ON THE MICROSCOPIC METHODS OF MEASURING THEDIMENSIONS OF THE BACTERIAL CELL

GEORGES KNAYSI

Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca, New York

Received for publication November 20, 1944

It has been shown by the author (Knaysi, 1941) that the dimensions of cellsmeasured on smears dried, fixed, and stained by the ordinary methods are con-siderably below those of cells in the living state and represent the dimensions ofthe shrunken protoplasm. More recently (Knaysi, 1944) the various methodsof microscopic measurements were discussed, and it was concluded that valuesapproaching true cell dimensions can be obtained either by measuring the livingcells or in smears especially stained to show the cell wall. Measurements fromelectron micrographs were considered satisfactory when the cell wall is visible.It was also stated: "Figures obtained from negative smears are too high becausethey include also the slime layer usually surrounding the cell. In the case ofcapsulated organisms, the relative error may be tremendous. There is also anadditional error due to the retraction, upon drying, of the coloredfilm inwhichthe bacteria are imbedded."Working with Bacillus megatherium, Dubin and Sharp (1944) compared the

dimensions of the same air-dried, unstained cells in photo and electron micro-graphs. They concluded: "Images in the electron micrographs indicate plainlythe presence of two structures constituting the bacterial cell, an inner dense sub-stance and an outer less dense substance. This outer substance of the bacterialcell is invisible in the light micrographs.... There is no significant differencebetween measurements of the size of unstained bacteria dried in air and photo-graphed with the light microscope and measurements of the comparable portion(inner dense substance) of the identical bacteria later photographed in the elec-tron microscope." Dubin and Sharp noted the agreement between their resultsand those of the author. However, their study of cell dimensions on negativepreparations (Benian's method) led them to the conclusion that they were "lessthan those of the total bacterial substance seen in the electron micrographs,"which is "not in agreement with Knaysi's observation that in negative-stainingpreparations the figures are even slightly higher than the true size because of theretraction of the colloidal film upon drying."

This apparent disagreement between the results of Dubin and Sharp and thoseof the author made it desirable to reinvestigate this elementary, but important,problem of bacterial morphology, the problem of correct measuring of celldimensions.

ORGANISMS AND METHODS

The organisms studied were strain C2 of Bacillus mycoides and strain C3 ofBacillus cereus previously classified in this laboratory by Lamanna (1940). We

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GEORGES KNAYSI

preferred these two organisms to Bacillus megatherium, the cells of which have atendency to curve or coil.Smears for cell wall and negative staining were prepared from the same sus-

pension of cells in distilled water from a 6-hour-old culture on beef infusion agarslants at 33 C. Living cells were studied in microcultures of the same strainson the same medium; measurements were made on photomicrographs preparedas previously described (Knaysi, 1940). In view of the well-knoNm wide varia-tion in cell length, only the width of the cells was measured. This dimensionisnearly the same for young cylindrical cells growing in nearly the same environ-ment (figures 1 and 6). The width at the median part of the photographicimage of a given cell was measured with dividers, and the value in microns wasobtained with the help of a scale similar to that previously used (Knaysi, 1929).In order to make this time-saving and very accurate scale better knowvn, we arereproducing it, with directions for its use and construction, in figure 8.

RESULTS AND DISCUSSION

The results of the present study are illustrated in figures 1-7.Living cells. The cells of figure 1 represent a microculture of Bacillus mycoides

in the early stages of development. They are not all the progeny of a singlecell and may be taken to represent what one may encounter in an actively grow-ing slant culture. Nevertheless, one can easily see that variations in width arerelatively slight. Careful measurement of twenty cells similar to those of figure1 shows that, regardless of the errors of measurement, the width of the variouscells falls within 1.13 and 1.20 ,, with an average of 1.17 ,u. Among the progenyof a single cell, the differences are even smaller; this is shown in figure 6for Bacillus cereus (see also Knaysi, 1941, and plates III and IV, 1944), where thewidth may be considered constant and equal to 1.68 ,u.

Cell wall method. Figure 2 shows young cells of Bacillus mycoides stained bythe author's cell wall method (Knaysi, 1941). Measurement of 14 cells similar tothose of figure 2 gave a range of 1.07 to 1.25 and an average of 1.16 , (comparedwith 1.17 , for living cells). The fluctuation here is 0.18 ,.t, about 2.5 timesgreaterthan that for the living cells. However, part of this fluctuation is undoubtedlydue to the fact that the cells on a slant culture are not growing in as uniform anenvironment as that of a microscopic field of a plane microculture; furthermore,there are probably differences in the degree of shrinkage and dye adsorption be-tween different cells. It must also be emphasized that those differences arebrought out here by the accuracy of the methods of measurement and wouldprobably pass unnoticed by the usual methods used by the bacteriologist. Thismethod gives for Bacillus cereus (figure 7) a width of 1.6 IA.

