OF WITH OFGram's choice of iodine, which he expected to formabackgroundcolor in his sections,...

THE GRAM STAIN

JAMES W. BARTHOLOMEW AND TOD MITTWER

Department of Bacteriology, University of Southern California, Los Angeles 7, California

CONTENTS

I. INTRODUCTION ............................................................... 1II. THE GRAM PROCEDURE....................................................... 2

1. The primary stain....................................................... 32. The mordant............................................................ 43. The decolorizer ......................................................... 44. The counterstain........................................................ 55. Other factors............................................................ 6

III. CORRELATION OF GRAM REACTION WITH PHYSIOLOGICAL CHARACTERISTICS...... 8IV. DEGREE OF GRAM POSITIVITY................................................ 10

1. Studies of decolorization time. Gram Dauer character.102. Conversion of gram positive cells to gram negative.123. Addition of gram positivity to cells previously rendered gram negative... 144. Conversion of cells normally gram negative to the gram positive state.... 14

V. THE MECHANISM OF GRAM DIFFERENTIATION ..151. Chemical theories of gram positivity.152. The isoelectric point concept....................................... 183. Theories involving permeability ...................................... 19

VI. THE SITE OF THE GRAM REACTION.20VII. CONCLUSIONS................................................................ 22VIII. REFERENCES................................................................. 23

I. INTRODUCTION

During the early days of bacteriology the detection of bacterial cells in tissueswas difficult since most of the staining methods used colored the tissue and thebacterial cells equally. Christian Gram (as a co-worker of Dr. Friedlander in themunicipal hospital of Berlin) attempted the development of a procedure whichwould differentially stain schizomycetes from tissue cells. Gram began his workwith pneumococci in the lungs of human pneumonia victims and with lung tissuefrom experimental animals. The first published mention of the now famous gramstaining procedure was in 1883, in a treatise by Carl Friedlander on the micro-cocci of pneumonia (59). This paper contained a brief description of Gram'sstaining procedure. The following year Gram published his staining method indetail (63).Gram's discovery was a peculiar mixture of accident and shrewd application

of a chance observation. During the course of his work in pathology he attemptedto obtain blue nuclei and brown cytoplasm in kidney sections by staining firstwith gentian violet and then with iodine-potassium iodide solution. This ap-plication of iodine, instead of the intended result, rendered destaining and clear-ing of the gentian violet, ordinarily very difficult, quite easy by the subsequentalcohol step. In Gram's words, "The experiments resulted from the accidentalobservation that aniline-gentian violet preparations of tissues, after treatmentwith iodine-potassium iodide, are completely and rapidly decolorized in al-

2 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16

cohol" (63). During this work Gram noticed the outstanding resistance of somebacterial cells to decolorization. The now famous, extremely useful, and enig-matic gram staining procedure was a natural development from these observa-tions.Although Gram did not present his procedure in the exact form in which it

is used today, its four fundamental steps are identical. During his work on lungtissue he used Ehrlich's aniline-gentian violet, Lugol's iodine, absolute alcoholfor decolorization, and Bismarck brown as a counterstain. Gram used his pro-cedure primarily to stain bacteria in tissues, but he also demonstrated its ap-plicability to smears of pure cultures. The counterstain was used to color thetissue cells, including their nuclei, differentially from the bacteria. AlthoughGram observed that certain types of bacterial cells such as typhoid bacilli weredecolorized by his procedure and took the color of the counterstain, he did notdivide bacteria into the now well-known gram negative and gram positive types.His failure to recognize the taxonomic values of his stain was probably due touncertainty resulting from his observation that while most of his pneumococciretained the gentian violet, some strains of pneumonia-producing bacteria weredecolorized. Probably the first diagnostic use to which Gram's stain was put wasin 1886 by Roux in connection with the gonococcus (115).Today Gram's staining procedure is generally recognized as a fundamental

contribution to biological science. In bacteriology it is the first and a very valua-ble step in diagnosis and classification. The ability to retain the primary dye inthe gram stain is a unique characteristic restricted to but a small fraction ofthe materials of nature. Most plant and animal cells stain gram negatively. Thegram positive characteristic is abundant only among the yeasts, the bacteria,and the molds. Certain other materials such as cell nuclei, certain protozoa,keratohyalin, virus protein, certain proteins from the ascarid gamete, and thepoliedes of the silkworm, have been reported as weakly to strongly gram posi-tive (22, 57, 67, 112, 137). Reports of extracellular gram positivity are rare,and in the opinion of the reviewers, the validity of the gram staining procedureinvolved can often be questioned.

In addition, the reaction to the gram procedure has been shown to correlatewith important chemical and physiological characteristics of the cell (see Sec-tion III). It is surprising that very few review articles exist on the gram stain.The best of the earlier review articles is that of Del6tang (45), but it is now outof print and difficult to obtain. In other papers where this stain has been con-sidered (36, 47, 77, 100, 118, 147) the review treatment of the subject is briefand incomplete. In the present review the authors will condense and bring upto date the historical and contemporary concepts of the gram stain.

II. THE GRAM PROCEDURE

The staining procedure as originally presented by Gram used Ehrlich's anilinegentian violet, an aqueous solution of iodine-potassium iodide, absolute alcoholas a decolorizer, and sometimes Bismarck brown as a counterstain. The methodis now fundamentally the same; however, a long series of important modifica-

1952] THE GRAM STAIN 3

tions has resulted in procedures which produce more reliable results, and whichare much more convenient than the original. The best comparisons of the variousproposed modifications have been conducted by Hucker and Conn (77). Thesemeticulous and extensive studies have given workers some scientific basis forthe choice of a gram stain method. Of the modifications suggested many gavegood, but three gave superior results. These were Hucker's modification (75,76, 77, 128), Burke's modification (24), and the Kopeloff-Beerman modification(89).The procedures used in the United States today usually have the following

features. The primary dye is crystal violet since it is a more definite and repro-ducible substance than the old dye mixtures called gentian violet. The primarydye is a stable solution, and it may contain a mordant such as ammonium oxalatefor constancy of action; or sodium bicarbonate may be added just before use tointensify the uptake of the color. The staining step and the iodine step may becarried out at an alkaline pH by addition of sodium bicarbonate or sodiumhydroxide to the dye, the iodine solution, or to both. This avoids poor resultswhich are sometimes due to the acid state of the organisms, their suspensionmedium (e.g., pus), or the reagents (24, 89, 124). Alcohol (95%) is most com-monly used as a decolorizer, although acetone and acetone-alcohol mixtures aresometimes used and are excellent (89, 100). The dilution of the alcohol withwater, by repeated use or by water remaining from a preceding wash step, is tobe avoided. Safranin is the most popular of the possible counterstains. Thesefeatures will be discussed in more detail in the following paragraphs. There isno gram procedure which can be referred to as the best for all laboratories andfor all situations. Hucker and Conn's recommendation (77) that the workeradopt at least two of the well-accepted methods, practice them until he isfamiliar with their characteristics, and use controls of organisms with known gramreactions is excellent counsel. This plan will give much better results than con-stant change of methods in the hope of finding one that is foolproof.

1. The primary stain. A difficulty of the original gram method was the lack ofstability of the Ehrlich's aniline gentian violet solution. This instability wasdue to a slowly formed precipitate which results from the interaction of the dyeand aniline. Among the attempts to increase this stability have been the sugges-tions of various nonprecipitate forming substitutes for aniline such as the slowaddition of phenol (41, 95, 96, 102, 110), boric acid (88), aniline sulfate or anilineoxalate (2), glycerol (78), oxalic acid (109), or even the complete omission ofthe mordant from the primary stain (24, 89, 90). The most widely used formulasin the United States are those of Hucker (75) and Kopeloff-Beerman (89).Hucker's crystal violet contains ammonium oxalate and is very stable. In addi-tion, Hucker and Conn demonstrated that ammonium oxalate in the formulagave better results than when the crystal violet was used alone (77). Burke (24)and Kopeloff and Beerman (89) added nothing to their stock solution of crystalviolet but added sodium bicarbonate to small quantities of stock solutions justbefore use, or even on the slide itself. The resulting alkaline pH for staining isknown to contribute to the clarity of the differentiation.


