RELATIONSHIP OF CELL WALL STAINING TO GRAM DIFFERENTIATION'

J. W. BARTHOLOMEW AND HAROLD FINKELSTEINDepartment of Bacteriology, University of Southern California, Los Angeles, California

Received for publication August 12, 1957

Bartholomew and Mittwer (1951) presentedphotographic evidence that the gram-positivestaining area of bacterial cells could be fittedinternally to the area demonstrated by the Dyarcell wall stain. Their conclusion was that thebacterial cell wall was not included in the gram-positive area. Lamanna and Mallette (1954)explained Gram differentiation of intact cellson the basis that crystal violet stained andformed a dye-iodine lake in the cell walls ofgram-positive cells, but did not stain and there-fore did not form a dye-iodine lake in the cellwalls of gram-negative cells. They reported alsothat if normally gram-positive cells were stainedwith crystal violet, and then were washed withtap water, the dye was removed from the cellwall and on subsequent completion of the Gramstain these cells were gram-negative. It was theirconclusion that the cell wall of intact cells mustbe stained with the primary dye for the cells toresult in gram-positive staining, and failure ofthe dye to be present in the cell wall was theexplanation of all gram-negative staining. Al-though the evidence presented in their paperwas primarily for yeast cells, they reported thatsimilar observations had been made for severalspecies of gram-positive and gram-negativebacteria. Hence, they believed that the abovebasis for Gram differentiation could be general-ized for all cases of gram-positive and gram-nega-tive staining.The present paper concerns the results of

additional studies on the relationship of cellwall staining to Gram differentiation for bacterialcells. Particularly studied was the effect of awash step following the primary dye on apparentcell size, and also the effect of this wash step on theresults obtained when the Gram stain procedurewas completed. It was hoped that an explanationcould be found for the conflicting viewpoints as

' This investigation was supported in part by aresearch grant (E 121) from the Institute of Al-lergy and Infectious Diseases, National Institutesof Health, United States Public Health Service.

to whether or not the cell wall was included inthe gram-positive staining area, and that infor-mation could be obtained concerning the validityof the Lamanna and Mallette concept of the basisfor Gram differentiation.

METHODS

Air dried and heat fixed bacterial smears onglass slides were prepared from 18 hr nutrientagar slant cultures incubated at 37 C. Sufficientslides were prepared at one time for all of thework reported in this paper. The steps used forthe Gram procedure were as follows. Kopeloff-Beerman crystal violet was applied for 3 min,Burke's iodine for 2 min, 95 per cent ethanolwas used as a decolorizer for 1 min, and Burke'ssafranin was applied for 10 sec as a counter-stain. A 2 to 3 sec distilled water wash was usedbetween each step, and all reagents includingthe wash water were kept in Coplin jars intowhich the slides were placed. When prolongedwash steps were used, the slide was progressivelytransferred at 30 sec intervals from one Coplinjar to another containing fresh distilled wateruntil the total indicated wash time had elapsed.For nigrosin-negative staining, a 2 per centsolution of water soluble nigrosin was used. Thiswas spread over the heat fixed smears by a bloodsmear technique in such a manner as to producea standard film thickness as judged by visualcomparison with a reference slide. Photographswere compared of the same bacterial field afterbeing stained by different procedures.An apochromatic Bausch and Lomb 1.4 N. A.

oil immersion objective with an aplanatic 1.4N. A. condenser was used for photomicrography.The photomicrographic camera was a Bauschand Lomb model L adapted to use 3}j by 4%Sin. Kodak Panatomic X film. An Ortho-Illumina-tor B (American Optical Co.) was used as alight source. Wratten filters, numbers 22 and 58,were used to increase photographic contrast forslides stained with crystal violet and safraninrespectively.