Of interest is a comparison between figures 5 and 7. In both figures thecells came from the same suspension. The cells of figure 5, however, were stainedwith Meyer's methylene blue (air-dried and mounted in oil) and obviously cor-respond to the central, cylindrical mass of shrunken cytoplasm of figure 7. Thewidth of this mass in figure 5 varies from 0.50 to 0.75 A with an average of 0.61 ,u;it compares with the width of the central mass of figure 7 and amounts to about

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R :S t., ;&iS . 4i _ _ a a 8 iFFIG. 1. BACILLUS MYCOIDES, STRAIN C2

Cells in a 3- to 4-lhour-ol( niCeroculture at 31 to 32.5 C oIn imeatphotographe( inldark field.

infusioni agar (p'1 7),

FIG. 2. BACILLI-S MYCOIDES, STRAIN C2Cells from a 6-hiour-old slant culture at 33 C on the medium: Imeat iinfusion (half concen-

tration) + 0.5't, tryptone + 0.5% glucose + 1.5%a agar; pH 7. C'ell wall mnetho(l. P'hoto-graphed using Wratten filters B58 + E22.

FIG. 3. BACILLUS MYCOIDESStrain andl culture as in figure 2. Negative preparation with water-soluble nigrosine.

Photographedl using Wratten filter B58.377

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Fir;. 4. BACIIL,US MYCOIDES (SEE CAPTION FOR FIGURE 3)F'IG. 5. BACILLUS CEREUS, STRAIN C3

Cells fromii a 6-b our-old slanit culture in miieat infusion agar (pH 7) at 33 C. Stained withllethleIlIe Ilue anil IllouItedl in oil. Photogr.aphed using Wratten filter A25.

11G. 6. BACILLUS CERIEIUS;, S1TRAIN C3Cells in a 7-hour-oldl culture at 31 to 32.5 C i'n imeat infusion agar (pR 7). Photographed

ill dark field.F11G. 7. BACILIlS CEREI-S

Straini anid culture as ill figure a. Cell w-all mietho(l. Photographe(d usinig Wratten1filters 1358 + E1*22.

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MICROSCOPIC MEASUREMENT OF BACTERIAL CELL DIMENSIONS

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FIG. 8. In part I, Side AB = DC of rectangle ABCD is equivalent to 10A. Sides AD andBC are divided into 10 equal parts, and the points of division are joined by the perpendicu-lars a', b' . . . i'. The diagonal AC, having a constant slope, intercepts on each of theperpendiculars a', b', . . . i', two segments. The lower ones, included between this diagonaland AD, have values respectively equivalent to 1, 2, . . . 9 ,A. If now from each point ofintersection a line is drawn parallel to AD, we would divide ABCD into 10 rectangles similarto AbcD in which the sides along AB and DC, such as Ab and Dc are each equivalent to 1 IA.The diagonals of these rectangle, such as Ac, intercept on each of the perpendiculars a', b',...i', two segments. In rectangle AbcD, the value of the lower one is, respectivelyequivalent to 0.1, 0.2, . .. 0.9 ,. The homologous values for the rectangle immediatelyabove are, 1.1, 1.2,. . .1.9,u; for the third rectangle, these values are 2.1, 2.2, . . . 2.9 ,u, etc.

In practice, only the diagonals of rectangles similar to AbcD are drawn by joining thenumbered points of division of sides AB and DC as follows: Point 0 (= A) on AB is joinedto point 1 on DC; 1 on AB to 2 on DC, etc., as in part II of the figure.

To find the value in, of a dimension measured with dividers, one point of the dividers isrun along AD (part II) until the other point rests exactly on one of the diagonals. Assum-ing that, in an instance, one point of the dividers was at 0.3 as the other point rested exactlyat point p of the diagonal 2, 3, the value of the dimension would be equivalent to 2.3 uA.For points between vertical lines, the values are estimated in centimicrons.

The length of A. is obtained from the image of a stage micrometer photographed at thesame magnification as the cells. The length of side A1J is arbitrary but usually made equalto 10 cm.