The specificity of crystal or gentian violet as the primary stain has long beena matter of dispute. The problem has recently been studied by Bartholomewand Mittwer (9, 10), who found that a large number of basic dyes, especiallyof the triphenylmethane-group, could be substituted for crystal violet. However,a study of 73 dye samples showed that no dye was superior or even equal tocrystal violet. Therefore, crystal violet, while not specific for the gram stain,is as good as or better than any other dye yet tested. Acid dyes, and basic dyeswith weak tinctorial properties, were found to be useless.

2. The mordant. In this section the term mordant is used to describe iodine-potassium iodide solution or a substitute for this solution as used in the gramprocedure. It is to be distinguished from substances added to the primary dyewhich are also called mordants. Gram's choice of iodine, which he expected toform a background color in his sections, was fortunate. No other reagent can besubstituted for the best and most reliable results (106). However, iodine is notabsolutely specific, and substitutes for it are possible. Suggested substitutes forthe iodine solution have included bromine, picric acid, any metallic iodide suchas that of Mg, Na, As, Al, Fe, Hg, or Zn, ammonium picrate, salts such as sodiumchloride, magnesium sulfate, and alum, any oxidizing agent such as KMnO4,H202, K2Cr2O7, or bleaching powder, colloidal iodine, a mixture of picric acid,HgCl2, and sodium acetate, and a mixture of iodine and picric acid. All of theseand others have recently been retested under standardized conditions (106).Since most give poor results or irregular results with the exception of picric acidand mercuric iodide or chloride, none of these suggested substitutes can berecommended.Many formulas for the iodine solution contain an alkalizing agent such as

sodium bicarbonate (124) or sodium hydroxide (89) because Sheppe and Con-stable (124) demonstrated that on storage the iodine solution develops sufficientacidity through oxidation to cause occasional errors in gram differentiation.In view of this work and the well-known influence of pH on a satisfactory gramstain (25), the use of an alkaline iodine solution can be recommended. That thisis not often a serious cause of error has been demonstrated, however, by thepresent reviewers, who acidified the iodine solution to pH's of less than 1 andobserved a slight increase in the percentage of the gram negative cells normallyfound in some smears of gram positive cells (6).A differentiation of bacteria similar to that of the gram differentiation can be

accomplished even without an iodine step, provided the technique be sufficientlycontrolled (14, 15, 17, 73, 91, 106). Although significant for research on thetheory, it would be practically impossible to determine the gram reaction of anunknown organism in this manner.

3. The decolorizer. The application of the decolorizer is the most critical stageof the gram procedure. There is danger from both over and under decolorization;therefore, the decolorizer and the technique should be as carefully standardizedas possible. Ethyl alcohol has been one of the most popular decolorizers. Accord-ing to Neide (108) addition of water to the alcohol increases its rate of de-colorization up to 40% alcohol; he considered 60% alcohol to be too rapid a


decolorizer for use in the gram stain and 80% alcohol as best. Others (17, 24,26, 27) have confirmed this effect of dilution. Hucker and Conn (77) found littlepractical difference between the decolorization effect of 95% and absolute al-cohol. These, however, gave more dependable results than alcohol solutions con-talning more water. Thus, it is necessary to conduct the procedure in such away that the alcohol does not change its dilution. Blotting dry before decoloriza-tion is recommended (128) and is probably desirable (45). The results reportedby Hucker and Conn (77) indicate that only 95% or absolute alcohol should beused when alcohol is applied as the decolorizer in the gram stain.

Other alcohols have been studied, and it has been reported that the morecomplex the alcohol, the slower the decolorization action. As the carbon chainlengthens, decolorization is slower. Kisskalt (84) found decolorization powerdecreasing in the following order: methyl, ethyl, propyl, butyl, and amyl al-cohol. Conn (40) found a mixture of equal parts of methyl and isopropyl alcoholsto have very similar decolorization properties to ethyl alcohol. In practice, how-ever, no known advantage can be gained by substituting the higher alcohols forethyl alcohol.The most often suggested substitute for ethyl alcohol is acetone (89, 100, 110),

a mixture of acetone and ether (24), or a mixture of acetone and ethyl alcohol(90, 110). Acetone is a more rapid decolorizer than alcohol (90, 110) and mustbe used with some care, but the validity of its use in the gram stain has beenamply demonstrated by Lillie (100) and others.

Other decolorizers suggested have included aniline (146) which was reportedto give more dependable results than ethyl alcohol because it was slower in actionand thus more easily controlled; chloroform (129), any surface tension depres-sant (15), dilute acids, acid alcohols (27, 63), sodium thiosulfate (101), and saltsolutions (41). In the hands of the reviewers, only alcohol, acetone, or mixturesof these two have given consistently good results. These latter decolorizers arealmost universally used, and the choice of one or the other is of less importancethan the strict standardization of the method chosen.The temperature of the decolorizer also affects the rate of decolorization (45,

98, 108) and should be standardized as much as is practical. Accurate determina-tions of the temperature effect are not now available, but it is established thatan increase in temperature results in more rapid decolorization (45). The evi-dence so far presented indicates that controlled decolorization temperature wouldbe of benefit in obtaining consistent results in critical research but probably notnecessary under ordinary laboratory conditions.

4. The counterstain. Some investigators of the gram stain have omitted thecounterstain in their work (132, 134). It is indeed possible to differentiate somebacteria entirely on the basis of the time necessary for complete or partialdecolorization (45, 98, 102, 108). However, this may or may not be related tothe true gram differentiation (9, 10, 26, 27, 77, 106). The ability of one dye toreplace another has been repeatedly demonstrated for years (12). This powerof replacement could be a means by which the counterstain might assume anactive role in the gram differentiation (77). Indeed, it is the opinion of some


authors (9, 10, 26, 27, 106) that for this reason the .application of the counter-stain is a fundamental and necessary step in true gram differentiation. If thisis so, then the factors influencing dye replacement assume importance in thegram procedure. Since the replacement tendency is in proportion to the concen-tration of the second dye and time of application (12), these two factors shouldbe controlled. The influence of the concentration of the counterstain on gramdifferentiation was shown by Lasseur and Schmitt (98) who reported that0.031% and 0.5% solutions of basic fuchsin gave greatly different results. The0.5% solution resulted in many gram positive cells appearing red since it hadsufficient replacement power to replace the primary dye remaining after de-colorization. Also, if no counterstain is used, gram negative cells may appeargram positive (9, 10, 106). Thus, the counterstain is a definite part of the gramprocedure. It is usually a weak solution of about 0.25% dye; its time of applica-tion should be carefully controlled (77, 90, 98).There appears to be no specificity in the type of the counterstain to be used.

The dyes used have included Bismarck brown (63, 75), dilute eosin (38, 77,110), rhodamine (143), basic fuchsin (38, 89), carbol fuchsin (129, 143), safranin(1, 24, 75), and neutral red (143). Some dye mixtures have been used such asBismarck brown plus basic fuchsin (20), neutral red plus carbol fuchsin (104),or pyronin Y plus methyl green (123).The extensive studies by Hucker and Conn (77) clearly demonstrate, however,

that not all basic dyes can be used as a counterstain. Some counterstains "areso powerful in their action that they tend to decolorize some of the gram positiveorganisms." Their experiments on the reliability of various counterstains indi-cated that "pyronin and Bismarck brown are the best counterstains, whileeosin and safranin are fair substitutes." As good contrast is also an importantfactor in practical work, this consideration led them to a personal preference forsafranin, a choice confirmed by the experiences of most workers.

