced more lively motility at 2000. than at 3700. Thebacteria of the intestinal group appear to be most often cited inobservations of this sort. Mironesco (1899) studied a typhoid-like bacillus which was absolutely non-motile and devoid offlagella at 380C., but actively motile at 2300. Kossel and Over-bech (1902) briefly noted that three strains of bacilli from pseudo-tuberculosis in guinea pigs were motile when grown at room tem-perature, but when left at 370 showed no motility. Nicolle andTrenel (1902) isolated from a guinea pig a typhoid-like bacillusthat was non-motile at 250 to 3500., but became motile whengrown at 180 to 2000. De'Rossi (1904) observed a coli-likebacillus which was actively motile when grown at 1500. but at370 almost completely non-motile. Neustadtl (1917), in study-ing motility in coli-like bacteria, found that some strains were mo-tile at one temperature, non-motile at another; in most instancesmotility was most pronounced at 370, but in three strains themotility was greater at 180 than at 37°. Braun and L6wenstein(1923-4) observed a dysentery-like bacillus, named by them B.inconstans, which was non-motile at 3700., motile at 220. Braunand Weil (1928) noted the occurrence in stools of bacilli culturallysimilar to the dysentery bacillus, but which were motile whengrown at 2200. although non-motile at 37°.

In spite of these scattered observations, the effect of tempera-ture on motility does not seem to have been generally recognizedby bacteriologists, and the usual "determination of motility"for diagnostic purposes is based on examination at one tempera-ture only.3

' See for example Committee on Bacteriological Technic of the Society ofAmerican Bacteriologists: Manual of Methods for Pure Culture Study of Bac-teria, Leaflet V, Fourth Edition, 1930, VJO-4.

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BACTERIAL MOTILITY

METHODS

For examination by the usual hanging drop method the cul-tures of about 40 strains of Salmonella Schottmulleri were grown instandard veal-infusion broth at 220 and 37°C., fresh young cul-tures (twenty to twenty-four hours) being always employed.

FIG. 1. S. SCHOTTMULLERI 209. INCUBATED 370C. TWENTY HOURS.NON-MOTILE COLONY TYPE

The plate shows approximately 219 colonies. There are 21 surface colonies and10 bottom colonies.

The percentage of motile cells may be roughly approximated bydrawing a ring with a wax pencil on the lower lens of the eyepieceand inspecting several fields of the drop within the ringed area.The hanging drop method has its limitations; the observer mayat times find it difficult to detect the presence of a few non-motile

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168 E. 0. JORDAN, M. E. CALDWELL AND D. REITER

organisms in a field of actively moving ones and it is impossibleto determine whether cells not showing motility are alive or dead.During the division period some cells have been observed to be-come non-motile. In an actively motile culture, an apparentlynon-motile bacillus with a deep constriction was watched by one

FIG. 2. S. SCHOTTMtJLLERI 379. INCUBATED 370C. TWENTY HoURS. MOTILECOLONY TYPE

of us (M. C.) for twenty minutes; soon after division was com-plete, both daughter cells exhibited motility.A semi-solid medium, similar to that used by Hiss (1902) and

by Li (1929), has also been employed in studying the property ofmotility. To a base of standard veal-infusion broth (pH 7.2 to7.3) 0.3 per cent agar and 4 per cent gelatin are added for 22°C.cultures, and 0.5 per cent agar and 8 per cent gelatin for 370C.

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BACTERIAL MOTILITY

cultures. A uniform and carefully adjusted consistency is im-portant since a medium that is too stiff does not permit adequatedifferentiation between the motile and non-motile colonies andan over-soft medium leads to blurring of the colony outlines.Overcrowding of the plate must be avoided. As pointed out byLi, the significant colonies on the poured plate are those embeddedin the substance of the medium; the colonies on the surface andthose at the bottom next the glass are spreading and often do notshow any characteristic difference. Figure 1 shows the non-mo-tile, and figure 2 the motile colony type. An interesting phe-nomenon, which might lead to confusion, is the change in colonytype on a semi-solid medium when plates seeded with an organismnon-motile at 370 but motile at 22° (see group 4) are first incu-bated at 370C. and then allowed to stand at 220; the non-motiletype of colony changes to the motile. One disadvantage of thesemi-solid medium is that if only a small proportion of motile cellsare present in the culture they may be entirely missed unlessmany plates are made.