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GEORGES KNAYSI

0.38 of the total width of the cell as obtained from the cell wall method. Thisratio is much below the 2/3 ratio found by various investigators (see Knaysi,1938), and even further below the ratios obtained from electron micrographs(Knaysi, 1942; Dubin and Sharp, 1944). This is probably due chiefly to theextreme youth of the cells used in the present experiments, and possibly some-what to further shrinkage due to fixing by heat.

Negative preparations. A glance at figures 3 and 4 reveals that negativepreparations, even in the absence of a thick slime layer, are unsuitable for thestudy of form and size of the bacterial cell. In negative preparations, the dimen-sions of the cell vary inversely with the thickness of the dye film. Measurementof 85 cells, in photomicrographs similar to those figures, shows cell widths rangingfrom 1.65 , in the thin places to 0.68 ,.s in the thick places, with an average of1.1 ,; this average is, indeed, slightly less than those given by the two othermethods but its value varies with the proportion of cells measured in thin andthick places. A glance at plate III of Dubin and Sharp's paper convinces onethat these workers must have used mostly smears of sufficient thickness to

TABLE 1Comparison between the values of cell width obtained by different methods

BACILLUS MYCOIDES BACILLUS CEREUSMETHOD .-

Range Average Range Average

Livinig cells in microcultures......... 1.13 to 1.20 1.17 1.68* 1.68Cell wall method ..................... 1.07 to 1.25 1.16 1.60* 1.60Negative-staining method............ 0.68 to 1.65 1.10Staining with Meyer's methylene blue. 0.5 to 0.75 0.61

* Constant, or nearly so.

cause reduction in cell size. Consequently, the conclusion of Dubin and Sharpis correct for films exceeding a certain thickness, and our previous conclusion iscorrect for films below that critical value. Obviously, a method which dependson such an uncontrollable factor as the thickness of the dye film is not suitablefor the study of size.

In view of the relation between thickness of film and cell dimensions, withwhich we have been familiar for many years, it becomes difficult to see how theexplanation given by Dubin and Sharp of the relation between cell and dye filmcould account for the observed facts. Those authors examined minute glasscylinders in a dilute solution of Congo red. They state: "It is seen at once thatdye tends to pile up or to be concentrated in the sulcus or angle formed where thecylinder lies on the slide. Under these conditions the negative image is boundedby dye lying beneath the actual cylinder and is therefore smaller than the cylin-der." Although this concept is adequate to explain reduction in size, it cannotexplain the experimental fact, clearly illustrated in figures 3 and 4, that when thedye film is below a certain thickness the cells appear larger than their true size.

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MICROSCOPIC MEASUREMENT OF BACTERIAL CELL DIMENSIONS

Our own conception based on the existence of a meniscus which retracts upondrying (Knaysi, 1944) accounts for both reduction and increase in the apparentdimensions of the cell. Evidently, the glass cylinder and the bacterial cell donot have an identical behavior with respect to Congo red films.

SUMMARY

A comparative study of the cell width of Bacillus mycoides and Bacillus cereusshows that the value obtained depends on the treatment of the cells measured.Measurements of the living cells in the medium in which they are growing agreewith those made on similar cells stained by a method showing the cell wall. Instained smears where the cell wall is not visible, the cells appear much smallerthan they really are and represent the shrunken masses of the cytoplasm. Innegative-staining smears, the cells may appear larger or smaller than their realsize depending on the thickness of the dye film surrounding the cells. Conse-quently, negative-staining smears are not suitable for the study of cell dimensions.

REFERENCESDUBIN, I. N., AND SHARP, D. G. 1944 Comparison of the morphology of Bacillus megathe-

rium with light and electron microscopy. J. Bact., 48, 313-330.KNAYSI, G. 1929 The cytology and microchemistry of Mycobacterium tuberculosis. J.

Infectious Diseases, 45, 13-33.KNAYSI, G. 1938 Cytology of bacteria. Bot. Rev., 4, 83-112.KNAYSI, G. 1940 A photomicrographic study of the rate of growth of some yeasts and

bacteria. J. Bact., 40, 247-253.KNAYSI, G. 1941 Observations on the cell division of some yeasts and bacteria. J. Bact.,

41, 141-154.KNAYSI, G. 1942 On the width and origin of bacterial flagella. Science, 95, 406-407.KNAYSI, G. 1944 Elements of bacterial cytology. Comstock Publishing Co., Inc.,

Ithaca, N. Y. 209 + xii p., 91 fig., 10 pl.LAMANNA, C. 1940 The taxonomy of the genus Bacillus. III. Differentiation of the large

celled species by means of spore antigens. J. Infectious Diseases, 67, 205-212.

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