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BARTHOLOMEW AND FINKELSTEIN

RESULTS AND DISCUSSION

Lamanna and Mallette (1954) used the abilityof the cell walls of organisms to be stained withcrystal violet as the primary basis for theirexplanation of the Gram differentiation. Theirevidence for staining of the cell wall was theappearance of cells in clusters. They consideredthat if the individuals in a group of stained cellsappeared to touch each other, the cell wallshad been stained. If the individuals in a groupof stained cells did not appear to touch, then thecell walls had not been stained. Using this astheir index of cell wall staining, they reportedthat crystal violet did not stain or form a dye-iodine lake in the walls of gram-negative cells,but did stain and form a dye-iodine lake in thewalls of gram-positive cells. Although theirobservations included 2 species of yeast, 4 speciesof gram-positive bacteria, and 2 species of gram-negative bacteria, almost all of the evidence forbacterial cells was omitted from their paper. Itwas thought that the implications of these obser-vations as the basis for explaining Gram differ-entiation were sufficiently important to warrantadditional studies on bacterial cells. The followingexperiments, therefore, were performed.Groups of Bacillus subtilis and Escherichia coli

cells were photographed and compared followingnigrosin-negative staining, and following eachstep in the Gram staining procedure. Representa-tive results for groups of B. subtilis cells are

presented in figure 1. This group of cells was

chosen for presentation because, as occasionallyoccurs when smears of B. subtilis are gramstained, it contained both gram-positive andgram-negative cells. The nigrosin-negative stainwas used to indicate the width of the unstainedcells. All of the individual cells appeared to havesimilar widths prior to the alcohol step. Thewidths observed following crystal violet stainingwere the same as indicated by the nigrosin-nega-tive stain. However, following the iodine step, allof the cells appeared slightly wider than indicatedby either the crystal violet or the nigrosin-nega-tive stain. Thus, it would seem that no discerniblearea of any of the cells was free of the dye-iodinelake. Although cells 1, 2, 3, and 5 touched andwere of similar widths following the crystalviolet or iodine steps, cells 1, 2, and 3 were gram-negative upon completion of the Gram procedure.It would not have been possible to observe theresults after the crystal violet cr after the iodine

steps and to foretell which of the cells in thisgroup would subsequently stain gram-negative.A similar series of photographs for cell groups

of E. coli is presented in figure 2. The cellstouched following both the crystal violet andthe iodine steps (see arrows in figure 2). Followingthe iodine step, these cells appeared slightlywider than indicated by the nigrosin-negativestain. It was evident that, as was observed withthe B. subtilis cells, no discernible area of theE. coli cells was free of the dye-iodine lake, andyet all of these cells were gram-negative oncompletion of the Gram procedure.These results failed to reveal any demonstrable

staining difference between gram-positive andgram-negative cells at any point prior to thedecolorization step of the Gram procedure. Bothgram-positive and gram-negative bacterial cellsappeared to be completely stained following theiodine step. Therefore, the results showed thatthere was no necessary correlation between theformation of a dye-iodine lake in any demon-strable outer portion of the bacterial cell and theresults obtained on completion of the Gramprocedure.Lamanna and Mallette (1954) reported in

their work that washing with distilled or tapwater of normally gram-positive cells followingcrystal violet staining resulted in the removal ofdye specifically from the cell wall. These cellswould then appear gram-negative on completionof the Gram procedure. However, little is knownconcerning the effects of such a wash treatmenton the dye distribution and visual appearanceof bacterial cells. Therefore, the next series ofexperiments was designed to obtain this informa-tion. Photographs were taken of the same B.subtilis cells following staining with crystalviolet, and after staining and exposure to asubsequent distilled water wash for differentperiods of time. Typical results for a single cellare shown in figure 3. A nigrosin-negative stainwas included for width comparisons. Whenstained with crystal violet and blotted dry, thecell was similar in width to that indicated by thenigrosin stain. When stained and then washedfor increasing periods of time, the apparent widthof the cell progressively decreased, and after 15min of washing, the cell had lost about one thirdof its apparent width. Although no visible orphotographic trace was present of the unstained

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CELL WALL STAINING AND GRAM DIFFERENTIATION

...

*:.:..:: :~~~~.::.::.. .. :;. ::.:.. :: ::.

:...,....::: '..~~.... ..

.........:........

*S:A:.F.R:A:N IN::.

(d-e)

(+)

(F)(-

Figure 1. Gram stain of Bacillus subtilis. The appearance of a group of cells following each step of theGram procedure (A-D), and nigrosin-negative staining (E). This group contained both gram-positiveand gram-negative cells. The dark appearing cells were gram-positive, the light appearing cells weregram-negative.

cell material, if the cell were restained with crys-tal violet and blotted dry, it then appearedidentical to its original width and shape. Thiswould indicate that the structure and staining

ability of the cell had not been changed by thewash procedure.The results shown in figure 3 demonstrate that

the effects of a wash step following staining were

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BARTHOLOMEW AND FINKELSTEIN

(8)