Forty-one strains of Salmonella Schottmiilleri, culturally andagglutinatively identical and all of the smooth type,4 were ex-amined in broth and in semi-solid medium: thirty-four of thesestrains were motile in approximately the same degree both at 220and 370, 4 strains were non-motile at both temperatures, 2 strainsproduced both motile and non-motile colony types on semi-solidagar and showed motile and non-motile cells in hanging droppreparations. One strain showed 100 per cent motile cells inbroth cultures grown at 220C. but in those at 370C. usually noneor at most 2 per cent; with this strain no colonies definitely of themotile type could be observed on the plates of semi-solid mediumincubated at 37°. Three single cell strains derived from theparent strain gave similar results.The four groups-all smooth strains-may be more particu-

larly described:1. Highly motile both at 220 and 370C. (34 strains). Observa-

tions in hanging drop indicated that from 95 to 100 per cent of4 The rough type is more irregular in behavior and for simplification is not

dealt with in this paper.

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170 E. 0. JORDAN, M. E. CALDWELL AND D. REITER

the celLs were motile. Marked gradations in colony size on thesemi-solid medium at 220 (forty-two hours) were observed rang-ing from 3 mm. in diameter to 26 mm.; two strains gave coloniesonly about 1 mm. in diameter although both showed predomi-nantly motile cells in hanging drop. Motility, therefore, cannotbe surely adjudged by mere inspection of colonies on semi-solidplates. There is no definite relation between the size of thecolony on semi-solid medium and the degree of motility observedwith the microscope: those strains producing colonies 3 mm. orless are, to the eye, as actively motile as those producing coloniesof 15 to 20 mm. In a general way, however, the highest propor-tion of motile cells, as observed by the microscope, occurred inthose strains giving the largest colonies: at 220, 4 strains were soactively motile that not a single non-motile cell could be observedin the hanging drop and all of these produced colonies 20 mm. indiameter or larger; at 370, 6 strains reckoned as "100 per centmotile" in the hanging drop gave colonies 15 to 25 mm. in di-ameter. The method of comparison is a rough one and greatvariations are observed. The degree of motility, as observed inhanging drop and as indicated by colony size, was practically thesame at 2200. and at 3700. for all these 30 strains.

2. Non-motile both at 220 and 370. (4 strains). These 4strains showed no motility in broth either at 220 or 370 and nomotile colonies on semi-solid medium.

3. Predominantly non-motile, but some motile cells usuallyobserved. Two strains (nos. 288 and 292) showed a few motilecells in broth culture and occasional motile colonies on semi-solidmedium, but the majority of the cells were non-motile. In oneseries, one colony out of 170 on semi-solid medium was of themotile type. The predominance of non-motile cells was manifestboth at 220 and 370C.

4. Predominantly motile at 220C., predominantly non-motile at370C. One strain (210),5 although motile at 220, has consistentlyremained almost completely non-motile when grown at 370, an

6 This strain was first isolated in 1916 from the blood of a patient in San An-tonio, Texas. Four single cell strains derived from the parent strain agree intheir temperature-motility relations.

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observation extending over two years. It is typically smoothand the colonies on ordinary agar are not different in appearancefrom those of the strains in group 1 (motile at both temperatures).Cultural and agglutination reactions are characteristic of theSchottmuller group.

FLAGELLAR STAINS

The results obtained by observations of the hanging drop andof the type of colony formation in semi-solid medium have beensupplemented by counts of flagellated and non-flagellated cells.The method used for staining flagella is that described by Saffordand Fleisher (1931) which, with slight modification, has givenexcellent results.