5. Other factors. Although many authors have observed that the culturemedium can influence the degree of gram positivity shown by an organism (18,45, 46, 98, 102), this effect has not been studied sufficiently to allow any exactstatement to be made. Most of the recorded variations have been observed onrather unusual media, such as the reported conversion of Bacillus subtilis to agram negative state by growth on cerium agar (126), the reported conversion ofEscherichia coli to the gram positive state by growth in a liquid medium con-taining a maximum amount of glucose, MgSO4, or NaCl (14, 15). On the otherhand, Lasseur and Schmitt (98) found it impossible to change the gram reactionof Micrococcus pyogenes var. aureus by changing its growth medium. It has beenreported that starvation of Bacillus cereus, i.e., placing the cells in distilled waterat 37 C for 48 hours, results in an increase in the number of gram negative cellsin a culture (135). Whether this is due to autolysis, leaching of materials fromwithin the cell, or to utilization of reserve food material in the cell, is not clear.Recently Knaysi and his collaborators (87) found that a small amount ofbenzimidazole added to Dubos' medium gradually rendered an avian strain ofMycobacterium tuberculosis gram negative as well as non-acid-fast. It is said that


benzimidazole inhibits the synthesis of ribonucleic acid. Experiments of thistype should in the future shed more light on the role of the medium in relationto the gram reaction. At present, though, little work is available to indicatewhether the influence of abnormal media would be greater or less than the in-fluence of normal variations in the technique used in the gram stain. It appearsfor the most part that the individual worker would not encounter serious errorif he used any medium well accepted for the culture of the species of organismat hand. However, in case of doubt as to the results obtained, two or three mediashould be used. More study of the influence of growth environment on the gramreaction is needed.The influence of temperature of incubation is not clear. Eisenberg (56) could

not change the gram reaction of Staphylococcus by 70 transfers incubated attemperatures very near the maximum for these organisms. There is no evidencethat incubation temperatures which vary only a few degrees from the optimumfor growth of the organism could seriously influence the results of the gramprocedure. However, since the influence of temperature is not well determined,it is undoubtedly wise to use incubation temperatures near or at the optimumfor the organism concerned.

All investigators agree that the age of the culture influences the degree ofgram positivity of the culture. According to Lasseur and Schmitt (98) there isa stage, which differs for each species of organism, in which the gram positivecharacteristic is most marked. The differences between young cultures of 24hours and old cultures of several days are easily demonstrated, and the old cul-tures are nearly always less gram positive (45, 90, 108). The difference between aculture of one or five hours, or twenty-four and forty-eight hours, however, ismuch less impressive. Lasseur and Schmitt (98) and Hucker and Conn (77)state that adult cells of approximately 48 hours of age are sometimes more grampositive than younger cells, whereas Del6tang (45) and Kopeloff and Cohen(90) state that 24 hour cells are more gram positive than 48 hour cultures.Benoit (18) in a careful study of Bacillus mesentericus and B. subtilis showedthat even at the stage of first sporulation these cells remained gram positiveafter decolorization for 10 minutes in 95% alcohol. The evidence shows that thecustom of using only an 18 to 24 hour culture for the determination of gramcharacteristics is unwise. Although serious errors due to use of a 12 or 48 hourculture probably would occur only in special instances, it would obviously bebetter to use at least three different ages of an unknown culture to determineits gram character as urged by Hucker and Conn on the basis of extensive studiesof this question (77).The thermophilic organisms must be given special consideration. DeBord (44)

showed that spore-forming thermophiles were gram negative by 24 hours andwere most gram positive at 8 hours, an understandable result in the light of thegreatly accelerated metabolism of the thermophilic species. Six to eight hourcultures, as well as the usual 18 to 24 hour cultures, therefore, should be used indetermining the gram reaction of thermophilic species.The degree of gram positivity of cells can be influenced considerably by the


method of fixation. Del6tang (45) clearly showed the great differences of de-colorization time between yeast cells fixed with alcohol-ether, Bouin solution(picric acid, acetic acid, formalin), acetic sublimate, Miller-formol (potassiumdichromate, sodium sulfate, neutralized formalin), and Champy's solution (os-mic acid, chromic acid, potassium dichromate). The differences are large, rang-ing from decolorization times of 10 hours to 40 hours. Del6tang's thorough workconfirmed a number of earlier reports (34). Since these fixatives are rarely usedin the ordinary gram procedure, they have little interest for those who use heat-fixed smears on glass slides. However, the work commands the attention of tech-nicians working with tissue sections. The common practice of using heat to fixslides for gram staining is supported by the work of Nikitin (111), who showedthat large variations in the heat fixing step did not influence the results ob-tained. A note of caution is created by the work of Neide (108), who demon-strated that prolonged heat fixation caused gram positive cells to stain gramnegatively, and by the variations in degree of gram positivity found by Del&tang (45) after various degrees of heat fixation. However, small differences inthe heat fixing step will not have a major effect on the results obtained.The technique of preparing the bacteriological smears on the slides is not

without influence on the results obtained. Lasseur and Schmitt (98) showedthat thick smears of Vibrio cholerae took 40 minutes to be decolorized completelywith acetic acid while thin smears took only 13 minutes. For critical research,standard amounts of standard suspensions of organisms should be spread overstandard areas of slides in order that individual cells be separated from eachother insofar as this is possible (9, 77). In all preparations large masses of groupedorganisms should be avoided.

Before staining, the slide should be protected against contact with other ob-jects. Any contact with another slide or hard object will create large or smallareas of gram negative cells in preparations which would otherwise be grampositive. This is due to the effect of crushing on the gram reaction of the cell(16, 17, 26, 27, 106).

III. CORRELATION OF GRAM REACTION WITH PHYSIOLOGICAL CHARACTERISTICS

Churchman (35) presented in a striking manner the correlation of the gramreaction of bacteria with their physiological characteristics. Since then manynew correlations have been demonstrated. Table 1 presents most of those knownat the present time. The table is necessarily incomplete both in subject contentand references. Any attempt to include all the material would prove too bulkyand would result in an unwieldy table containing much unreliable and con-flicting data.

In perusing the material in table 1 it must be kept in mind that almost allof the differences shown are of degree rather than absolute. This is true even forthe differential nature of the gram staining procedure itself. It is impossible tocompile a table which will give characteristics applicable to all organisms. Forinstance, the organisms in the genus Neisseria are usually classified as gramnegative, but their physiological reactions are more like those of the gram posi-


tive group (7, 127). Thus, table 1 applies to organisms in general but does notnecessarily apply to specific organisms or strains.Some attempts have been made to correlate electrophoretic velocity with the

gram characteristic of cells. Although Lasseur and his collaborators (97) claimsuch a correlation, other investigators have not confirmed them (23, 28, 150).

TABLE 1

Correlation of gram reaction with physiological characteristics*t

Gram positive cells are more resistant to digestive enzymes such as trypsin (22), pepsin(127), pancreatic juice (43).

Gram positive cells are more resistant to lysis or death from the action of alkalies (116,117, 127).

Gram negative cells are more resistant to lysis from action of HCl, H2SO4, or acetic acid(116, 117).

Gram positive cells are more susceptible to death or growth inhibition by:Anionic detergents in general (5, 52)Cationic detergents in general (5, 52)Higher alkyl sulfates (42)Aniline, methyl aniline, phenol, cyclohexol, ethyl alcohol, toluol, benzol, xylol, chloro-form, ethyl ether, butyl ether, diphenyl ether, ethyl acetate, ethyl butyrate (83a)

Antibiotics such as penicillin, actinomycin A, gramicidin (52)Iodine (19)Basic derivatives of cholane and norcholane (131)Sulfa drugs (52, 71)Basic dyes (31, 32, 33, 37, 85, 133).