VIOLETCRY L

(D)

._

S:{

-.9U:. t

_ -_141.._

IODINE, SAFRANIN

Figure 2. Gram stain of Escherichia coli. The appearance of a group of cells following nigrosin-nega-tive staining (A), and each step of the Gram procedure (B-D). The appearance of the group followingthe alcohol step has been omitted because the cells were decolorized and difficult to photograph at thispoint. All of the cells were gram-negative following the safranin step. The darkness of the safraninstained cells was due to the filter used for photomicrography.

not restricted to only the cell wall. After a 15min wash the cell was reduced in apparent widthby approximately 0.3 ,u. If the value of 0.02 Iu isaccepted as representing the thickness of thebacterial cell wall (Chapman and Hillier, 1953),it becomes evident that dye was lost from a

much greater area than just the cell wall alone.This would be expected since several workershave reported that water or salt solutions are

capable of completely decolorizing the stainedbacterial cell (McCalla, 1940; Bartholomew et al.1950; Finkelstein, 1957).One can conclude that a tap water or distilled

water wash following staining would affect thetotal dye content of the entire cell, and not justremove dye specifically from the cell wall. Theseresults also would explain the observations ofLamanna and Mallette (1954) concerning the

appearance of demonstrable unstained areas

between bacterial cells in groups, following a

wash step. It is apparent that the clear areas

which they observed were not due to a loss ofdye from the cell wall alone, but were due todye loss from the cell as a whole.The wash step following the crystal violet

staining also had an effect on the results obtainedon completion of the Gram stain procedure. WhenB. subtilis cells were stained with crystal violetand then washed for 5 min, the cells were nor-

mally gram-positive on completion of the Gramprocedure. This gram-positive result was ob-tained despite the fact that the crystal violet hadbeen removed from a demonstrable outer area

of the cell representing about 25 per cent of thetotal cell width. If, however, the wash stepfollowing crystal violet staining was extended to

(C)

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CELL WALL STAINING AND GRAM DIFFERENTIATION

iI A

II I

....... AI II I

.. .~I

NIGR 0 SIN

BLOT

E WASH 5sec

w It I min-J

-J

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ll lOmin

>- ~ 10min

.5m

15 min,: ~~I i

i ... ..I.Figure S. The effect of a wash step on apparent cell size. This Bacillus subtilis cell was stained for 3

min with Kopeloff-Beerman crystal violet, followed by a blot dry or various periods of washing in

distilled water. In order to facilitate size comparison, a nigrosin-negative stain was included and thearea of the cell indicated by the dotted lines was enlarged and placed end to end on the right side ofthe figure.

15 min, most of the cells were gram-negativeupon completion of the Gram procedure.2The wash times needed to produce a gram-

negative result for B. subtilis cells in theseexperiments were considerably longer than the 1

2 The present authors believe that the wash stepbetween the crystal violet and iodine is verycritical and that its importance in the Gram stainprocedure has not been adequately appreciated.The directions given are often vague except forthose procedures which completely omit the wash

min tap water wash reported by Lamanna andMallette (1954) for yeast cells. This would indi-cate that the distilled water wash used in thepresent study was a milder decolorization pro-cedure than the tap water wash. Finkelstein(1957) has shown that tap water is a stronger

step. If the slide is washed following the crystalviolet, as indicated in the Hucker modification,this treatment should be rigidly controlled andlimited to 2 or 3 sec. Care at this point contributesmarkedly to a successful Gram differentiation.

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BARTHOLOMEW AND FINKELSTEIN

NIGROSIN '7SAFRANIN

BLOT WASH 5 sec WASH

S,._I-I- . .

Figure 4. Burie's safranin stain, 1 min, of Bacillus subtilis. The effect of a 5 sec and a 5 min washstep on the apparent size of cells, as compared to safranin staining with no wash and nigrosin-negativestaining.

decolorizer for basic dyes than distilled waterdespite the lower pH of the latter. The decoloriza-tion power of tap water is due to the presence

of dissolved cations which actively replace thebound basic dye in the cells.