Method. Organisms from the water of condensation of twenty-hour veal infusion agar slants were transferred to a small volume,1 to 2 cc., of sterile distilled water, until a barely perceptible tur-bidity was produced. The aqueous emulsion was then incubatedfor thirty to sixty minutes. After incubation the suspension wasplaced on clean slides and dried in the air.When dry the preparation was covered with freshly prepared

fixative, heated to steaming and the fixative allowed to act forone to two minutes. The fixative6 was then washed off withdistilled water and the slide allowed to dry, after which the slidewas covered with Fontana spirochaete stain7 and heated to steam-ing. The stain was allowed to act for one to two minutes, and theslide then washed with distilled water and dried.Twenty-hour cultures were uniformly used in making the

flagellar stains. Only those slides or sections of slides werestudied which were well stained and showed an even distributionof organisms.For control of the staining technique, a known motile organism

was treated on each slide.6 The fixative was prepared as follows: 100 cc. one-fourth saturated (aqueous)

solution of picric acid (temperature of saturation, 220C.); 5 grams tannic acid;7.5 grams ferrous sulphate (FeSO4-7H20).

I The Fontana spirochaete stain must be freshly prepared as follows: To 25cc. of 2 per cent AgNOa add ammonium hydroxide, CP, diluted 1 to 3 till the pre-cipitate which forms redissolves. Then add more AgNOs till faint turbidityresults. A clear solution is useless.

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172 E. 0. JORDAN, M. E. CAIDWELL AND D. BEITER

TABLE 1Flagella stains

22C. 370C.

sNmA Num- Number Per Per cent Num- Number Per Perber non-fiagel- Total cent non- ber non-flael- Total cent cent

flagel- lated flgel- flagel- flagel- lated flagel- non-lated lated lated lated lated flal-

late

Group 1

12 1,112 152 1,264 87.97 12.03 968 86 1,05491.8 8.2149 1,170 131 1,301 89.9 10.1 1,405 438 1,843 76.2 23.8169 1,054 156 1,210 87.1 12.9 626 60 686 91.3 8.7211 963 51 1,01495.0 5.0 436 89 52583.05 16.95303 1,130 78 1,208 93.5 6.5 1,034 82 1,116 92.7 7.3308 966 140 1,106 87.3 12.7 576 127 703 81.9 18.1559 1,172 98 1,270 92.3 7.7 1,068 88 1,156 92.4 7.6652 1,194 104 1,298 92.0 8.0 1,071 132 1,203 89.03 10.97684 1,014 89 1,103 91.9 8.1 1,098 70 1,168 94.0 6.0707 912 80 992 91.9 8.1 1,040 84 1,124 92.5 7.5

Group 2

175 0 1,060 1,060 0 100 0 1,000 1,000 0 100209 0 1,300 1,300 0 100 0 4,228 (1)* 4,229 0 100209S 0 1,004 (5)* 1,009 0 100 0 1,032 1,032 0 100221 0 1,008 (6)* 1,014 0 100 0 2,959 2,959 0 100316 0 1,349 1,349 0 100 0 2,000 (3)* 2,003 0 100

Group 3

288 80 976 1,056 7.6 92.4 132 3,066 3,198 4.1 95.9292t 186 3,456 3,642 5.1 94.9 63 954 1,017 6.2 93.8

Group 4

210 772 465 1,237 62.4 37.6 56 1,858 1,914 2.9 97.1

210-S3 723 283 1,006 71.9 281f 0 2,500 2,500 0 100IO-Sll7231283 1,006 91 2101 1,509 1,71912.2t 87.8

* Probably all non-flagellated organisms. There was, however, a slight haze,perhaps due to precipitated stain, around a few bacteria, the number indicated inparentheses, so that it was impossible to be perfectly certain that flagella werenot present.

t The figures for this strain are subject to error since the flagellated organismswere not evenly distributed over the field, but occurred in clumps. There was adecided predominance of non-flagellated organisms.

t This figure is unusually high. It represents that portion of the slide whichshowed the maximum number of flagellated organisms. Many fields showed onlynon-flagellated forms. 210-S3 is like 292 in that the flagellated organisms werelimited to certain parts of the slide.

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BACTERIAL MOTILITY

The results of counting flagellated and non-flagellated cells areshown in table 1. While, on the whole, they are in harmony withthe observations on hanging drop and semi-solid medium, theybring out several additional points of great interest. It appearsthat non-flagellated cells occur in cultures of all motile strains invarying proportions. On the other hand, in some instances anoccasional flagellated cell may be observed among a large numberof non-flagellated cells (group 3). The temperature at which theculture is grown has a marked effect upon the proportion of flagel-lated cells in one strain (group 4).