Gram negative cells are more susceptible to death or growth inhibition by:Azides, tellurites, bichromates, arsenites (52, 122)Oxidizing agents such as iodine, potassium permanganate, potassium dichromate, sodium

sulfate, potassium ferro- and ferricyanide (135a)Auxins (53)Quinone (62).

Gram positive cells are less able to synthesize essential amino acids (60).Gram positive cells concentrate in the cell certain amino acids such as arginine, glutamic

acid, histidine, lysine, and tyrosine (60, 138).Gram positive organisms are resistant to lysis under influence of sudden release of C02

after 120 atmospheres pressure (94).Gram positive cells are more readily made photosensitive by such dyes as methylene blue,

eosin, and mercurochrome (140, 141).Gram negative cells are more readily made photosensitive by safranin (140, 141).Gram positive cells have a lower isoelectric point than do gram negatives (132).

* The differences shown are general rather than absolute; see text.t Numbers refer to references at end of review.

The evidence for both contentions leads to the impression that such a correlationdoes not exist, and that the results of Lasseur might be explained on some otherbasis, such as a chance choice of bacterial species. In any event, if a correlationbetween gram character and electrophoretic velocity does exist, it is sufficientlysmall so as to be missed by several investigators.A similarity in mechanisms of the gram stain and the acid-fast stain has long


been suspected (16, 17). This suspicion is based on the many similarities betweenthe two procedures. Aniline gentian violet, which can be used for the gram stain,can also be used to replace the carbol fuchsin in the acid-fast stain (92). Bothprocedures require a mordant; iodine for the gram stain and phenol for the acid-fast stain. Both can use alcohol as a decolorizer, and both use a counterstain.Both acid-fast and gram positive characteristics are lost when the cell is ruptured(16, 17, 26, 27, 125), and conversion of acid-fast cells to a non-acid-fast state isoften accompanied by conversion from the gram positive to the gram negativestate (6, 92). Media and conditions of growth influence both reactions (87).Since all of these similarities exist, possibly the mechanism of these two dif-ferential staining reactions is based on similar principles.

IV. DEGREE OF GRAM POSITIVITY

Gram differentiation, whether on a staining basis or a physiological basis,represents a wide scale on which any two organisms may be widely or narrowlyseparated. If the gram negative and gram positive groups as determined bystaining are imposed on a scale representing physiological differences, thenoverlapping between gram positive and gram negative species would oftenoccur. Churchman and Siegel (37) presented such overlapping for dye sensitiv-ity. Smith (127) demonstrated a similar overlapping for sensitivity to electro-lytes and digestion by trypsin or alkali. The best acknowledgment of this grada-tion between gram positive and gram negative characteristics is the commonobservation that a reliable gram stain is more the result of a technique than of astep by step procedure.The causes of gram variability can be grouped under two headings: (a) varia-

bility due to the staining technique used, and (b) variability inherent in theorganisms themselves. The variability attributable to the technique has beenconsidered in an earlier section. It has been shown, however, that even carefulstandardization of the technique will not prevent conflicting results (77). Someorganisms such as yeasts are so gram positive that few errors occur; others suchas the diphtheria organism, various aerobic spore-formers, or the gonococcus,often give variable results (72). The gram positive character varies from strongto weak, not only among species but also among different cultures of the samespecies grown under different conditions (39). Thus, often the true gram reactionof an organism can be obtained only after a considerable period of observationinvolving repeated gram stains under various growth conditions of the culture(77).Three terms are often applied to describe the gram character of a culture:

gram positive, gram negative, and gram variable. The latter term was intro-duced to describe those organisms which were not consistent in their reactionto the gram stain. However, the addition of the term gram variable, while useful,has not solved the problem of terminology. Some workers prefer to retain theold gram positive or gram negative groupings.

1. Studies of decolorization time. Gram Dauer character. Neide (108), clearlyrecognizing the weakness of a strict gram positive or gram negative terminol-


ogy, tried to establish a more definite criterion which he called the Gram Dauercharacteristic of an organism, defined as the time necessary to decolorize thecells under standardized conditions after the application of the primary dye andthe iodine. Thus, no cell is absolutely gram positive, i.e., it will be decolorizedif the alcohol is allowed to act long enough. Neide found that the decolorizationtime was a constant for each species or strain under standard conditions, andthat the decolorization time was sufficiently different between species to makethis a valuable differential characteristic. Lasseur and Schmitt (98) and Del6-tang (45) extensively studied the Gram Dauer characteristic and proposed modi-fied techniques to increase the reproducibility of results.

In the final analysis the Gram Dauer character demonstrates a gradation ofthe decolorization times of different species of bacteria. It does not result ingrouping of the organisms but shows that all bacteria lie on a broad scale withreference to their degree of gram positivity (or resistance to decolorization).For many reasons the Gram Dauer is as yet of little use as a substitute for theusual gram differentiation. A true decolorization end point is difficult to establish.Cells vary in decolorization rate in any one culture, and a plot of percentage ofcells decolorized vs. time results in a rather gently-sloping S-curve (45). Varia-tions in concentration, temperature, and time of application of the reagents, aswell as the cultural conditions of the organism, strain used, and the like, allinfluence the results. Del6tang's method (45) is reproducible under carefullystandardized conditions. It has provided valuable data and has demonstratedthe variation in degree of gram positivity as a function of species, culture me-dium, fixatives used, and other factors. It is unfortunate that the procedure ismuch too painstaking and laborious a process to be used routinely since thisquantitative measure of gram positivity would be extremely useful in classifica-tion and in research of many kinds. If a simple method of quantitatively deter-mining gram positivity could be found, a real service would be rendered to bac-teriology.

Another confusing variability in the gram procedure is the presence of grampositive granules in cells otherwise gram negative. An interesting example ofthis is Thiobacillus thiooxidans (86). It is well known that an overdecolorizationof strongly gram positive cells results in a spotty appearance of the cells (33, 67)and that weakly gram positive organisms, such as the diphtheria organism,regularly show strongly gram positive areas (30). In this connection, it is pos-sible that an explanation may lie in the recent findings of Mudd and Smith (107)that bacteria possess "vesicular nuclei containing chromatin." Also Knaysi andhis collaborators (87) found certain deeply staining type A (nuclear) bodies in M.tuberculosis to possess a dense outer shell. These type A bodies were seen toremain gram positive after the cells themselves had been rendered gram nega-tive. However, very little work has been done on the nature of gram positivegranules in general. It may well be that their gram positive property rests on amechanism involving factors in addition to those which determine the gramcharacteristic of the cell as a whole (10). The confusion occasioned by thesespots is reduced if one determines the gram character of an organism only from


the appearance of the cells as a whole, and only after an extensive study of thegram character of the organism involved.