Figures 1 and 2 show that the gram-negativelystained (i. e., safranin stained) cells appearedmuch smaller than when stained with crystalviolet alone, or with the dye-iodine lake. Thissize difference has been observed previously(Churchman, 1927; Bartholomew and Mittwer,1951; Lamanna and Mallette, 1954). The lastnamed authors interpreted this staining differenceas representing the inability of safranin to stainthe bacterial cell wall. However, due to thethinness of the bacterial cell wall this interpreta-tion would seem improbable as an explanationfor the marked size differences as observed infigures 1 and 2 of the present paper. It was shown(figure 3) that washing in distilled water, fol-lowing crystal violet staining, greatly reducedobservable cell size. Since a wash step usually isused following safranin staining, it could bepossible that this has a bearing on the apparentsmall size of safranin stained cells. Experimentswere conducted therefore, to determine thecomparative appearances of cells stained withsafranin, both without and after the applicationof a subsequent wash step. The results are shownin figure 4. When blot dried the safranin stained

cells were comparable in size to the nigrosin-negative stained cells. However, a 5 sec washfollowing the safranin was sufficient to producean apparent reduction of about one third incell width. When crystal violet was used (figure3), a prolonged wash step of 15 min was necessary

to result in a similar size reduction. It was con-

cluded, therefore, that the reason for the smallapparent size of safranin stained cells was therapidity with which the dye was lost during thewash step which usually is used following a

staining procedure. There was no indication thatcrystal violet and safranin differed in theirability to be present in any single area of the cellsuch as the cell wall. However, it appeared thatthere was a difference in the affinity of thesedyes for the cell material.On the basis of the results presented in figures

1-4, it is perhaps now possible to postulate an

explanation for reconciling the conflicting view-points of Bartholomew and Mittwer (1951) andLamanna and Mallette (1954) concerningwhether or not the cell wall must be included inthe gram-positive staining area of the cell. It was

shown in figures 1 and 2 that no difference in thestaining of cell areas could be seen between gram-

positive and gram-negative cells following thecrystal violet or the iodine steps, and thatdifferentiation occurred primarily during thesubsequent decolorization. The results of figures

5 min

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CELL WALL STAINING AND GRAM DIFFERENTIATION

EShE ..... :.

(A) (B).5 sec -.WASH 5 min WASH

Figure 5. Gram stain of Bacillus subtilis. Comparison of the effect of a 5 sec wash (A) and a 5 minwash (B) between the crystal violet and iodine steps on the apparent size of, and dye distributionwithin, cells staining gram-positively.

3 and 4 showed that the extent of the washfollowing staining greatly influenced the observ-able size of the cells. The extent of the washfollowing crystal violet influenced also the Gramresult of gram-positive cells. If such cells wereonly mildly washed, they appeared gram-positive;if washed extensively, they appeared gram-negative. Therefore, the possibility existed thatthe size of the gram-positive staining area wasdependent, not specifically on the staining ofthe cell wall, but on the extent of the wash stepbetween the crystal violet and iodine steps. Thispossibility is particularly pertinent if one accepts0.02 Iu as the approximate thickness of the bac-terial cell wall. The optical microscope would notbe able to differentiate between two cells dif-fering only in whether or not the cell wall wasstained, even if these cells were in contact.Therefore, this possibility was tested by Gramstaining a single group of B. subtilis cells, andcomparing the final apparent cell width whentwo different wash times were used. A 5 secwash and a 5 min wash were chosen since it wasknown that such treatments would not causethe cells to be gram-negative on completion ofthe Gram procedure. The results of this experi-

ment are shown in figure 5. Although the cellswere gram-positive with either of the washtreatments, a considerable difference could beseen in the apparent size of the cells, and alsoin the dye distribution within the cells. Theshorter wash resulted in cells appearing to be incontact (see arrows in figure 5), and therefore,the entire cell apparently containing the dye-iodine lake. The longer wash treatment resultedin cells which were not in contact. This indicatedthat the cells had lost considerable dye and hadfailed to form the dye-iodine lake in the outer cellarea. The apparent size reduction from the longerwash was about one fourth of the cell width asobserved following the 5 sec wash. Hence, thewidth reduction was much greater than could beaccounted for if dye were lost only from the cellwall. These results demonstrated that cells canlose dye from the outer area of the cell and yet begram-positive. They are in agreement withprevious results where following a 5 min washbetween the crystal violet and the iodine, B.subtilis had lost 25 per cent of its apparent widthbut still stained gram-positive on completion ofthe Gram procedure.The different conclusions reached by Barthol-