SUMMARY AND DISCUSSION

Within a given bacterial "species" carefully studied and de-limited by cultural and agglutination absorption tests (E. 0. J.)different degrees of "motility" exist. Some strains, under ordi-nary conditions of cultivation, consist practically entirely of motilecells, other strains show no motility in hanging drop or in semi-solid medium and no flagellated cells can be found in stainedfilms. Non-motile strains of species predominantly motile areprobably more common among bacteria than generally supposed.Four out of about 41 strains of S. Schottmiilleri examined haveremained consistently non-motile over a period of several years.The percentage of motile cells in cultures of a given strain

appears usually to be quite constant but may occasionally showconsiderable variation, as in strain 288, which in May, 1930 wascomposed of motile and non-motile cells in about the same pro-portion and again in April, 1931 showed a similar mixture. Ex-amination in January, February and May, 1932, however, in boththe hanging drop and semi-solid medium showed a very smallpercentage of motile cells (2 to 3 per cent) and this was confirmedby the relatively small proportion of flagellated cells seen instained preparations. Throughout this period, the strain re-mained smooth so far as could be determined by repeated ex-amination of broth cultures and of colonies on agar plates.

In one instance (strain 210 and single-cell derivatives) a definitetemperature relation was found to exist over a three-year periodof observation. Although little or no motility was manifested

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by the cells from cultures of this organism grown at 37°C., two-thirds or more of the cells grown at 220 were motile. This rela-tion was further confirmed by flagella stains which showed 60 to70 per cent of flagellated cells in 22°C. cultures and a much smallernumber in 370 cultures. In one series of slides of 370 culturesnot a single flagellated cell was found in 2500 examined (table 1).The following conclusions appear warranted:In general, the proportion of motile cells in a given strain re-

mains fairly constant when particular attention is given to condi-tions of growth and testing. Certain strains, however, may showconsiderable variation in the proportion of motile celLs even whencultures are maintained and handled in as uniform a manner aspossible.Environmental conditions affect motility in some strains. A

strain actively motile -when grown at 22°C. may be practicallynon-motile when grown at 370; the motility of other strains re-mains apparently uninfluenced by changes in temperature.

Motility is not a character that can be used for exact delimitation of species or varieties of bacteria. Standard cultural char-acters and biochemical reactions do not appear to be correlatedwith the presence or absence of motility.

REFERENCESBRAuN, H., AND L6WENSTEIN,_P. 1923-4 Centralbl. f. Bakt., I, Orig., 91, 1.BRAUN, H., AND WEIL, A. J. 1928 Centralbl. f. Bakt., I, Orig., 109, 16.COLQUHOUN, D. B., AND KIRKPA-TRICK, J. 1932 Jour. Path. and Bact., 35, 367.DE'RossI, GINO 1904 Centralbl. f. Bakt., I, 36, 685; 37, 107.GARDNER, A. D. 1929 System of Bacteriology in Relation to Medicine (Med.

Res. Council), IV, p. 163.HISS, P. H. 1902 Jour. Med. Res., 8, 148.KOSSEL, H., AND OVERBECH 1902 Arb. a.d.k. Ges., 18, 114.LENTZ AND PRIGGE 1931 Kolle and Wassermann, Handbuch, III, p. 1399.LI, C. P. 1929 Jour. Exp. Med., 50, 245; 255, 767.MATZUSCHITA, T. 1901 Centralbl. f. Bakt., II, 7, 209.MIGULA, W. 1897 System der Bakterien, 1, 130.MIRONESCO, T. G. 1899 Hyg. Rundsch., 9, 961.NEUSTADTL, R. 1917 Munch. med. Wchnschr., 64, 1507.NICOLLE, C., AND TRENEL, M. 1902 Ann. de l'Inst. Past., 16, 562.SAFFORD, C. E., AND FLEISHER, M. S. 1931 Stain Technology, 6, 43.SMITH, T., AND REAGH, A. L. 1903 Jour. Med. Res., 10, 89.

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