2. Conversion of gram positive cells to gram negative. Almost as soon as the grampositive state was recognized, attempts were made to determine its cause by theuse of methods of pretreatment which convert normally gram positive cells to agram negative state. These attempts have been numerous and often successful.However, they leave behind them a bewildering array of methods used and con-flicting results and interpretations. The influence of media and temperature ofincubation have been mentioned in an earlier part of this review and need notbe discussed further.The simplest of the conversion methods consists of suspending the cells,

either living or killed, in water at temperatures ranging from 20 to 52 C, fortimes of from two hours to several days (1, 36, 79, 135, 139). The success ofthis method has been limited to certain species and culture strains, and positiveresults have not been experienced at all times, but there is no doubt that it issometimes successful. The results obtained could be due to simple leaching ofmaterial from the cell, activity of endorespiratory enzymes within the livingcell (79, 135), activity of autolytic enzymes in general within the cell followingdeath (36, 139), or activity of specific autolytic ribonucleases from the cellsthemselves (3, 51, 82).A more specific and, therefore, more enlightening method of gram conversion

has been the use of enzyme preparations. In a study of the natural conversionof pneumococci from a gram positive to a gram negative state following death,Avery and Cullen (3) found that they could isolate an autolytic enzyme whichwas active against pneumococci of all types, but not against other species oforganisms. Later Dubos (51) showed that the active enzyme was a ribonuclease,and crude preparations of this enzyme were prepared. Bartholomew and Um-breit (13) took advantage of the newly available pure crystallized preparationsof ribonuclease to show that this enzyme had a general ability to convert grampositive bacteria to a gram negative state. The work of Avery, Cullen, and Duboswas carried into extremely interesting phases by Jones, Stacey, and Webb (82)when they isolated species specific factors from Clostridium welchii and staphylo-cocci. These factors, combined with the nucleinases of the cell, converted onlyone species of organism from gram positive to gram negative. The participationof ribonucleates in the gram positive state appears to be well established. Never-theless, good evidence indicates that the gram positive state involves more thanjust the presence or absence of ribonucleates.

It is important to note that other enzymes have also been shown to result ina conversion from the gram positive to the gram negative state. Webb (145)demonstrated this ability for lysozyme. Lysozyme was shown to act only on thepolysaccharides or mucopolysaccharides of the cells; however, both polysaccha-rides and ribonucleic acids left the cell upon the action of the lysozyme. Webbsuggested that both ribonucleic acids and polysaccharides are involved in thegram positive state. Others (148) have also reported the gram conversion powerof lysozyme. Some workers (29) using very crude preparations reported that


such proteolytic enzymes as pepsin and trypsin could convert gram positiveorganisms to the gram negative state, but others (51, 82, 145) have been unableto confirm this observation for trypsin.The gram conversion of organisms by chemical pretreatment is less spec-

tacular than the use of enzymes, and more confusing. It is often difficult toseparate the action of the chemicals used from the action of autolytic enzymesnormally present in the cells and from the action of nonspecific factors such asthe boiling temperatures sometimes used during exposure of cells to the reagents.Some success has been obtained with such fat solvents as boiling ether (50),chloroform (7), benzol, toluol, acetone, and tetrachlorethylene (113). However,Eisenberg (55) reported negative results for chloroform, and Schumacher (120),for alcohol and ether. Success has been reported with mixtures or successiveapplication of acid alcohol, ether, and benzyl chloride (61, 121, 136). Trommsdorff(139) reported that reversal could be accomplished if the cells were killed bychloroform but not if killed by boiling water. None of these reports is veryhelpful since the methods used, such as prolonged boiling with ether, could pro-duce a multitude of changes within the cell. The same can be said for conversionby hydrolysis with HCl (144), strong acetic acid or alkalies (27), the conversionby the action of dyes such as acriviolet (32, 33), the action of oxidizing agents(64), or the action of hydrogen under pressure (94).More helpful, and of much greater interest, are those investigations which

have attempted to associate the loss or gain of the gram positive character withsome specific extractable material. Many workers have associated a change ingram character with the fatty components of the cell. Dreyer, Scott, and Walker(50) extracted staphylococci with ether and on their conversion from gram posi-tive to gram negative isolated a lipoid from the extract. By ether extraction ofgram positive bacteria Tamura (136) isolated a lipoid which he claimed was it-self gram positive. Schumacher (121) isolated a fatty acid from yeast which ac-cording to his reports stained gram positively and which rendered protein grampositive. Other work has associated the gram positive to gram negative conver-sion with the nucleic acids of the cell. Deussen (47, 48, 49) used acid and al-kaline hydrolysis to convert gram positive cells to gram negative. On conversionhe observed the release of nucleoproteins and ribonucleic acids from the cell.Henry and Stacey (68) exposed C. welchii and yeast cells to 2% bile at 60 Cfor 12 to 144 hours and on conversion to the gram negative state noted that mag-nesium ribonucleate was the principal substance released from the cells. Somepolysaccharide and protein were also observed. The polysaccharides have alsobeen associated with gram positivity as shown by the action of lysozyme (145),and by their presence in the material extracted by bile (68), and in the presenceof reducing sugars in the extract obtained by Dubos (51).Of the foregoing work, that associating gram positivity with ribonucleic acids

has been the most convincing because of the specificity of the reagents used, therestoration to the gram positive state by these substances, and the reproducibil-ity of the results (103). There is no doubt that ribonucleates are involved in thegram positive state, but it would be unwise to ignore completely the evidence

14 JAMES W. BARTHOLOMEW AND TOD MI'VrWER [VOL. 16

pointing to other factors such as lipoids, polysaccharides, and the cell membraneitself.One of the quickest and easiest methods to convert a gram positive cell into a

gram negative state is to crush the cell (16, 17, 26, 27, 106). These experimentsare easily reproducible in a few minutes in any laboratory, and they presentgreat difficulties to any strictly chemical concept of the gram positive state.However, as we will see later, the strictly chemical theories and the cell mem-brane permeability theories are not necessarily incompatible since permeabilityis undoubtedly associated with the chemical nature of the membrane.

Other reported methods of gram conversion include the action of immuneserum (114), the action of antibiotics (54), and mutation (133). A very simpleconversion method is exposure to ultraviolet light of heat-fixed smears (Bartholo-mew and Mittwer, forthcoming publication).

3. Addition of gram positivity to cells previously rendered gram negative. Themost outstanding work on the chemical nature of the gram positive state hasbeen the demonstration that the gram negative cells produced by the removalof ribonucleic acids could be returned to the gram positive state by the additionof the removed material. This was first accomplished by Deussen (47, 48, 49),who used acids and alcohols to obtain the gram negative state, and who thenrestored the gram positive state by exposure of the cells to ribonucleic acid.Henry and Stacey (68) removed a magnesium salt of ribonucleic acid from thecells of C. welchii and Saccharomyces cerevisiae, thus, converting these cells tothe gram negative state. They then showed that magnesium ribonucleate fromany source could restore the gram positive state to the converted cells providedthese cells were kept in a reducing medium. Desoxyribonucleic acids, nucleo-sides, or nucleotides did not restore gram positivity, and the sodium salt ofribonucleic acids had but weak action. This observation was soon extended to avariety of species of microorganisms (69) and was soon confirmed in variousaspects by other investigators (13). Magnesium ribonucleate failed, however,to make truly gram negative organisms such as E. coli gram positive (4, 7) al-though some success was obtained for an obviously intermediate organism suchas members of the genus Neisseria. Thus, it is obvious that the cells must becapable of receiving the magnesium ribonucleate in a certain manner in order tocreate the gram positive state. Henry and his collaborators (70) have suggestedthat the cause of the gram positive state is the combination of magnesium ribo-nucleate with certain basic and novel types of proteins present only in grampositive cells. Webb (145) found that cells which were converted to the gramnegative state by lysozyme could not be rendered gram positive by exposure tomagnesium ribonucleate alone. This suggests that polysaccharides are part ofthe gram positive complex.

4. Conversion of cells normally gram negative to the gram positive state. Evennormally gram negative bacteria such as E. coli and Aerobacter aerogenes havebeen reported to stain gram positively when grown on butter agar (29) or ex-posed to lecithin (50) or petrolatum (74). This work lacks conviction since theauthors themselves report irregularities in results obtained. Bartholomew (7)

19521 THE GRAM STAIN 15

reported success in converting Neisseria but not E. coli into a gram positive statewith magnesium ribonucleate; however, irregularities in results occurred. Bakerand Bloom (4) reported that a viscous preparation of desoxyribonucleic acidcaused E. coli to stain gram positively, but this gram positive state could easilybe removed by washing with water. At present, it appears that there is no de-pendable way to convert normally gram negative cells to a gram positive state.Those agents which are successful in restoring the gram positive state to nor-mally gram positive cells do not affect normally gram negative cells. This sug-gests again that the fundamental differences between the normally gram positiveand gram negative types of cells are more than the simple presence or absence ofsuch substances as magnesium ribonucleate or fatty material.