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BARTHOLOMEW AND FINKELSTEIN

omew and Mittwer (1951) and Lamanna andMallette (1954), therefore, could be explained onthe basis of the effects of a wash step betweenthe crystal violet and iodine steps of the Gramprocedure. That is, if a short or no wash stepwere used the gram-positive staining area wouldappear to include the entire cell. However, if alonger wash step were used, a sufficient amount ofdye could be lost from the cell and thereby resultin a definite reduction in the apparent width ofthe gram-positive staining area. Hence, the outerareas of the cell would appear not to be includedin the gram-positive staining material. Theconflicting results obtained by the above authorsthen, can be explained on the basis of differencesin experimental procedure.One could conclude from the above results

that Gram differentiation is not necessarily dueto the presence or absence of crystal violet inany part of the cell, such as the cell wall. Rather,it appears that gram-positivity is dependent inpart upon the presence of a certain minimalamount of dye-iodine lake in the normallygram-positive cell in order for it to resist thedecolorization treatment. This does not implythat Gram differentiation is due to a greater totalcrystal violet content in gram-positive cells thanin gram-negative cells. On the contrary, it hasbeen shown that E. coli actually could bind morecrystal violet per g cell weight than B. subtilisor Saccharomyces cerevisiae cells (Bartholomewand Finkelstein, 1954). In the opinion of thepresent authors, Gram differentiation of intactcells is based on the differences in the rate of dyeloss during the decolorization step betweengram-positive and gram-negative cells. It isbelieved that this rate difference is primarily dueto permeability factors.

SUMMARY

The results presented here have demonstratedthat there is no necessary correlation betweenthe staining of the cell wall area of a cell and itssubsequent Gram result. Bacillus subtilis cellscould be stained gram-positively whether or notthe crystal violet had been washed out of the cellwall area. Conversely, Escherichia coli cells whichappeared to be completely stained by the crystalviolet and the dye-iodine lake, were gram-nega-tive on completion of the Gram procedure. Itwas concluded that Gram differentiation wasdependent upon more complex factors thansimply the staining or failure to stain the cellwall area.

The conflicting statements in the literature,concerning whether or not the cell wall was in-cluded in the gram-positive staining area, wereshown to be due to differences in experimentalmethods. If little or no wash were used betweenthe crystal violet and iodine reagents, the gram-positive area might include the cell wall. If alonger wash step were used at this point, the cellwall area might not be included but the cell stillwould stain gram-positively. If excessive washingwere used, normally gram-positive cells stainedgram-negatively.An explanation was presented for the often

observed smaller size of safranin stained cells ascompared to cells stained with crystal violet. Itwas found that safranin was more easily washedfrom the outer areas of the cell as compared tocrystal violet. For safranin stained cells a 5 secwash resulted in an apparent size reduction ofabout one third. It took 15 min of washing in dis-tilled water to achieve a similar apparent sizereduction for cells stained with crystal violet.Thus, the apparent difference in cell size resultingfrom safranin as compared to crystal violet stain-ing, was due to the ease with which safranin waswashed from the cell following the staining step.

REFERENCESBARTHOLOMEW, J. W. AND FINKELSTEIN, H. 1954

Crystal violet binding capacity and the gramreaction of bacterial cells. J. Bacteriol., 67,689-691.

BARTHOLOMEW, J. W. AND MITTWER, T. 1951Cell structure in relation to the gram reactionas shown during lysis of Bacillus subtilis. J.Gen. Microbiol., 5, 39-45.

BARTHOLOMEW, J. W., ROBERTS, M. A., ANDEVANS, E. 1950 Dye exchange in bacterialcells, and the theory of staining. StainTechnol., 25, 181-186.

CHAPMAN, G. B. AND HILLIER, J. 1953 Electronmicroscopy of ultra-thin sections of bacteria.I. Cellular division in Bacillus cereus. J.Bacteriol., 66, 362-373.

CHURCHMAN, J. W. 1927 The structure of B.anthracis and the reversal of the gram reac-tion. J. Exptl. Med., 46, 1007-1029.

FINKELSTEIN, H. 1957 Quantitative investiga-tions of dye uptake by bacterial cells. Ph.D.Dissertation. University of Southern Cali-fornia, Los Angeles.

LAMANNA, C. AND MALLETTE, M. F. 1954 Thecytological basis for the role of the primarydye in the gram stain. J. Bacteriol., 68,509-513.

MCCALLA, T. M. 1940 Cation adsorption bybacteria. J. Bacteriol., 40, 23-32.

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