V. THE MECHANISM OF GRAM DIFFERENTIATION

It was only natural that the great development of interest in the gram stainingprocedure was also accompanied by a widespread interest in the reasons for thedifferentiation observed. Despite the efforts of many investigators, none hasyet been able to describe definitively the mechanism although numerous in-teresting and significant experimental facts have been presented. Not only is itcertain that the gram mechanism is much more complex than was at first be-lieved, but it is possible that gram positive substrates vary as to the mechanismresponsible for their gram positive state.For the purposes of discussion the various proposed theories of the mechanism

of gram differentiation can be grouped under three headings. First, the chemicaltheories which seek to show that gram positivity is a character attributable tosome specific chemical substance or chemical complex within the cell. Second,the isoelectric point concept, which attributes a greater degree of dye-retentionby gram positive organisms to the more acid state, e.g., lower isoelectric point,of their protoplasm. Third, the permeability theories, which explain grampositivity on the basis of particular permeability characteristics of the cell orcell membrane to such substances as alcohol, iodine, or the dye-iodine precipi-tate.

Churchman's concept, associating gram positivity with the presence of agram positive cell cortex (32, 33, 36), might also be referred to as an additionaltheory. However, since Churchman made no attempt to explain the gram posi-tive state of the cortex, we shall discuss this concept in a following sectionconcerning the site of the gram reaction.

1. Chemical theories of gram positivity. One of the earliest chemical theorieswas that proposed by Unna (142), who stated that an alcohol resistant dye-iodine-cell complex was formed in gram positive organisms, but not in gram nega-tives. This simple theory, however, offered no proof of the real existence, or thechemical nature, of this gram positive complex.Many workers have correlated gram positivity with the fatty acid, lecithin,

or lipoprotein fractions of the cell substances. Guerbet, Mayer, and Schaeffer(64) thought that gram positivity was due to the formation of a dye-iodine-unsaturated fatty acid complex which was resistant to dissociation by the action

16 JAMES W. BARTHOLOMEW AND TOD MITMWER [VOL. 16

of alcohol. Schumacher (121) reported the isolation of a gram positive fatty acidfrom yeast. The removal of this fatty acid by hydrolysis in acid alcohol renderedthe yeast cell gram negative, and the gram positive state could be restored tothis cell by exposure to an alcoholic solution of the cell substances removed.Thus, many of the earlier workers looked hopefully for an explanation of grampositivity on the basis of unsaturated fatty acid content of the cells. However,Jobling and Peterson (80), trying to support the unsaturated fatty acid concept,showed that the affinity of iodine (iodine number) for cells of B. subtilis andClostridium tetani was almost identical with iodine affinity for such cells as E.coli and Salmonella typhi. This result, of course, would not be expected if grampositive cells contained unusual amounts of unsaturated fatty acids. Breinl(19) reported that, in general, gram negative cells took up more iodine than grampositives. Furthermore, Williams, Bloor, and Sandholzer (149) showed that ninestrains of gram negative enteric bacilli contained considerable amounts of un-saturated fatty acids. Thus the role of unsaturated fatty acids in the gram posi-tive state has not been rigorously demonstrated.Some workers (50) have believed that lecithin or lecithin-like substances were

responsible for the gram positive state. Others reported isolating a lipid extractwhich stained gram positively (136). Generally, however, the work involvinglecithin and lipoid extracts as the gram positive material has fared no better inits reproducibility than the work with the fatty acids. It has proved difficult orimpossible for some workers to convert gram positive organisms to a gramnegative state by the use of fat solvents such as alcohol, ether, or chloroform,even when used in the boiling state (55). The restoration of the gram positivestate by exposure to lecithin or lipid has also proved difficult to reproduce (47,48, 49). It appears that while lecithin or lipids are not the "gram positive" sub-stances they might serve some role, perhaps minor, in the gram differentiation.The work indicating that nucleoproteins are involved in the gram positive

state is much more convincing, and the results have proved to be reproduciblein the hands of various workers. Deussen (47, 48, 49) first presented extensiveexperiments which showed that acid or basic hydrolysis of gram positive cellsconverted them into a gram negative state. He also showed that gram positivitycould be restored by reintroducing nucleic acids into the extracted cells. LaterHenry and Stacey (68, 69) converted cells to a gram negative state by theaction of bile salts and observed that the substances given off during this conver-sion were magnesium ribonucleate, inert polysaccharides, and traces of protein.They also found that of the substances given off, magnesium ribonucleate couldbest restore gram positivity to the cells. Bartholomew and Umbreit (13) firstused specific crystalline enzymes to show that magnesium ribonucleate played adefinite role in gram positivity. Following the lead of Henry and Stacey, theyfound that crystalline ribonuclease converted all types of gram positive bacteriainto the gram negative state and also confirmed the ability of magnesium ribo-nucleate to restore gram positivity to these cells. However, gram positivity isnot due entirely to the presence or absence of ribonucleates since these havebeen demonstrated in gram negative forms, and it has been shown that gram


positive organisms are converted to a gram negative state even though only afraction of the ribonucleic acids is removed from the cell (70, 81, 93, 105). Ithas also been shown that ribonucleic acids from gram negative organisms suchas E. coli can be used to restore gram positivity to organisms such as C. welchiiwhich have previously been rendered gram negative by the action of bile salts(81). Henry and his collaborators (68, 69, 70) have suggested that gram posi-tivity involves the combination of ribonucleates with proteins of a very basicand novel type, and have reported the isolation of a gram positive nucleoproteinfrom gram positive organisms while they could not isolate it from gram negativeorganisms (70). This nucleoprotein could be hydrolyzed into gram negativenucleic acids and a gram negative protein, and its gram positivity restored by arecombination into a nucleoprotein. Stacey (130) has suggested that anothercontributing factor to gram positivity might be the ratio of ribonucleic acid(RNA) to desoxyribose nucleic acid (DNA). In C. welchii and the streptococcithe RNA/DNA ratio was about 8.1 (70) whereas that for some gram negativeorganisms was reported as 1.3 (130). However, this concept of ratio was notwell supported by published evidence, and the analyses of Mitchell and Moyle(105) demonstrated that no such relationship existed.

All data purporting to show a cell-free gram positive substance are open tosome objection unless a clear statement of the gram staining method used isgiven. This is true since the decolorization times of all stainable material arebound to vary, and thus many substances may appear to be gram positive, butonly in comparison with very easily decolorized substances. Henry, Stacey, andTeece (70) made no comparison of the gram positive nature of their cell-freesubstances with such controls as Neisseria, E. coli, and yeast. It would seem thatthe reports of cell-free gram positive substances should be accepted only withreservations until much additional work has been done.

Mitchell and Moyle (105) have suggested another possibility as to the natureof the gram positive substance. They found that determining nucleic acids inprotein by analysis of the phosphate present sometimes gave much higher valuesthan when the nucleic acids were determined by a pentose method. They showedthat for M. pyogenes var. aureus the phosphate method gave a phosphate contentof 30% over that accountable for on the basis of tetranucleotide structure. Thisexcess was present mostly in the ribonucleic acid fraction, and Mitchell andMoyle attributed it to an unknown phosphoric ester "XP". Mitchell and Moylethen suggested that there was a direct correlation between this "XP" esterand the gram positive state. They backed this claim with an analysis of 16different organisms. Their data do show a general correlation between "XP"ester and gram positivity but become less convincing on an examination ofdetails. For instance, if "XP" ester is responsible for gram positivity, it shouldbe present in all gram positive organisms, but the data of Mitchell and Moyleshow it to be absent in C. welchii. Also, some unmistakably gram negative or-ganisms such as E. coli, A. aerogenes, and Neisseria catarrhalis contained as muchor more of "XP" ester than gram positive forms such as bakers' yeast or B.aubtilis. Certainly "XP" ester is interesting and may constitute part of the reason

18 JAMES W. BARTHOLOMEW AND TOD MIX'rWER [VOL. 16

for gram positivity, but little evidence exists as yet that it is the gram positivesubstance.A recent report by Panijel (112) stated that a gram positive protein exists in

the male gamete of Ascaris megalocephala. His electrophoretic, spectrographic,and physicochemical studies showed the substance to be considerably differentfrom the gram positive substance of Henry and Stacey since it was an acid pro-tein of high nitrogen content and was not a nucleoprotein. The gram stainingprocedure was not given in this report, and as in other cases of extracellulargram positivity which have been reported, the reader is left uncertain as to thedegree of gram positivity involved.The evidence that certain nucleoproteins have an important role in gram

positivity is good. However, there is no reason to believe that they constitute agram positive substance in themselves, nor is there reason to believe that theyconstitute the sole factor producing gram positivity in the cell. Polysaccharidematerial has also been associated with gram positivity (68, 145), but as yet itsrole has not been experimentally determined.

2. The isoelectric point concept. Steam and Steam (132, 134) proposed a dif-ferent concept of the relationship between cell chemistry and the gram positivecharacteristic. They did not seek a gram positive substance but sought to cor-relate the over-all isoelectric point (or isoelectric range) of the cell substancewith the gram staining properties of the cell. They determined isoelectric pointson the basis of cell affinity for acid and basic dyes at various pH levels. Theirisoelectric point was the pH at which the cell showed equal affinity for both acidand basic dyes. They showed that increasing amounts of basic dyes were takenup as the pH increased over the isoelectric point, and increased amounts of aciddyes were taken up as the pH decreased from the isoelectric point. Using thismethod, they reported that gram positive organisms generally possessed iso-electric points somewhat below those of gram negative organisms. Steam andSteam reasoned that since the gram stain is carried out at a pH near 7, the grampositive cells would take up more dye than the gram negative and that they alsowould retain the dye with greater tenacity. Hence, the gram negative cells wouldbe easier to decolorize during the alcohol step. Their concept of the function ofiodine was that it simply increased the range of the differences between theisoelectric points of the gram positive and gram negative cells, thus makingthe differentiation more marked. They believed that the action of iodine was tooxidize certain substances in gram positive cells, thus rendering them moreacidic and lowering their isoelectric point, and that any oxidizing agent wouldhave the same effect as iodine and could be substituted for it.

This concept sounds logical in many respects, but it encounters serious diffi-culty in view of several known experimental facts. In the first place, manyworkers have not been able to confirm Steam and Steam's contention that anyoxidizing agent could replace iodine in the gram stain (26, 27, 106). Secondly,if this theory were correct, it should be possible to obtain a good gram differen-tiation even though the iodine is applied before the dye, and this has been shownnot to be true (26, 27, 64, 106). Also, if this theory were correct, it should be


applicable to crushed cells as well as intact cells, but as has been pointed outearlier, crushed cells do not stain gram positively. Levine (99) showed that themethods used by Steam and Steam to determine isoelectric points were in-capable of actually determining a true isoelectric point, and that results some-times varied more than two pH units. Lastly, Steam and Stearn did not use acounterstain in their work, and, hence, in the opinion of several workers (26, 27,106) they did not study the true gram differentiation. Steam and Steam'swork was valuable in that it contributed to general knowledge of staining phe-nomena; however, their conclusions regarding the mechanism of gram differen-tiation can hardly be accepted in the light of more recent knowledge.

3. Theories involving permeability. The possibility that gram differentiationcould be due to differences in the permeability of the cell as a whole, or of thecell membrane, has always received considerable consideration. These theorieshave been objected to on the basis that it is problematic that a killed cell retainsany selective permeability characteristics (45). However, the suggested per-meability differences are not necessarily of the selective type. The differencesmight be simply in permeability to substances in general.

Fischer (58) believed that the cell substance of gram positive bacteria is lesspermeable to penetrating substances than that of gram negative bacteria, hence,the greater difficulty in the decolorization of gram positive cells. However,Fischer did little experimentation to confirm his observation although he didconduct some experiments designed to show that the protoplasm of gram posi-tive bacteria consisted of coarser granules than that of gram negative bacteria.Brudny (21) expanded the concept of Fischer and proposed that the differencebetween gram positive and gram negative bacteria was permeability of the cellas a whole to iodine. He believed that a dye-iodine precipitate was formed in theinterstitial spaces between the large protoplasmic granules proposed by Fischer.This precipitate would then be difficult to remove. Gram negative bacteria,he claimed, had such a fine texture that the iodine could not penetrate into theinterstitial spaces. Thus, the dye-iodine precipitate was formed on the outsideof the cell and was easy to remove. The specific concepts of Fischer and Brudnydid not solve the problem of the mechanism of gram differentiation. However,their general idea of the significance of permeability is becoming accepted moreand more through later experiments.Benians (16, 17) furthered the permeability concept by suggesting that gram

differentiation was due to the permeability characteristics of the intact cellmembrane. His experiments were impressive in that they showed that a crushedgram positive cell always stained gram negatively. This experimental fact iseasily reproducible (26, 27, 106, 125) and must be considered by anyone advo-cating a strict chemical concept of gram positivity. Benians presented goodevidence against a strictly dye-iodine-cell substance complex when he showedthat stained cell debris actually decolorized easier after iodine treatment thanbefore. In his first paper (16) he theorized that iodine rendered the membranesof gram positive bacteria impermeable to alcohol, but he presented no directproof to substantiate this idea. In his later paper (17) he had altered his views

20 JAMES W. BARTHOLOMEW AND TOD M17TWER [VOL. 16

and divided all bacteria into the following three types: First, the regular grampositive organisms in which the dye and iodine penetrate into the cell and thereform a large molecule which does not easily pass out of the cell during decolor-ization. Second, the regular gram negative organisms in which the primary dyedoes not penetrate into the cell; therefore, no dye-iodine precipitate is formed.Third, the gonococcal type of gram negative organism in which both the dye andiodine penetrated into the cell, but the dye-iodine precipitate easily passed outof the cell during decolorization.Many of the conclusions of Benians are easily criticized. Steam and Steam

(134) showed that the addition of iodine to the dye molecule did not increaseits size sufficiently to influence its permeability. Benians based his conclusionthat the primary dye does not enter gram negative cells on the observation thatcells of E. coli were colorless when centrifuged down from a suspension in methylviolet. He ignored the fact that E. coli readily takes the stain on a heat fixedslide. Benians' work is impressive mostly for his observation on the effect ofcrushing the cell, and that iodine did not fix dye to cell protein, but rather de-taches it therefrom, thus explaining Gram's original observation that gram nega-tive substances are more easily decolorized after iodine treatment than before.Kaplan and Kaplan (83) presented evidence concerning the importance of

permeability of the cell membrane to iodine in alcoholic solution in the gramreaction. Their experiments showed that iodine in the decolorizer had a drasticretarding effect on decolorization. They concluded that two factors contributedto gram positivity. One was the low alcohol solubility of the dye-iodine com-pound, and the other was the lower permeability of gram positive cells to iodinein alcoholic solution. Thus, on decolorization the dye-iodine complex would bemuch slower in dissolution and in leaving the cell in gram positive bacteria.This concept was given additional weight by the observation of Mittwer, Bar-tholomew, and Kallman (106) that gram positive cells took up alcoholic iodinemore slowly than gram negative cells, and that only dye-iodine precipitates withcertain solubility characteristics could give a gram differentiation.

VI. THE SITE OF THE GRAM REACTION

Churchman (32, 33, 36) believed that all gram positive cells possessed agram positive external cortex layer which surrounded a gram negative internalmedulla. He supported this concept by showing that acriviolet conversion ofBacillus anthracis to a gram negative state was accompanied by a 40% reduc-tion in cell size as measured by the filar micrometer. This size difference wasobvious even upon visual examination of his preparations under the microscope.Similar visual evidence could also be seen when yeast was converted to a gramnegative state by holding in water suspensions at high temperatures. In addition,Churchman reported a 50% weight loss when cocci were converted to a gramnegative state.The validity of Churchman's concepts has never really been proved or dis-

proved. Certainly everyone who has studied gram stains has noticed occasionalsmall thin gram negatively staining cells in a chain of larger gram positively


staining cells. This would appear to confirm Churchman's concepts. However,a gram positive cell can be made to stain gram negatively by starvation, or ex-posure to ribonuclease, without an apparent size reduction (11, 135). Therefore,Churchman's cortex itself could not be the cause of gram positivity since it canbe made gram negative by the loss of chemical substances. Other workers haveconfirmed that reduction of cell size may occur when gram positive cells areconverted to a gram negative state (79, 106), but this size reduction may followby some hours the change in gram staining character (106). No proof is availableto show that Churchman's medulla is gram negative when surrounded by thegram positive cortex, although it is gram negative when the cortex is removed.Henry, Stacey, and Teece (70) have presented pictures of yeast partially con-verted to the gram negative state by the action of bile salts. These pictures sug-gest that an area analogous to Churchman's medulla is still strongly gram posi-tive even though the outer area has been made gram negative. Benians (16, 17)believed that all of the cell substances stained gram positively since debris ofgram positive cells crushed after gram staining was all gram positive.The relationship between the work of Churchman and Gutstein (65, 66) and

that of Eisenberg (55) is not clear, since they demonstrated an external "ecto-plasm" layer simply by a staining technique. Churchman disclaims any con-nection, but Gutstein reported that this ectoplasm layer disappeared whenyeast cells were converted to a gram negative state. However, it has been shown(66, 106) that this method would also stain an ectoplasm area on such gramnegative cells as E. coli.The work of Gutstein and Churchman has created considerable interest in

the site of the graim reaction. Churchman's work indicates that all of the grampositive material is located in an outer cortex area of the cell and that the pres-ence or absence of this cortex is directly associated with the gram posi-tive characteristic. Gutstein's work suggests that the gram positive material isassociated with the ectoplasm area, but not necessarily identical with it, sincegram negative cells also possessed this area. Lamanna and Mallette (93) statedthat the gram positive material included the cell wall as well as the cell cyto-plasm, a conclusion based on the observations that adjacent yeast cells could beshown to be in contact by both the gram and a cell wall stain. They also ob-served that in chains of gram positive cells containing an occasional gram nega-tive cell the gram negative cells took the cell wall stain whereas the cell wall areaof the gram positive cells was covered by the gram stain.Bartholomew and Mittwer (11) believed that the gram positive staining

material was interior to the cell wall area. In a series of photographs of the samecells, stained successively by gram and a cell wall stain, they found that thegram stained cell could be fitted within the area left unstained by the cell wallmethod. Cell measurements confirmed this concept except for a small area ofoverlapping.Some attempts have been made to observe the site of gram positivity by cell

sectioning methods. Schumacher (119) cut frozen sections of yeast at 5 ,u thick-ness. He found that all sectioned cells stained gram negatively so he could not


observe the site of gram positive material. Bartholomew (6) applied the newmethods of cutting thin sections of 0.05 ;i, but few definite conclusions could beobtained from the sections.

It appears then that the site of the gram reaction is still in doubt and it canbe spoken of only in general terms. Indications are that the gram positive areais certainly throughout the interior of the cell, and that it may include the cellwall either completely or partially.

VII. CONCLUSIONS

In summary, the gram positive state cannot yet be ascribed to any one uniquechemical compound or complex present in gram positive, but absent in gramnegative organisms. This is true even though several compounds have beenshown to be closely associated with the gram positive state. It also seems thatcell membrane permeability must be given an important role since crushed ormutilated cells always stain gram negatively. It is probable that chemical com-position and permeability are jointly involved in gram differentiation.The conversion of cells from gram positive to gram negative by removal of a

single specific chemical compound does not mean that that compound is solelyresponsible for gram positivity; especially since removal of a number of verydifferent compounds has been shown to result in loss of gram positivity; theseinclude magnesium ribonucleate, lipids, polysaccharides, fatty acids, nucleo-proteins, and unknown phosphoric esters. Likewise, the list of reagents or proc-esses which convert cells to a gram negative state is long: water, ribonucleases,lysozyme, boiling ether, fat solvents, acids, alkalies, acriviolet dye, oxidizingagents, bile salts, crushing, immune serum, antibiotics, ultraviolet radiation.It is likely that removal of lipoids, ribonucleates, polysaccharides, or other com-pounds results in a more permeable cell membrane through the disruption of itsmolecular architecture and thus affects the gram reaction.Through their own research and a study of the literature, the reviewers regard

the following brief description as probably what happens in a gram differentia-tion:

Primary stain. Gram positive and gram negative cells take up the primarystain by an ionic bond between the basic groups of the dye and the acidic groupsof the cell (8). It is possible that both types of cells take up approximately equalamounts of dye (105).

Iodine. The iodine, in aqueous solution, enters both types of cells. Here, itforms a precipitate with the dye present, either by competitive removal of thedye from the protein, or by addition to the dye in situ.

Alcohol. It is in this step that the actual differentiation takes place. In gramnegative cells the alcohol freely passes the cell membrane, dissolves and disso-ciates the dye-iodine precipitate or complex, and washes it away, leaving the cellcolorless. In gram positive cells the alcohol, the iodine in alcoholic solution, orboth pass through the membrane only with difficulty. Consequently, dissolutionof the dye-iodine compound is slow and the washing-out process is even slower.After the usual decolorization time, therefore, most of the dye-iodine compound


is left in the cell and the cell retains its color. It should be emphasized that thispermeability characteristic varies greatly on a wide scale, resulting in decolor-ization times of a few minutes to many hours. All gram positive cells will de-colorize if exposed long enough to alcohol. If various substances such as mag-nesium ribonucleate, mucopolysaccharides, or lipoproteins are removed from thecell membrane, the latter becomes more permeable to the decolorizer, and thecell reacts to the gram stain in the same manner as do normal gram negativecells.

Counterstain. The counterstain freely stains the gram negative cells whichhave been restored in the alcohol step to their original colorless condition.

For practical gram staining the reviewers wish to quote and emphasize therecommendation of Hucker and Conn (77) that "to determine the tendency of anorganism with regard to the gram stain, more than one staining procedure beused, and that preparations of the culture be prepared at various stages ofgrowth." Also, as recommended by Burke (24), control organisms should beplaced on the same slide to determine the validity of the gram stain. It is ouropinion that before a substance be called gram positive it should be able towithstand a decolorization step sufficient to render gram negative the Neisseriaspecies and also the nuclei of plant and animal cells as these two substances havebeen commonly accepted as being gram negative.

There is an enormous literature on the gram stain, and an immense amount ofresearch has been performed to elucidate its mechanism. More will undoubtedlybe done in the future. Much misinterpretation of results and much needless du-plication of effort could be avoided if a further counsel of Hucker and Conn(77) is followed: "It must be urged that all authors publishing results dependingin whole or in part on the gram stain describe their staining method in consider-able detail ... their results will have more significance in other quarters if theexact steps of the staining procedure are published."

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